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Elucidation of host and symbiont contributions to peptidoglycan metabolism based on comparative genomics of eight aphid subfamilies and their Buchnera [1]

['Thomas E. Smith', 'Department Of Integrative Biology', 'University Of Texas At Austin', 'Austin', 'Texas', 'United States Of America', 'Yiyuan Li', 'Julie Perreau', 'Nancy A. Moran']

Date: 2022-07

Pea aphids (Acyrthosiphon pisum) are insects containing genes of bacterial origin with putative functions in peptidoglycan (PGN) metabolism. Of these, rlpA1-5, amiD, and ldcA are highly expressed in bacteriocytes, specialized aphid cells that harbor the obligate bacterial symbiont Buchnera aphidicola, required for amino acid supplementation of the host’s nutrient-poor diet. Despite genome reduction associated with endosymbiosis, pea aphid Buchnera retains genes for the synthesis of PGN while Buchnera of many other aphid species partially or completely lack these genes. To explore the evolution of aphid horizontally-transferred genes (HTGs) and to elucidate how host and symbiont genes contribute to PGN production, we sequenced genomes from four deeply branching lineages, such that paired aphid and Buchnera genomes are now available for 17 species representing eight subfamilies. We identified all host and symbiont genes putatively involved in PGN metabolism. Phylogenetic analyses indicate that each HTG family was present in the aphid shared ancestor, but that each underwent a unique pattern of gene loss or duplication in descendant lineages. While four aphid rlpA gene subfamilies show no relation to symbiont PGN gene repertoire, the loss of aphid amiD and ldcA HTGs coincides with the loss of symbiont PGN metabolism genes. In particular, the coincident loss of host amiD and symbiont murCEF in tribe Aphidini, in contrast to tribe Macrosiphini, suggests either 1) functional linkage between these host and symbiont genes, or 2) Aphidini has lost functional PGN synthesis and other retained PGN pathway genes are non-functional. To test these hypotheses experimentally, we used cell-wall labeling methods involving a d-alanine probe and found that both Macrosiphini and Aphidini retain Buchnera PGN synthesis. Our results imply that compensatory adaptations can preserve PGN synthesis despite the loss of some genes considered essential for this pathway, highlighting the importance of the cell wall in these symbioses.

Throughout evolution, animals have sometimes gained novel abilities by acquiring bacterial genes through horizontal gene transfer. For some insects harboring bacterial symbionts, horizontally-transferred genes may enable hosts to regulate symbiosis by influencing symbiont cell wall metabolism. While mealybug horizontally-transferred genes work collectively to synthesize the symbiont cell wall, the role of aphid horizontally-transferred genes in symbiont cell wall metabolism is unclear. We examined whether different aphid horizontally-transferred genes co-occur with symbiont genes underlying cell wall metabolism across different aphid lineages, indicative of linked function. We included 17 aphid species representing eight distantly related lineages, four of which we sequenced for this study. We found that two of the three horizontally-acquired gene families are present only when symbionts possess cell wall pathway genes, while the third shows no correlation. These results reveal that despite their putative involvement in symbiont cell wall synthesis, aphid horizontally-acquired genes operate independently from one another and likely have lineage-specific functions. Furthermore, we observed that symbiont cell wall synthesis is maintained in one aphid lineage despite loss of genes considered essential for producing the cell wall, implying that other adaptations preserve the cell wall in aphid species with incomplete cell wall synthesis pathways.

Data Availability: Sequencing data can be found at NCBI (PRJNA758084). Genome annotations and other data generated during the current work can be found at Zenodo ( https://zenodo.org/record/5517159 ). Scripts and code used in this study can be found at GitHub ( https://github.com/lyy005/peptidoglycan_related_genes_in_basal_aphids ). All accession numbers and URLs to data repositories can be found in the main text of the manuscript.

