Coordination of Host and Symbiont Gene Expression Reveals a Metabolic Tug-Of-War Between Aphids and Buchnera

Coordination of host and symbiont gene expression reveals a metabolic tug-of-war between aphids and Buchnera

Thomas E. Smitha,1 and Nancy A. Morana

aDepartment of Integrative Biology, University of Texas at Austin, Austin, TX 78712

Edited by Éva Kondorosi, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary, and approved December 19, 2019 (received for review September 27, 2019) Symbioses between animals and microbes are often described as Buchnera genome is highly reduced, retaining only ∼540 protein- mutualistic, but are subject to tradeoffs that may manifest as shifts coding genes, with many metabolic pathways for the biosynthesis in host and symbiont metabolism, cellular processes, or symbiont of essential nutrients fragmented or absent (8). However, Buchnera density. In pea aphids, the bacterial symbiont Buchnera is confined and bacteriocyte metabolic capabilities are complementary, such to specialized aphid cells called bacteriocytes, where it produces that together, host and symbiont can synthesize all EAAs required essential amino acids needed by hosts. This relationship is dy- for their survival (9, 10). Aphids regulate this shared metabolism to namic; Buchnera titer varies within individual aphids and among maximize the benefits of symbiosis via selective amino acid trans- different clonal aphid lineages, and is affected by environmental port (11–13), control of symbiont degradation (14, 15), and regu- and host genetic factors. We examined how host genotypic vari- lation of the number and size of bacteriocytes (16–18). On the other ation relates to host and symbiont function among seven aphid hand, Buchnera shows a limited capacity to respond to changing clones differing in Buchnera titer. We found that bacteriocyte gene conditions, displaying a reduced heat-shock response (19) and little expression varies among individual aphids and among aphid response to shifts in host diet (20, 21), although it exhibits small clones, and that Buchnera gene expression changes in response. transcriptomic changes during host development (22–24). Collec- Buchnera By comparing hosts with low and high titer, we found tively, these observations support the view that regulation of this Buchnera that aphids and oppositely regulate genes underlying symbiosis lies mostly with the host. MICROBIOLOGY amino acid biosynthesis and cell growth. In high-titer hosts, both Previous results showed that Buchnera titer varies significantly bacteriocytes and symbionts show elevated expression of genes among clonal lines of the pea aphid Acyrthosiphon pisum, with underlying energy metabolism. Several eukaryotic cell signaling high-titer lines exhibiting slightly reduced reproductive rates pathways are differentially expressed in bacteriocytes of low- ver- under laboratory conditions (25). Differences in titer occur even sus high-titer hosts: Cell-growth pathways are up-regulated in between aphid lines derived from the same maternal clone and low-titer genotypes, while membrane trafficking, lysosomal pro- thus sharing the same Buchnera haplotype, implying that titer is cesses, and mechanistic target of rapamycin (mTOR) and cytokine dependent on host genotype (25). The variation in titer among pathways are up-regulated in high-titer genotypes. Specific Buchnera functions are up-regulated within different bacteriocyte environments, with genes underlying flagellar body secretion and Significance flagellar assembly overexpressed in low- and high-titer hosts, respectively. Overall, our results reveal allowances and demands Symbioses between animals and microbes often involve host made by both host and symbiont engaged in a metabolic “tug- control of symbionts to harness a benefit. In aphids, Buchnera of-war.” symbionts produce amino acids for their hosts, enabling these insects to survive solely on plant sap. Buchnera abundance symbiosis | RNA-seq | host–symbiont interactions | aphids | Buchnera varies among aphid lineages, suggesting that distinct host– symbiont relationships exist among host genotypes. We found that Buchnera gene expression varies among aphid lineages, ymbioses between unrelated species can confer the ability to indicating that Buchnera also contributes to the host–symbiont use novel resources or withstand environmental stress or S relationship. Aphids and Buchnera inversely up-regulate amino natural enemies, enabling the expansion of ecological range (1–3). acid biosynthesis and cell-proliferation genes, and both aphid Symbioses are ubiquitous in eukaryotes, ancient in origin, and and Buchnera genes implicated in host–symbiont interactions often perpetuated by vertical transmission from mother to off- were differentially expressed, indicating crosstalk. Overall, we spring, and can involve one partner living intracellularly within a demonstrated that Buchnera alters its gene expression in re- host. Such close association comes with risks; symbiotic partners sponse to the host genetic background, and this likely affects are so specialized for living together that they are typically un- host gene expression. able to survive apart. Obligate symbioses require regulatory

feedback so that symbiont populations are contained and per- Author contributions: T.E.S. and N.A.M. designed research; T.E.S. performed research; form required functions. However, this regulation is complicated T.E.S. analyzed data; and T.E.S. and N.A.M. wrote the paper. by evolutionary conflicts of interest between partners, each of The authors declare no competing interest. which has the potential to evolve so as to promote its own fitness This article is a PNAS Direct Submission. at the cost of its partner’s fitness (4–7). Thus, the host–symbiont Published under the PNAS license. relationship is complex and dynamic, involving highly specific Data deposition: The processed transcriptomic data reported in this paper have been adaptations and counteradaptations. deposited in GenBank, http://www.ncbi.nlm.nih.gov/bioproject (BioProject ID no. In one of the best-studied examples of symbiosis, aphids obtain PRJNA564856). All custom scripts are available through GitHub at https://github.com/ essential amino acids (EAAs) from their bacterial symbionts, smit4227/aphid-Buchnera-RNAseq. Buchnera aphidicola, allowing these insects to feed exclusively on 1To whom correspondence may be addressed. Email: [email protected]. nutrient-poor plant sap. Buchnera resides within specialized aphid This article contains supporting information online at https://www.pnas.org/lookup/suppl/ cells called bacteriocytes that facilitate nutrient exchange between doi:10.1073/pnas.1916748117/-/DCSupplemental. host and symbiont. As a result of an endosymbiotic lifestyle, the First published January 21, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.1916748117 PNAS | January 28, 2020 | vol. 117 | no. 4 | 2113–2121 Downloaded by guest on September 26, 2021 A. pisum lines provides an opportunity to explore 1) how bac- ρ = 0.70, P = 9.3e-4) (Fig. 1). This result suggests that at least teriocytes vary among aphid genotypes, and 2) whether Buchnera part of the observed variation in titer can be attributed to vari- responds to this variation. ation in the number of Buchnera cells per bacteriocyte, and that We studied the transcriptomes of bacteriocytes from seven high-titer lines have both more Buchnera and correspondingly A. pisum genotypes, collectively representing a wide range in more Buchnera transcripts per bacteriocyte. symbiont titer, to look for correlations between host and symbiont gene expression. We found that Buchnera gene expression Test for Differential Expression. We next quantified the number of varies among host genotypes and among individual hosts, and reads mapped to each gene, normalized gene counts across that Buchnera gene expression is strongly correlated with aphid samples, and tested for differential gene expression using gene expression within bacteriocytes. Our results demonstrate DESeq2 (26). Buchnera gene expression was expected to show – that bacteriocyte metabolism and function differ greatly between relatively slight variation based on previous studies (19 21). We aphid genotypes with low and high symbiont titer, and that thus performed the likelihood ratio test (LRT) for A. pisum and Buchnera responds to these differences by altering its own gene Buchnera independently so that subtle changes in Buchnera gene expression. Specifically, we found evidence of a metabolic “tug- expression could be detected without being masked by the higher of-war” between host and symbiont, in which bacteriocytes and variance in expression of A. pisum genes. Of the 15,682 A. pisum genes detected from bacteriocytes, 4,395 (28.0%) were differ- Buchnera oppositely up-regulate amino acid biosynthesis and < cell-proliferation processes. entially expressed across all aphid genotypes (based on P 0.05 following adjustment for false discovery) (Dataset S1). Of the Results 616 annotated Buchnera genes detected, 100 genes (16.2%) were < On average, we obtained 16,500,112 high-quality 74-bp reads per differentially expressed across aphid genotypes, at P 0.05 individual pooled sample, with 61.0% of these reads mapping to (Dataset S2). Principal-component analysis of A. pisum gene the A. pisum genome and 33.4% to the Buchnera genome, with expression showed distinct grouping of samples by titer, with an average of 94.5% of reads mapped per sample (SI Appendix, AL1 and parental genotypes (intermediate titer) grouped be- Table S1). The mapped reads represent 15,682 out of 20,687 tween TL1/TL2 (low-titer) and AL3/AL4 (high-titer) genotypes SI Appendix A B (75.8%) and 616 of 616 (100%) uniquely annotated A. pisum and ( , Fig. S1 and ). Our analysis initially included two Buchnera clusters, each consisting of near-identical haplo- Buchnera genes, respectively. types (25, 27), with one cluster represented by only a single aphid Symbiont titer was previously measured in whole insects as the line (LSR1) (SI Appendix, Fig. S1C). To remove any influence of number of Buchnera genome copies per aphid genome copy by Buchnera haplotype on our downstream analyses, we repeated measuring single-copy gene abundance using qPCR (25). These the LRT excluding that line. The remaining samples showed sig- measures of titer differ significantly among clones, implying nificant overlap, but were roughly separated by parentage (maternal variation in the number of symbionts per bacteriocyte, the genotype) (SI Appendix,Fig.S1D). Overall, these results show that number of bacteriocytes per aphid, or a combination. Using a host genotype affects Buchnera gene expression but has a greater subset of the same aphid clones, we observed a positive corre- effect on aphid gene expression within bacteriocytes. lation between these previous measures of Buchnera titer for A limitation of this analysis is the small sample of aphid whole aphids and the proportion of Buchnera reads among ’ genotypes, causing variation in Buchnera titer to be confounded mapped reads for bacteriocyte samples (Pearson s correlation; with other genetically based variation. For example, all low-titer genotypes originating from A. pisum-Tucson mothers are green, while all high-titer genotypes originating from A. pisum-Austin mothers are red, so gene expression differences related to color or parentage may manifest as significant in the LRT. Indeed, the top A. pisum hit from the LRT is the tor gene, responsible for producing a red carotenoid, which is completely absent from the genomes of green A. pisum clones (28).

Correlation of Host and Symbiont Gene Expression. A. pisum clones differ in average symbiont titer, and titer also varies among in- dividuals within clones due to developmental and environmental variation. To examine the links between host and symbiont gene expression, independent of aphid genotype and titer estimates, we analyzed both host and symbiont transcriptomes within samples, consisting of bacteriocytes collected from five 7-d-old aphids (fourth-instar nymphs) harvested simultaneously from the same plant. For this analysis, we included all genes determined to be differentially expressed by the LRT as well as those displaying a high level of variation among samples (5,415 A. pisum and 225 Buchnera genes). We performed all possible pairwise correlations of host and symbiont gene expression, and then clustered host genes that were correlated with the same symbiont genes, and vice versa (SI Appendix, Fig. S2). The resulting correlation matrix shows that host genes and symbiont genes can each be distributed to two major groups, with each host group Fig. 1. Relationship of Buchnera titer in whole aphids to Buchnera repre- (A and B) being inversely correlated with the two Buchnera groups sentation in transcriptome reads across A. pisum lines. The Buchnera-to- (1, 2) (Fig. 2 and Datasets S3 and S4). For each gene included in aphid ratio of mapped transcriptome reads, calculated for bacteriocyte samples from each aphid line, increases with the average titer of each aphid this analysis, the module membership data (the correlation of an line, measured previously by qPCR as the ratio of Buchnera genome copies to individual gene with its module eigengene) are provided in aphid genome copies for whole aphids (25). Error bars indicate ±SD, while Datasets S1 and S2. We used the similarity profile (SIMPROF) the data were fit by linear regression (R2 = 0.485, P = 9.31e-4). permutation test to examine the possibility that the observed

2114 | www.pnas.org/cgi/doi/10.1073/pnas.1916748117 Smith and Moran Downloaded by guest on September 26, 2021 A MICROBIOLOGY

Fig. 2. Heatmap of host–symbiont gene correlations clustered by cocorrelation. For each sample, sequencing reads were mapped to host and symbiont genomes. Normalized, log-transformed gene expression data were filtered to exclude nonvariable genes. Each remaining A. pisum gene (5,415; 34.3%) was correlated with each remaining Buchnera gene (225; 36.5%) to yield a correlation matrix (Pearson’s correlation; ρ) of host–symbiont gene pairs. For both host and symbiont, genes with similar patterns of correlation were clustered, resulting in two primary groups of genes: groups A and B for A. pisum, and groups 1 and 2 for Buchnera. Arbitrary colors were assigned to designate modules of similarly correlated genes. Groups A and B consist of host genes underlying proliferation and energy metabolism, respectively. Groups 1 and 2 consist of symbiont genes underlying amino acid biosynthesis and energy metabolism, respectively.

pattern of gene clustering was due to random chance (29) and body color (ρ = 0.76, P = 1e-4) and parentage (maternal geno- found that all assigned aphid and symbiont gene modules were type; ρ = 0.76, P = 1e-4). Of the 2,213 genes in this module, 388 statistically valid (α < 0.01) (SI Appendix, Fig. S3). We then ex- genes were grouped within host–symbiont correlation analysis amined how host and symbiont gene modules correlated with group A (15.3%) and 728 in group B (32.9%) modules, while each other and with the average titer measured for each aphid 1,097 genes (49.6%) were excluded from the correlation analysis. clone (SI Appendix, Table S2). For both host and symbiont, gene For Buchnera, the three modules most strongly correlated with modules consisting of more strongly correlated genes were also titer were not statistically significant (ρ = 0.34 to 0.40, P = 0.3 to more strongly correlated with titer (SI Appendix, Fig. S4). Thus, 0.08) (SI Appendix, Fig. S5B and Dataset S2). Between these two despite the independence of this analysis from measures of titer, methods, many of the same host genes were found to be corre- titer is linked to the observed variation in gene expression. lated with titer, indicating that the methods largely agree on We also performed an analysis to directly correlate groups of which genes covary. coexpressed host or symbiont genes with titer, using weighted gene correlation network analysis (WGCNA) (30, 31), including Functional Roles of Host and Symbiont Genes with Correlated Expression. all genes for which expression was detected. For A. pisum, seven We next sought to identify functional roles for genes for which gene modules correlated with titer (average ρ = 0.59, P < 0.05) expression is correlated between host and symbiont. Using Gene (SI Appendix, Fig. S5A and Dataset S1). Of these, one module Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and stood out as the most strongly and significantly correlated (blue; Genomes) enrichment analyses for each host and symbiont gene ρ = 0.93, P = 5e-9); this module was also correlated with aphid module, we found stark differences in the metabolism and cell