In this work, we introduce draft genomes of aphid species from four additional aphid subfamilies: Geopemphigus sp. (Fordinae), Stegophylla sp. (Phyllaphidinae), Chaitophorus viminalis (Chaitophorinae), and Pemphigus obesinymphae (Pemphiginae). We applied phylogenetic profiling across the Aphidoidea superfamily to evaluate whether certain host and symbiont PGN genes co-occur across different aphid species, supporting a potential functional relationship. For the 17 annotated aphid genomes, we assayed the incidence (presence/absence) of host genes with putative functions in PGN metabolism, including homologs of the A. pisum HTGs ldcA, amiD, rlpA, and bLys, as well as PGN pathway genes from the corresponding primary symbiont of each host species. For the latter, we used a manually-curated database of primarily E. coli and Buchnera genes representing known PGN genes and members of the cell division and flagellar basal body (FBB) pathways, both of which may interact with or depend on cell wall metabolism [ 35 – 37 ]. We show that the PGN gene repertoire of each aphid species varies for both host and symbiont. The pattern of aphid ldcA and especially amiD gene loss correlates with the absence of PGN pathway genes in their respective symbiont(s), while rlpA genes, though variable in the incidence and abundance of four distinct paralogous groups, are conserved among all aphids. These observations imply that aphid HTGs are not all involved in symbiont PGN metabolism and likely function independently of each other, suggesting that the encoded enzymes do not cooperate to complement a missing multi-step pathway in symbionts as proposed for mealybug HTGs [ 25 ]. Furthermore, a clear difference in gene repertoire exists between two tribes of the Aphidinae subfamily. Relative to tribe Macrosiphini, Aphidini aphids lack amiD and their symbionts lack 2–3 genes central to lipid II biosynthesis (murF, murC, and/or murE). Thus, gene repertoires suggest that Aphidini Buchnera cannot synthesize cell walls at all. In contrast with this hypothesis, we provide experimental evidence for functional cell wall synthesis in an Aphidini species, Rhopalosiphum maidis, demonstrating that 1) novel adaptations have evolved within the PGN biosynthesis pathway to preserve the cell wall in this species, and 2) genomic analysis alone is an insufficient predictor of symbiont capabilities.

Despite the apparent relationship between aphid HTGs and symbiont cell wall metabolism, it is unclear from their putative functions alone how they might contribute to this process. The A. pisum amiD and rlpA1-5 genes may be functionally redundant with Buchnera amiB and emtA or mltA, respectively, and Buchnera completely lacks the PGN recycling pathway to which aphid LdcA putatively belongs ( Fig 1 ) [ 21 ]. Recent evidence shows that aphid LdcA instead acts on PGN within or shed from the Buchnera cell wall [ 22 ]. One way to establish whether genes are functionally linked is to determine whether they are preserved or lost together throughout evolution using phylogenetic profiling—the correlated presence of genes within genomes of distinct evolutionary lineages can provide a signal of functional interactions [ 23 , 24 ]. Similarly, if host and symbiont genes interact, they should co-occur in paired host and symbiont genomes across diverse lineages, whereas genes that do not interact will be gained or lost independently of one another. Such an approach has been used previously to detect host-symbiont collaborations among various mealybug species [ 25 ]. Gene distributions across species have also been used to correlate the lack of class A penicillin-binding proteins with an intraceullar lifestyle among distantly related bacteria [ 26 ]. Within the aphid superfamily Aphidoidea, Buchnera symbionts vary widely in genome size and content, including PGN genes [ 27 ], providing a natural source of variation to test whether host HTGs coincide with certain symbiont PGN genes or pathways. Furthermore, in a few aphid lineages, Buchnera has been lost and replaced with a novel symbiont type [ 28 , 29 ] or has been joined by additional symbionts [ 30 , 31 ]. While many Buchnera genomes are available from diverse aphid species [ 27 , 30 – 34 ], only 13 aphid genomes have been sequenced, annotated, and published to date, and most are concentrated in a single subfamily, the Aphidinae ( S1 Table ).

PGN metabolism is divided into four pathways: PGN recycling and lipid II synthesis in the cytoplasm, and cell wall synthesis and remodeling in the periplasm. Arrows depict enzymatic reactions, with black, gray, and white colors indicating complete, sporadic, or no conservation of the responsible gene(s) among ten Buchnera genomes. Light and dark green arrows denote incomplete and complete conservation of host HTGs. For PGN cartoons, dark and light brown indicate GlcNAc and MurNAc glycans, respectively, while black-ended light brown indicates anhydro-MurNAc. Stem peptides consist of l-Ala (dark blue), iso-d-Glu (green), meso-diaminopimellic aicd (mDap) (maroon), and d-Ala (light blue) amino acids. For lipid II, the phosphate groups of undecaprenyl-diphosphate are shown in pink.