Smith and Moran PNAS | January 28, 2020 | vol. 117 | no. 4 | 2115 Downloaded by guest on September 26, 2021 Fig. 3. Differential expression of bacteriocyte and Buchnera metabolism. A schematic representation of how bacteriocyte, Buchnera, and mitochondrial metabolism differ between host proliferative (P; blue) and metabolic (M; red) states, characteristic of aphid genotypes with low and high Buchnera titer, respectively. Arrows represent enzymatic reactions or the direction of small-molecule transport. Arrow color indicates up-regulation of the corresponding gene in either the proliferative (blue) or metabolic state (red), or the absence of differential gene expression (black). The large yellow rectangle represents a bacteriocyte, while Buchnera is depicted in gray as two concentric circles, representing the symbiosomal membrane (gray) and the Buchnera double membrane (black). A mitochondrion is depicted as a blue rectangle with double membrane (black). Broken light gray lines designate metabolites transported into or exported from Buchnera. GenBank accession numbers or Buchnera gene names are provided for chemical reactions (arrows) catalyzed by encoded enzymes. All labeled A. pisum genes are differentially expressed (P < 0.05 after adjustment for false discovery), while labeled Buchnera genes represent those with either P < 0.05 or inner-quartile range > 0.1. The 87 and 106 aphid genes involved in oxidative phosphorylation and transporter activity, respectively, represent genes belonging to KEGG pathway 00190 and GO:0005215, respectively.

2116 | www.pnas.org/cgi/doi/10.1073/pnas.1916748117 Smith and Moran Downloaded by guest on September 26, 2021 biology of both partners between low- and high-titer lines demonstrated that cystathionine contributes to Met production (Dataset S5). For A. pisum, cell-signaling and cell-cycle functions in Buchnera, suggesting that A. pisum CBS may operate in re- are up-regulated for low-titer genotypes, while energy metabo- verse to produce HCys. This hypothesis is supported by our data; lism and transmembrane transport are up-regulated for high- a SAM-dependent DNA methyltransferase (103308886) is also titer genotypes, suggesting that bacteriocytes exhibit distinct overexpressed for low-titer genotypes, indicating that the di- cellular states among aphid lines. Henceforth, we refer to host rection of metabolic flow runs from cystathionine to HCys, the and symbiont genes of groups A and 1, respectively, as charac- putative substrate for Met production in Buchnera (Fig. 3). Al- teristic of the host “proliferative” state, while host and symbiont ternatively, CBS is capable of a number of other reactions (34), genes of groups B and 2, respectively, characterize the host many of which generate hydrogen sulfide, a key signaling mol- “metabolic” state. Mapping of host genes onto individual KEGG ecule involved in aging in Drosophila melanogaster (35). pathways implicated specific cellular pathways as being associ- Genotypic differences in host and symbiont energy metabolism. Bac- ated with each titer state: the canonical Wingless (Wnt), Notch, teriocytes of high-titer aphid genotypes exhibit increased metabo- Hippo, Hedgehog, and TGFβ cell signaling pathways with low lism relative to those of low-titer genotypes, overexpressing titer, and the planar cell polarity (PCP)/Wnt and mechanistic genes underlying gluconeogenesis, nucleotide biosynthesis, and target of rapamycin (mTOR) signaling pathways as well as in- trehalose metabolism, as well as a number of mitochondrial tracellular vesicular transport and lysosomes with high titer pathways, including pyruvate metabolism, the tricarboxylic acid (Dataset S6). (TCA) cycle, oxidative phosphorylation, the glycine cleavage For Buchnera, the small number of genes limited the useful- complex, and proline metabolism (Fig. 3 and SI Appendix, Table ness of functional enrichment analyses (Dataset S5) but mapping S3). Likewise, in the host metabolic state, Buchnera is more genes to KEGG pathways (Dataset S6), aided by manual in- metabolically active than Buchnera of the host proliferative state, spection, indicated that Buchnera genes involved in energy me- up-regulating glycolysis (fba), the pentose phosphate pathway tabolism and stress response are up-regulated in high-titer (pgl, tktB), oxidative phosphorylation (nine genes), cofactor genotypes, and nearly all amino acid biosynthesis genes are up- biosynthesis (eight genes), and fatty acid biosynthesis (fabB, regulated in low-titer genotypes. Strikingly, almost all A. pisum fabI), for which Buchnera lacks the complete biosynthetic ma- genes involved in amino acid biosynthesis are up-regulated in the chinery (Fig. 3) (8). In bacteriocytes, gluconeogenesis is pro- high-titer state; that is, amino acid biosynthesis is oppositely posed to supply glucose for Buchnera, as well as intermediates regulated between host and symbiont. In order to better un- for uracil (32) and polyol biosynthesis (10). The increased ex- derstand these differences, we manually inspected several hun- pression of trehalase and 10 genes encoding trehalose trans- dred genes for each host and symbiont gene group, compiling a porters implies that trehalose is also a significant source of MICROBIOLOGY short list of genes belonging to enriched GO/KEGG pathways, or glucose for bacteriocytes (Fig. 3). Bacteriocyte mitochondria related to genes and gene functions previously implicated in the have been proposed to initiate gluconeogenesis by the synthesis aphid–Buchnera symbiosis (SI Appendix, Tables S3 and S4). The and export of phosphoenolpyruvate (PEP), consume Buchnera- results of our inspection are summarized below. derived pyruvate in the TCA cycle, and regulate folate metabolism Genotypic differences in host and symbiont amino acid biosynthesis. Both viatheglycinecleavagecomplex(32). Our data indicate that mi- aphid and Buchnera genes are required to synthesize the EAAs tochondria are also important for proline metabolism, as evidenced Phe, Ile, Leu, Val, and Met, as well as the nonessential Cys and by the up-regulation of proline dehydrogenase (PRODH; 100160914), Tyr, while Buchnera alone can make the remaining five EAAs: pyrroline 5-carboxylate reductase (P5CR; 100167377), and orni- His, Lys, Arg, Trp, and Thr (8). Among Buchnera EAA bio- thine aminotransferase (OAT; 100168809) in the metabolic state synthetic genes, we observed differential expression of genes (Fig. 3 and SI Appendix,TableS3), which are collectively involved underlying biosynthesis of Leu, Ile, Val, Trp, Lys, Thr, and His; in oxidative stress (36). Taken together, the host proliferative state 16 of the 20 genes in these pathways were associated with the is characterized by elevated EAA biosynthesis by Buchnera,while host proliferative state (Fig. 3 and SI Appendix,TableS4). the host metabolic state is characterized by increased energy Conversely, differentially expressed aphid genes involved in AA metabolism in both host and symbiont. biosynthesis were almost all up-regulated in the host metabolic Host and symbiont cometabolism. We found evidence for two in- state (Fig. 3). We also observed up-regulation of genes encoding stances of host–symbiont cobiosynthesis that were previously numerous small-molecule and ion transporters in the metabolic only hypothesized in silico. Based on their gene repertoire, state (Fig. 3), including two transporters recently implicated in aphids lack the capacity to regenerate nucleotides by salvaging regulating nutrient exchange within bacteriocytes, the Gln free nucleobases (37), while Buchnera is predicted to produce transporter ApGLNT1 (12) and the AA transporter and nutrient adenine via polyamine biosynthesis using host-derived ornithine, sensor AAAP-536 (13). a product of Arg and Pro metabolism (37). We observed up- Despite the tendency for up-regulation of bacteriocyte AA regulation in the host metabolic state of both host and symbiont production in the host metabolic state, aphid genes encoding glu- genes underlying these processes, including ornithine decarboxylase tamine synthetase (GS; 100574604) and cystathionine β-synthase (100161119), speD, speE, pfs, adenine phosphoribosyltransferase (CBS; 100166111) were more highly expressed in the proliferative (100162276, 100161922), IMP and GMP synthases (100167425 and state (Fig. 3). In bacteriocytes, GS recycles ammonia waste into Gln 100165217, respectively), and 5′ nucleotidase (100167522), reflect- as part of the glutamine oxoglutarate amidotransferase (GOGAT) ing an increased demand for guanosine, for which Buchnera is cycle. Aphids reared on nitrogen-poor plants up-regulate GS auxotrophic (Fig. 3) (37). Additionally, we detected up-regulation of expression in bacteriocytes (32), suggesting that low-titer states host genes involved in the synthesis and transport of polyols, in- might also reflect nitrogen limitation. Two aphid GS genes cluding two putative xylulose reductases (100145846, 100144913), (100160139 and 100165282) exhibit bacteriocyte-specific ex- three aldo-keto reductases (100158792, 100164695, 100161529), pression (9), and our analysis implicates a third, distinct GS gene and the entomoglyceroporin ApAQP2 (100168499), which, unlike in bacteriocyte AA metabolism. CBS synthesizes cystathionine most aquaporins, can transport large polyols (Fig. 3) (38, 39). All of from homocysteine (HCys) in the transsulfuration (TS) pathway these genes are more abundant in bacteriocytes relative to other that converts Met to Cys. Our data show that CBS expression is aphid tissues (9, 10, 40). Polyols act as osmoprotectants, shielding oppositely regulated relative to other TS pathway genes, in- bacteriocytes from osmotic stress potentially linked to the host diet cluding cystathionine γ-lyase (CGL; 100159197, 100168016, (41). Polyol biosynthesis is also important for symbiosis in weevils 100159560) and HCys S-methyltransferase (100168557). Though (42), suggesting that adoption of this environmental stress response CBS reactions are typically unidirectional, Russell et al. (33) for endosymbiosis is widespread. The Buchnera genome contains

Smith and Moran PNAS | January 28, 2020 | vol. 117 | no. 4 | 2117 Downloaded by guest on September 26, 2021 the mtlA and mtlD genes, encoding mannitol permease and B (100166823), respectively, and members of the Hippo, Hedgehog, mannitol-1-phosphate-5-dehydrogenase, respectively; the latter is canonical Wnt, Notch, Decapentaplegic (Dpp)/TGFβ, insulin, and overexpressed in the metabolic state (Fig. 3). These results sup- semaphorin cell signaling pathways (Fig. 4A and SI Appendix,Table port the hypothesis that host-derived polyols are used by Buchnera S3). Many of these genes are up-regulated in bacteriocytes in re- as a carbon source (39). sponse to EAA limitation; bacteriocytes increased in number and Genotypic differences in host and symbiont cell biology. Bacteriocytes size, suggesting that bacteriocytes may regulate their growth in of the host proliferative state show up-regulation of genes underlying order to increase EAA production by accommodating additional cell growth and proliferation, including those involved in chromatin Buchnera (16). We also detected overexpression of homeobox remodeling and DNA replication, various transcription factors, the transcriptionfactorsknowntoplay a role in bacteriocyte develop- G1-, G2-, and M-phase cyclins D (100167546), A (100160996), and ment, including ultrabithorax (Ubx; 100160569) and two engrailed

Fig. 4. Bacteriocyte and Buchnera cell biology in host proliferative and metabolic states. A schematic representation of how cell biology differs in (A) bacteriocytes and (B) Buchnera, between host proliferative (P; blue) and metabolic (M; red) states, characteristic of aphid genotypes with low and high Buchnera titer, respectively. Cartoons represent structural features, proteins, or other biomolecules occurring within Buchnera cells, while arrows indicate protein–protein interactions or cellular processes. Colored cartoons/arrows indicate differential expression (P < 0.05 after adjustment for false discovery, or inner-quartile range > 0.25 for A. pisum or 0.1 for Buchnera) or the absence of differential gene expression (black/gray). DNA and RNA are represented by orange lines. Full gene names for differentially expressed A. pisum and Buchnera genes are provided in SI Appendix, Tables S3 and S4, respectively. A. pisum genes not differentially expressed but depicted in the figure include mishappen (Msn, 100162642), slipper (Slpr, 100161755), hemipterous (Hep, 100165019), basket (Bsk, 100163276), kayak (Kay, 100164827), jun-related antigen (Jra, 100161138), Drosophila TNF receptor-associated factor 2 (dTRAF2, 100575177), hippo (Hpo, 100168569), warts (Wts, 100161589), Van Gogh/strabismus (Stbm, 100166671), disheveled (Dsh, 100166217), fused (Fu, 100169043), short neuropeptide F (NPF, 100575917), and tuberous sclerosis complex (TSC1, 100163135; TSC2, 100164018).