Aphids are sap-feeding insects that rely on their Buchnera symbionts to provide essential amino acids lacking in their diet. The pea aphid (Acyrthosiphon pisum) Buchnera genome is missing many genes considered to be essential in free-living relatives like Escherichia coli [ 18 ], including most PGN pathway genes. Pea aphids contain eight HTGs with putative functions in PGN metabolism: ldcA, amiD, rlpA1-5, and bLys [ 12 , 13 ], of which all but bLys show greatly elevated expression in bacteriocytes, the specialized host cells that harbor Buchnera, relative to other aphid tissues [ 13 ]. The putative functions of the remaining seven HTGs correspond to specific steps in bacterial PGN metabolsim: amiD and rlpA putatively encode an amidase and lytic translgycosylase (LT), respectively, both of which are involved in cell wall remodeling, while ldcA encodes a putative l,d-carboxypeptidase of the PGN recycling pathway ( Fig 1 ). Aphids also contain invertebrate-type (i-type) lysozymes that are highly expressed in bacteriocytes[ 19 ], especially during late adulthood when symbionts and bacteriocytes are degraded [ 20 ]. Thus, aphid hosts may have the enzymatic potential to control Buchnera by influencing its cell wall structure.

Horizontal gene transfer (HGT) has played a pivotal role in eukaryotic evolution, granting novel functions to its recipients and providing the means to expand to new ecological environments [ 1 – 7 ]. HGT has been particularly relevant in the evolution of eukaryotes that live in close association with bacteria [ 8 , 9 ]. At the extreme, the majority of mitochondrial and plastid genes have been transferred to the host genome [ 10 , 11 ], affording hosts complete control of their bacterial symbionts and the benefits they provide. In contrast, transfer of symbiont genes to the host genome is absent or limited in a number of insect endosymbioses [ 12 – 15 ]. In these systems, host genomes may instead harbor genes anciently acquired from bacteria other than the current symbiont(s) that have obtained roles in maintaining extant symbioses through nutrient provisioning [ 15 , 16 ] or the metabolism of peptidoglycan (PGN), the building block of bacterial cell walls [ 12 – 14 ]. For example, the genome of the mealybug Planococcus citri contains horizontally-transferred genes (HTGs) encoding enzymes of the PGN synthesis pathway that perfectly complement the PGN gene repertoire of the symbionts [ 14 ] and contribute to the construction of the symbiont cell wall [ 17 ].

A) Pea aphids, both cured (Austin-c; left column) and uncured (Austin; middle column) of secondary Serratia symbionts, and R. maidis (Rma-NM-1; right column) were fed ELA (left-side) or EDA (right-side) via their host plants as described in Fig 4A . Embryos removed from 4 th instar larvae were fixed, treated with azide-linked Alexa Fluor 488 (AF488), hybridized with Buchnera- and Serratia-specific Cy5- and Cy3-labeled riboprobes, respectively ( S10 Table ), and counter-stained with DAPI. B) Enlarged cutout images (white boxes in A) of EDA-labeled, uncured A. pisum show overlapping AF488 fluorescence (i) with Buchnera cells (ii) and, to a minor extent, Serratia cells (iii). A bacteriocyte containing Serratia is highlighted with a dashed line in each image. The white scale bars represent 20 μm. Images were falsely colored to aid visualization.

Next, we applied the same cell wall labeling strategy to R. maidis aphids (tribe Aphidini) and pea aphids either cured of or containing the facultative symbiont Serratia symbiotica. Symbiont identities were confirmed using diagnostic FISH microscopy alongside the clickable cell wall probes ( Fig 5 ). As expected, no cell wall fluorescence was observed in symbionts fed ELA. However, Buchnera cell walls were labeled with EDA in both aphid species ( Fig 5A ), demonstrating that even species lacking aphid amiD and Buchnera murC, murE, and murF can synthesize a cell wall. Additionally, pea aphids harboring Serratia show only minor incorporation of EDA into Serratia symbionts relative to Buchnera ( Fig 5B ), suggesting that Serratia differs from Buchnera in their primary source of d-Ala.

A) Aphids were reared on plant leaves immobilized in agar containing ethynyl-d-Ala (EDA) or the control l enantiomer from birth until the 4 th instar larval stage, after which Buchnera cells were purified from aphid homogenate. B) Cells were fixed and stained with azide-linked Alexa Fluor 488 to label PGN stem peptides (PGN stem peptides), wheat germ agglutinin CF-640R conjugate to label the PGN glycan backbone (PGN glycans), and the counter-stain DAPI to label DNA (DNA counter-stain). Hen eggwhite lysozyme (HEWL; PGN glycan-targeting) and E. coli AmiD (PGN stem peptide-targeting) were used to demonstrate incorporation of EDA within Buchnera PGN stem peptides. Images were falsely colored to aid visualization. The white scale bar represents 5 μm. C) For each combination of enzyme and probe, the average fluorescence intensity (AFI) per area was measured for each of n Buchnera cells using ImageJ software ( S9 Table ). Significant differences between means are indicated by compact letter displays above each boxplot—means with no letters in common are significantly different by the Dunn’s Multiple Comparison test (p-value < 0.01 following adjustment for false discovery).