2118 | www.pnas.org/cgi/doi/10.1073/pnas.1916748117 Smith and Moran Downloaded by guest on September 26, 2021 paralogs (En; 100161671, 1001670540) (Fig. 4A) (43, 44). Genes increased Buchnera turnover and nutrient/protein transport asso- mediating lipid transfer from hemolymph-circulating lipophorin, ciated with elevated oxidative stress and metabolism, respectively. including apolipophorin (100159010) and a number of low-density Genotypic differences in host and symbiont stress responses. Another lipophorin receptor-related proteins (LDLRs) (100168886, signature of the host metabolic state is activation of immunity or 100168992, 100568980, 100169728), were also up-regulated in the stress-response pathways in both host and symbiont. The up- proliferative state (Fig. 4A). Signal transduction via LDLR-type regulation of host cytokine pathways, including Jak/STAT (dome- proteins also overlaps with Wnt, Hedgehog, Dpp, and mTOR less, hopscotch), JNK (eiger, alphabet, raw), and Toll (spätzle) signaling pathways (45, 46). Buchnera of proliferative-state bac- pathway constituents, indicates an activated immune response (Fig. teriocytes showed up-regulation of genes involved in carbohydrate 4A and SI Appendix,TableS3). In a previous study, domeless transport (ptsH), glycolysis (gpmA), DNA synthesis (holB, nrdA), (100163919) was overexpressed in bacteriocytes of EAA-starved DNA repair (sbcB), transcription (rpoB, rho, nusG, topA), trans- aphids, which are also believed to contain a high Buchnera abun- lation (infA, infC, prfA), and many tRNA and ribosomal genes dance (16). Thus, the activation of cytokine pathways could repre- (Fig. 4B and SI Appendix,TableS4). Buchnera shows up-regulation sent a host response to increased bacterial load. We detected up- of putative transcription factors fis, ihfA/himA,andcsrA,thelatter regulation of genes encoding lysosomal enzymes and membrane ofwhichisknowntoactivateglycolysis (47), as well as ebfC/ybaB, proteins, suggesting a higher rate of symbiont turnover, potentially encoding a nucleoid-associated protein believed to bind DNA and via the same mechanisms documented previously in aging aphids regulate gene expression (Fig. 4B) (48). Overall, this pattern of (14, 15). Buchnera also up-regulates stress-response genes in the expression suggests that the proliferative state is characterized by host metabolic state (ibpA, grpE, mutS, nfo, mfd), suggesting ele- larger and more numerous bacteriocytes, with Buchnera heavily vated oxidative phosphorylation activity (Fig. 4B). In this state, invested in gene expression and protein synthesis, likely to enhance Buchnera also overexpresses RNA-processing genes (rnpA, rnpB, production of EAA biosynthetic proteins. rne) and several tRNA-maturation genes (queA, miaA, yeaZ, yheL, We observed overexpression of multiple vesicular trafficking ygjD, cca), many of which modify tRNAs, which themselves are also pathways in bacteriocytes associated with the host metabolic overexpressed (Fig. 4B and SI Appendix,TableS4). The increase in state. Vesicular trafficking has been implicated in the degrada- genes underlying tRNA modification may reflect a need for in- tion of Buchnera cells in aging aphids, as evidenced by the ac- creased translational fidelity (56). Finally, several of the few trans- cumulation of Rab7 GTPase at the host-derived symbiosomal port proteins encoded by the Buchnera genome are up-regulated membrane that envelopes Buchnera (15). Overexpression of (57), including the Rnf electron transport chain (rnfD, rnfE)(58),an Rab7 (100161383) and its binding partners (100160179, 100572849, ABC transporter involved in detachment of outer-membrane lipo- 100168146), which together mediate endosome-to-lysosome traf- proteins from the inner membrane (lolC-E) (59), the outer- MICROBIOLOGY ficking (49), suggests increased Buchnera turnover in the metabolic membrane porins ompA and ompF,andtheZnimporterznuB state (Fig. 4B and SI Appendix, Table S3). Rab7 also mediates (Fig. 4B). All of these functions could contribute to or help mitigate trafficking away from endosomes by interaction with the retromer oxidative stress. Finally, aphids up-regulate several uncharacterized complex (50), a process that depends on actin for sorting cargo genes that could contribute to stress or immunity signaling path- within endosomes (51). We detected overexpression of genes ways: neuropeptide F receptors (100570116, 10016184, 100161936, encoding retromer proteins (100160254, 100166208, 100168020), 100166824), plexin-A3 semaphorin receptors (100575173, 100568670), as well as components of the Arp2/3 (100166913, 100159878) and and putative odorant-binding proteins (OBPs) (100158928, 100162203) WASH complexes (100165971, 103311828, 100165614), which (Fig. 4A), of which the latter two are significantly enriched in regulate actin polymerization (51) (Fig. 4B and SI Appendix,Table bacteriocytes relative to other aphid tissues (9, 10, 40). In tsetse S3). Up-regulation of the retromer complex could indicate recy- flies, gut-associated symbionts induce expression of OBPs and cling of Buchnera and symbiosomal compartments following lyso- stimulate crystal cell differentiation, implicating a role in host somal degradation. Additionally, we detected overexpression of immunity (60). genes underlying pathways mediating trans-Golgi network (TGN)- Genotypic differences in host–symbiont interactions. We observed dis- to-plasma membrane (PM) trafficking (100159221, 100164636, crepancies in the expression of both host and symbiont genes 100160609, 100167300, 100163694, 100166962) and endoplasmic that mediate host–symbiont interactions. We found that ex- reticulum (ER)-to-TGN trafficking (100159072, 100571150, pression of Buchnera flagellar basal body (FBB) genes varied 100159596) (SI Appendix,TableS3) (49, 50), indicative of among host genotypes (Fig. 4B). Buchnera FBBs are functional increased membrane protein synthesis and recycling. type III secretion systems (61) that are abundant on the Buchnera The host metabolic state also shows up-regulation of signaling cell surface (62) and are believed to secrete one or more as yet pathways with the potential to regulate membrane trafficking. unidentified Buchnera effector proteins. Buchnera genes encoding The mTOR complex 1 (mTORC1) is believed to sense Buchnera- the export apparatus (fliI, fliP, fliR), rod protein fliE,andan derived EAAs within bacteriocytes via vATPase (10 genes) and associated cell wall-degrading enzyme (flgJ) were up-regulated SLC38A9 (100158916) (11), while the PCP/Wnt pathway, in- in the host metabolic state, while genes encoding additional rod cluding Daam1 (100160941) and RhoA (100160039), mediates proteins (flgC, flgF) and secreted components of the hook (flgD, cytoskeletal rearrangement (Fig. 4A and SI Appendix, Table S3) flgE, fliK) were up-regulated in the host proliferative state. Thus, (52, 53). Genes underlying lipid biogenesis, which likely affect FBB assembly is up-regulated in the metabolic state, while membrane trafficking, are also up-regulated; these include genes active secretion is up-regulated in the proliferative state. In- encoding torsin (100572759), lipin (100164616), and choline- terestingly, the only putative Buchnera transcription factor up- phosphate cytidylyltransferase (100160383) (SI Appendix, Table regulatedinhigh-titer genotypes, cspE, regulates expression of S3) (54). Membrane trafficking and regulation thereof could be stress-response and flagellar genes in Salmonella enterica (63), necessary to house a large population of Buchnera. Indeed, suggesting that CspE may control FBB assembly in Buchnera. Buchnera shows up-regulation of DNA replication genes (dnaA) We also observed up-regulation of peptidoglycan (PG)-degrading and cell-division genes ftsA and minE. Based on experimental genes associated with high-titer genotypes, including two aphid studies in Escherichia coli, MinE, when in complex with MinD, lysozymes (100167742, 100160909) and ldcA, amiD,andrlpA1-5, initiates cell division by displacing the inhibitor MinC at the which are genes horizontally acquired by aphid ancestors from cleavage site (Fig. 4B) (55), indicating that Buchnera is actively bacterial genomes (Fig. 4B and SI Appendix,TableS3) (64, 65). It dividing in the host metabolic state. Collectively, our data sug- has been proposed that the encoded enzymes protect Buchnera gest that up-regulation of the cellular machinery required for from the host immune system by degrading Buchnera-derived lipid membrane trafficking in bacteriocytes may be associated with II (66). Supporting this proposal, RNAi knockdown of ldcA and

Smith and Moran PNAS | January 28, 2020 | vol. 117 | no. 4 | 2119 Downloaded by guest on September 26, 2021 amiD resulted in reduced symbiont abundance (66). However, does maintain some limited capacity for gene regulation within aphids lack the canonical PG recognition proteins that bind lipid bacteriocytes. For example, the Buchnera genome encodes a II and elicit an immune response (67), indicating an alternative number of putative transcriptional regulators (ihfAB, dksA, fis, role for PG degradation in the aphid–Buchnera system. For ex- pepA, hns, csrA, and ybaB/ebfC) (8) and has recently been shown ample, PG degradation is required for cell division (68). Buchnera to encode a number of small RNAs (23), at least some of which encodes a complete cell-wall synthesis pathway but lacks many of appear to regulate expression of specific Buchnera metabolic the genes involved in cell-wall degradation (69). Host provisioning genes, including carB of the Arg biosynthesis pathway (24). How- of key enzymes in PG degradation could enable direct control of ever, Buchnera gene expression shows little response to host diet symbiont proliferation. The mealybug genome contains seven (20, 21), and the small transcriptional changes observed lack any horizontally acquired PG synthesis genes (70) encoding enzymes clear link to nutritional demands (74). In contrast, our results imply that were recently shown to contribute to cell-wall synthesis in its that Buchnera gene expression responds to variation in host geno- bacterial endosymbiont (71), which lacks the complete PG syn- type. One explanation for why Buchnera might respond to host thesis pathway, suggesting that host control of cell-wall metabo- genotype but not to host diet is that bacteriocytes maintain ho- lism could be more widespread. meostasis, and thus a constant Buchnera environment, under dietary Discussion changes, whereas different host genotypes impose sufficiently dif- Clonal lines of A. pisum differ in Buchnera titer, and these differ- ferent environments within bacteriocytes that Buchnera must adjust ences at least partly reflect differences in symbiont density within its gene expression to compensate. bacteriocytes (Fig. 1). Indeed, we found substantial differences in In summary, our results demonstrate that Buchnera regulates aphid gene expression within bacteriocytes among seven full- and its own gene expression in response to host genotypic variation. half-sibling clonal lines of A. pisum representing a wide range in The bacteriocyte state for a given aphid genotype derives from Buchnera titer. Furthermore, we found that Buchnera gene ex- the specific combination of host alleles, for which Buchnera pression varies among the aphid clones, even for the same Buchnera optimizes its own gene expression (within its ability) and host– haplotype. By correlating host and symbiont gene expression, two symbiont interactions. Regardless of whether change in Buchnera major gene groups were recognized for both host and symbiont, gene expression leads to a shift in titer or vice versa, Buchnera is each of which describes a unique cellular and metabolic state within able to take advantage of its host in favorable environments (high bacteriocytes or Buchnera. These states illustrate tradeoffs between titer) or compensate for its host in poor environments (low titer). host and symbiont; Buchnera of low-titer genotypes invests more Thus, the relationship between aphids and Buchnera is dynamic, heavily in amino acid biosynthesis, an activity that benefits the host, resembling a tug-of-war in which both host and symbiont exert while bacteriocytes of high-titer genotypes invest more heavily in allowances and demands on each other. providing and processing metabolites to and from Buchnera,atcost to the host (Fig. 3). Titer and reproductive rate show a weak but Materials and Methods significant negative correlation in laboratory-bred A. pisum clones Aphid Rearing, Dissections, and RNA Extractions. Clonal lines of A. pisum were (25). This observation is corroborated by our gene expression data; reared in cup cages on broad bean (Vicia faba), with three sublines per the up-regulation of cell pathways underlying growth and pro- clone. RNA was extracted from bacteriocytes dissected from five individual liferation could explain the slightly faster reproductive rate of low- aphids per subline. More details are provided in SI Appendix, Materials titer clones, while the increased metabolism and concomitant in- and Methods. duction of stress-response pathways align well with the slightly slower reproductive rate of high-titer clones. Overall, our results demon- RNA-Seq Library Construction. Libraries were constructed from bacteriocyte strate that the host–symbiont relationship can vary substantially RNA using the Illumina Ultra II Directional RNA Library Kit (New England among aphid clones. Biolabs) with random priming after digestion of genomic DNA and removal of ribosomal RNA. More details are provided in SI Appendix, Materials While Buchnera titer is affected by host genotype, it is also and Methods. affected by environmental factors, including the host’s diet (18, 72). Many aphid species feed on multiple host plants that differ RNA Sequencing and Analysis. Libraries were sequenced on an Illumina in nutritional content, including AA composition (73), poten- NextSeq500 instrument. Raw data were processed and mapped to the tially contributing to the change in Buchnera titer that can occur A. pisum and Buchnera genomes (assembly Acyr_2.0 and str. APS, respectively, when aphids are moved between plant species (18). Individual downloaded from the NCBI). Differential gene expression was calculated using aphids of the same clonal lineage are also variably affected by DESeq2 with the likelihood ratio test (26). Correlation of host and symbiont host plant species (18). Aphids also alter the volume and number genes was performed using custom R scripts. A data-transformation workflow of bacteriocytes in response to host diet (16), presumably as a is provided in SI Appendix,Fig.S2. The results were validated using the means of regulating symbiont capacity. Previous research showed SIMPROF permutation test (29). For WGCNA, we used customized scripts that bacteriocyte growth is accompanied by up-regulation of following instructions recommended by the authors (31). More details are many of the same genes and pathways that are overexpressed in provided in SI Appendix, Materials and Methods. low-titer aphid genotypes (16). Collectively, these observations suggest that aphids respond to nutritional variability by regulat- Data Availability. Processed transcriptomic data were uploaded to GenBank ing Buchnera abundance. The observed differences among host under BioProject ID no. PRJNA564856. All custom scripts are available through genotypes in bacteriocyte gene expression parallel those in re- GitHub at https://github.com/smit4227/aphid-Buchnera-RNAseq. sponse to changes in environmental conditions, implicating the ACKNOWLEDGMENTS. We thank Kim Hammond for help with aphid cultures same underlying mechanisms in both scenarios. and the Genome Sequencing and Analysis Facility at the University of Texas at The aphid host appears to take primary responsibility for Austin for sequencing services. This work was supported by NIH Award regulating the metabolic output of bacteriocytes, but Buchnera 5F32GM126706 (to T.E.S.) and NSF Award 1551092 (to N.A.M.).

1. P. Baumann, Biology bacteriocyte-associated endosymbionts of plant sap-sucking in- 4. G. M. Bennett, N. A. Moran, Heritable symbiosis: The advantages and perils of an sects. Annu. Rev. Microbiol. 59, 155–189 (2005). evolutionary rabbit hole. Proc. Natl. Acad. Sci. U.S.A. 112, 10169–10176 (2015). 2. J. J. Wernegreen, Mutualism meltdown in insects: Bacteria constrain thermal adap- 5. J. R. Garcia, N. M. Gerardo, The symbiont side of symbiosis: Do microbes really ben- tation. Curr. Opin. Microbiol. 15, 255–262 (2012). efit? Front. Microbiol. 5, 510 (2014). 3. L. V. Flórez, P. H. Biedermann, T. Engl, M. Kaltenpoth, Defensive symbioses of animals 6. P. J. Keeling, J. P. McCutcheon, Endosymbiosis: The feeling is not mutual. J. Theor. with prokaryotic and eukaryotic microorganisms. Nat. Prod. Rep. 32, 904–936 (2015). Biol. 434,75–79 (2017).

2120 | www.pnas.org/cgi/doi/10.1073/pnas.1916748117 Smith and Moran Downloaded by guest on September 26, 2021 7. C. D. Lowe, E. J. Minter, D. D. Cameron, M. A. Brockhurst, Shining a light on ex- 41. T. Wilkinson, D. Ashford, J. Pritchard, A. Douglas, Honeydew sugars and osmoregu- ploitative host control in a photosynthetic endosymbiosis. Curr. Biol. 26, 207–211 lation in the pea aphid Acyrthosiphon pisum. J. Exp. Biol. 200, 2137–2143 (1997). (2016). 42. A. Heddi et al., Molecular and cellular profiles of insect bacteriocytes: Mutualism and 8. S. Shigenobu, H. Watanabe, M. Hattori, Y. Sakaki, H. Ishikawa, Genome sequence of harm at the initial evolutionary step of symbiogenesis. Cell. Microbiol. 7, 293–305 the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407,81–86 (2005). (2000). 43. C. Braendle et al., Developmental origin and evolution of bacteriocytes in the aphid- 9. A. K. Hansen, N. A. Moran, Aphid genome expression reveals host-symbiont co- Buchnera symbiosis. PLoS Biol. 1, E21 (2003). operation in the production of amino acids. Proc. Natl. Acad. Sci. U.S.A. 108, 2849– 44. Y. Matsuura, Y. Kikuchi, T. Miura, T. Fukatsu, Ultrabithorax is essential for bacter- 2854 (2011). iocyte development. Proc. Natl. Acad. Sci. U.S.A. 112, 9376–9381 (2015). 10. A. Poliakov et al., Large-scale label-free quantitative proteomics of the pea aphid- 45. E. M. Gleixner et al., V-ATPase/mTOR signaling regulates Megalin-mediated apical Buchnera symbiosis. Mol. Cell. Proteomics. 10, M110.007039 (2011). endocytosis. Cell Rep. 8,10–19 (2014). 11. E. B. James, H. Feng, A. C. C. Wilson, mTOR complex 1 implicated in aphid/Buchnera 46. J. R. Lindner et al., The Drosophila Perlecan gene trol regulates multiple signaling host/symbiont integration. G3 (Bethesda) 8, 3083–3091 (2018). pathways in different developmental contexts. BMC Dev. Biol. 7, 121 (2007). 12. D. R. Price et al., Aphid amino acid transporter regulates glutamine supply to in- 47. J. Timmermans, L. Van Melderen, Post-transcriptional global regulation by CsrA in tracellular bacterial symbionts. Proc. Natl. Acad. Sci. U.S.A. 111, 320–325 (2014). bacteria. Cell. Mol. Life Sci. 67, 2897–2908 (2010). 13. H. L. Lu, C. C. Chang, A. C. Wilson, Amino acid transporters implicated in endocytosis 48. B. L. Jutras et al., EbfC (YbaB) is a new type of bacterial nucleoid-associated protein of Buchnera during symbiont transmission in the pea aphid. Evodevo 7, 24 (2016). and a global regulator of gene expression in the Lyme disease spirochete. J. Bacteriol. 14. K. Nishikori, K. Morioka, T. Kubo, M. Morioka, Age- and morph-dependent activation 194, 3395–3406 (2012). of the lysosomal system and Buchnera degradation in aphid endosymbiosis. J. Insect 49. A. Wandinger-Ness, M. Zerial, Rab proteins and the compartmentalization of the – Physiol. 55, 351 357 (2009). endosomal system. Cold Spring Harb. Perspect. Biol. 6, a022616 (2014). 15. P. Simonet et al., Bacteriocyte cell death in the pea aphid/Buchnera symbiotic system. 50. Z. Li, G. Blissard, The vacuolar protein sorting genes in insects: A comparative genome – Proc. Natl. Acad. Sci. U.S.A. 115, E1819 E1828 (2018). view. Insect Biochem. Mol. Biol. 62, 211–225 (2015). 16. S. Colella et al., Bacteriocyte reprogramming to cope with nutritional stress in a 51. M. N. Seaman, A. Gautreau, D. D. Billadeau, Retromer-mediated endosomal protein phloem sap feeding hemipteran, the pea aphid Acyrthosiphon pisum. Front. Physiol. sorting: All WASHed up! Trends Cell Biol. 23, 522–528 (2013). 9, 1498 (2018). 52. R. Gombos et al., The formin DAAM functions as molecular effector of the planar cell 17. T. L. Wilkinson, D. Adams, L. B. Minto, A. E. Douglas, The impact of host plant on the polarity pathway during axonal development in Drosophila. J. Neurosci. 35, 10154– – abundance and function of symbiotic bacteria in an aphid. J. Exp. Biol. 204, 3027 10167 (2015). 3038 (2001). 53. T. Matusek et al., The Drosophila formin DAAM regulates the tracheal cuticle pattern 18. Y. C. Zhang, W. J. Cao, L. R. Zhong, H. C. J. Godfray, X. D. Liu, Host plant determines through organizing the actin cytoskeleton. Development 133, 957–966 (2006). the population size of an obligate symbiont (Buchnera aphidicola) in aphids. Appl. 54. M. Grillet et al., Torsins are essential regulators of cellular lipid metabolism. Dev. Cell – Environ. Microbiol. 82, 2336 2346 (2016). 38, 235–247 (2016). 19. J. L. Wilcox, H. E. Dunbar, R. D. Wolfinger, N. A. Moran, Consequences of reductive 55. Z. Hu, J. Lutkenhaus, Topological regulation of cell division in Escherichia coli involves – evolution for gene expression in an obligate endosymbiont. Mol. Microbiol. 48, 1491 rapid pole to pole oscillation of the division inhibitor MinC under the control of MinD 1500 (2003). and MinE. Mol. Microbiol. 34,82–90 (1999). 20. N. A. Moran, G. R. Plague, J. P. Sandström, J. L. Wilcox, A genomic perspective on 56. A. K. Hansen, N. A. Moran, Altered tRNA characteristics and 3′ maturation in bacterial

nutrient provisioning by bacterial symbionts of insects. Proc. Natl. Acad. Sci. U.S.A. MICROBIOLOGY symbionts with reduced genomes. Nucleic Acids Res. 40, 7870–7884 (2012). 100 (suppl. 2), 14543–14548 (2003). 57. H. Charles et al., A genomic reappraisal of symbiotic function in the aphid/Buchnera 21. N. A. Moran, H. E. Dunbar, J. L. Wilcox, Regulation of transcription in a reduced symbiosis: Reduced transporter sets and variable membrane organisations. PLoS One bacterial genome: Nutrient-provisioning genes of the obligate symbiont Buchnera 6, e29096 (2011). aphidicola. J. Bacteriol. 187, 4229–4237 (2005). 58. E. Biegel, S. Schmidt, J. M. González, V. Müller, Biochemistry, evolution and physio- 22. J. Bermingham et al., Impact of host developmental age on the transcriptome of the logical function of the Rnf complex, a novel ion-motive electron transport complex in symbiotic bacterium Buchnera aphidicola in the pea aphid (Acyrthosiphon pisum). prokaryotes. Cell. Mol. Life Sci. 68, 613–634 (2011). Appl. Environ. Microbiol. 75, 7294–7297 (2009). 59. S. Narita, H. Tokuda, An ABC transporter mediating the membrane detachment of 23. A. K. Hansen, P. H. Degnan, Widespread expression of conserved small RNAs in small bacterial lipoproteins depending on their sorting signals. FEBS Lett. 580, 1164–1170 symbiont genomes. ISME J. 8, 2490–2502 (2014). (2006). 24. M. W. Thairu, S. Cheng, A. K. Hansen, A sRNA in a reduced mutualistic symbiont 60. J. B. Benoit et al., Symbiont-induced odorant binding proteins mediate insect host genome regulates its own gene expression. Mol. Ecol. 27, 1766–1776 (2018). hematopoiesis. eLife 6, e19535 (2017). 25. R. A. Chong, N. A. Moran, Intraspecific genetic variation in hosts affects regulation of 61. A. Moya, J. Peretó, R. Gil, A. Latorre, Learning how to live together: Genomic insights obligate heritable symbionts. Proc. Natl. Acad. Sci. U.S.A. 113, 13114–13119 (2016). into prokaryote-animal symbioses. Nat. Rev. Genet. 9, 218–229 (2008). 26. M. I. Love, W. Huber, S. Anders, Moderated estimation of fold change and dispersion 62. K. Maezawa et al., Hundreds of flagellar basal bodies cover the cell surface of the for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). endosymbiotic bacterium Buchnera aphidicola sp. strain APS. J. Bacteriol. 188, 6539– 27. N. A. Moran, H. J. McLaughlin, R. Sorek, The dynamics and time scale of ongoing 6543 (2006). genomic erosion in symbiotic bacteria. Science 323, 379–382 (2009). 63. C. Michaux et al., RNA target profiles direct the discovery of virulence functions for 28. N. A. Moran, T. Jarvik, Lateral transfer of genes from fungi underlies carotenoid – production in aphids. Science 328, 624–627 (2010). the cold-shock proteins CspC and CspE. Proc. Natl. Acad. Sci. U.S.A. 114, 6824 6829 29. K. R. Clarke, P. J. Somerfield, R. N. Gorley, Testing of null hypotheses in exploratory (2017). community analyses: Similarity profiles and biota-environment linkage. J. Exp. Mar. 64. N. Nikoh et al., Bacterial genes in the aphid genome: Absence of functional gene Biol. Ecol. 366,56–69 (2008). transfer from Buchnera to its host. PLoS Genet. 6, e1000827 (2010). 30. B. Zhang, S. Horvath, A general framework for weighted co-expression network 65. N. Nikoh, A. Nakabachi, Aphids acquired symbiotic genes via lateral gene transfer. analysis. Stat. Appl. Genet. Mol. Biol. 4, Article 17 (2005). BMC Biol. 7, 12 (2009). 31. P. Langfelder, S. Horvath, WGCNA: An R package for weighted correlation network 66. S. H. Chung, X. Jing, Y. Luo, A. E. Douglas, Targeting symbiosis-related insect genes by – analysis. BMC Bioinformatics 9, 559 (2008). RNAi in the pea aphid-Buchnera symbiosis. Insect Biochem. Mol. Biol. 95,5563 32. D. Kim, B. F. Minhas, H. Li-Byarlay, A. K. Hansen, Key transport and ammonia recycling (2018). genes involved in aphid symbiosis respond to host-plant specialization. G3 (Bethesda) 67. N. M. Gerardo et al., Immunity and other defenses in pea aphids, Acyrthosiphon 8, 2433–2443 (2018). pisum. Genome Biol. 11, R21 (2010). 33. C. W. Russell, S. Bouvaine, P. D. Newell, A. E. Douglas, Shared metabolic pathways in a 68. R. Priyadarshini, D. L. Popham, K. D. Young, Daughter cell separation by penicillin- coevolved insect-bacterial symbiosis. Appl. Environ. Microbiol. 79, 6117–6123 (2013). binding proteins and peptidoglycan amidases in Escherichia coli. J. Bacteriol. 188, 5345–5355 (2006). 34. S. Singh, R. Banerjee, PLP-dependent H2S biogenesis. Biochim. Biophys. Acta 1814, 1518–1527 (2011). 69. C. Otten, M. Brilli, W. Vollmer, P. H. Viollier, J. Salje, Peptidoglycan in obligate in- 35. H. Kabil, O. Kabil, R. Banerjee, L. G. Harshman, S. D. Pletcher, Increased trans- tracellular bacteria. Mol. Microbiol. 107, 142 –163 (2018). sulfuration mediates longevity and dietary restriction in Drosophila. Proc. Natl. Acad. 70. F. Husnik et al., Horizontal gene transfer from diverse bacteria to an insect genome Sci. U.S.A. 108, 16831–16836 (2011). enables a tripartite nested mealybug symbiosis. Cell 153, 1567–1578 (2013). 36. J. M. Phang, W. Liu, C. N. Hancock, J. W. Fischer, Proline metabolism and cancer: 71. D. C. Bublitz et al., Peptidoglycan production by an insect-bacterial mosaic. Cell 179, Emerging links to glutamine and collagen. Curr. Opin. Clin. Nutr. Metab. Care 18, 703–712.e7 (2019). 71–77 (2015). 72. T. L. Wilkinson, R. Koga, T. Fukatsu, Role of host nutrition in symbiont regulation: 37. J. S. Ramsey et al., Genomic evidence for complementary purine metabolism in the Impact of dietary nitrogen on proliferation of obligate and facultative bacterial en- pea aphid, Acyrthosiphon pisum, and its symbiotic bacterium Buchnera aphidicola. dosymbionts of the pea aphid Acyrthosiphon pisum. Appl. Environ. Microbiol. 73, Insect Mol. Biol. 19 (suppl. 2), 241–248 (2010). 1362–1366 (2007). 38. R. N. Finn, F. Chauvigné, J. A. Stavang, X. Belles, J. Cerdà, Insect glycerol transporters 73. J. P. Sandström, J. Petterson, Amino acid composition of phloem sap and the relation evolved by functional co-option and gene replacement. Nat. Commun. 6, 7814 (2015). to intraspecific variation in pea aphid (Acyrthosiphon pisum) performance. J. Insect 39. I. S. Wallace et al., Acyrthosiphon pisum AQP2: A multifunctional insect aqua- Physiol. 40, 947–955 (1994). glyceroporin. Biochim. Biophys. Acta 1818, 627–635 (2012). 74. N. Reymond et al., Different levels of transcriptional regulation due to trophic con- 40. S. Shigenobu, D. L. Stern, Aphids evolved novel secreted proteins for symbiosis with straints in the reduced genome of Buchnera aphidicola APS. Appl. Environ. Microbiol. bacterial endosymbiont. Proc. Biol. Sci. 280, 20121952 (2013). 72, 7760–7766 (2006).

Smith and Moran PNAS | January 28, 2020 | vol. 117 | no. 4 | 2121 Downloaded by guest on September 26, 2021