The observation that Buchnera of most Aphidini aphids lack murF, murC, and/or murE ( Fig 3 ), representing three critical steps in lipid II biosynthesis ( Fig 1 ), raises the question of whether these symbionts are capable of synthesizing a cell wall at all [ 26 ]. The loss of these particular genes may be coincidental, reflecting the breakdown of cell wall synthesis altogether rather than evidence of functional linkage. To test this hypothesis, we developed a method to fluorescently label Buchnera PGN via incorporation of ethynyl-d-alanine (EDA), a d-alanine analogue compatible with click chemistry [ 58 , 59 ], by adapting a host feeding approach previously used to label symbiont PGN in the mealybug system [ 17 ]. Aphids were reared from birth on individual leaves immobilized in agarose gel containing EDA until reaching seven days of age ( Fig 4A ). To validate our approach, Buchnera cells were isolated from pea aphids (tribe Macrosiphini) reared on EDA, fixed, and stained with azide-linked Alexa Fluor 488 (AF488) via click reaction. Buchnera derived from hosts fed EDA exhibited green fluorescence at the cell periphery while control aphids raised on ethynyl-l-alanine (ELA) did not ( Fig 4B ), indicating 1) transport of EDA from host plant xylem to phloem, and 2) selective incorporation of EDA from plant phloem into Buchnera cell walls. Furthermore, the observed fluorescence was definitively linked to Buchnera cell wall stem peptides by treating cells with PGN-degrading enzymes prior to fixation and counter-staining with the PGN backbone-specific probe wheat germ agglutinin CF-640R conjugate (WGA640). Hen eggwhite lysozyme (HEWL) and E. coli AmiD target distinct PGN features: the backbone and stem peptides, respectively. We observed a decrease in fluorescence of both EDA-AF488 and WGA640 following HEWL treatment and of EDA-AF488 alone for AmiD ( Fig 4B and 4C and S9 Table ), firmly associating AF488 fluorescence with PGN stem peptides.

Aphid HTGs present in species harboring symbionts undergoing gene loss in the PGN pathway or lacking it altogether may have evolved alternative functions through natural selection. Because ldcA is present in species with varying degrees of PGN pathway degradation, we were able to test which conditions might lead to positive selection. The most complete PGN pathways are found in tribe Macrosiphini, followed by tribe Aphidini—PGN pathways are near-absent in subfamily Hormaphidinae and entirely absent in all other subfamilies with Buchnera. We tested for lineage-specific diversifying selection in the aphid ldcA gene family using BUSTED [ 57 ], selecting different sets of test lineages based on the criteria described above. For example, we asked whether diversifying selection has influenced ldcA in members of the tribe Aphidini, where the loss of aphid amiD and Buchnera murF, murC, and/or murE may signal the absence or modification of stem peptides from Buchnera PGN in this lineage, leading to a potential shift in LdcA function, which also putatively targets stem peptides. We found evidence for positive selection only when all lineages or Aphidini-specific ldcA genes were selected as the test group ( Table 1 ), suggesting that host ldcA is undergoing adaptive evolution in at least one member of the Aphidini. These results are especially interesting in the light of our recent biochemical characterization of A. pisum LdcA, which shows that the aphid enzyme is multifunctional and plays a novel role in PGN degradation relative to its homolog in E. coli [ 22 ]. In contrast, we did not detect evidence for positive selection in ldcA when test groups included: all Macrosiphini species, species with symbionts lacking PGN genes and lacking secondary symbionts (P. obesinymphae and E. lanigerum), species with intermediate PGN gene loss (H. cornu), and species in which ldcA has been duplicated (Sitobion miscanithi, Rhopalosiphum padi, C. cedri, and P. obesinymphae) ( Table 1 ). Additionally, we were unable to detect evidence for positive selection of amiD when all genes or only duplicated genes were selected as test groups ( Table 1 ). Thus, tribe Aphidini stands out in showing evidence of positive selection for ldcA. However, the lack of evidence for positive selection in some of our BUSTED tests may reflect lack of power due to small sample sizes.

We also evaluated the symbiont gene repertoire of pathways related to but outside of PGN metabolism. Cell division is closely coordinated with cell wall synthesis and remodeling [ 34 ], while assembly of flagella basal bodies FBBs involves interaction with the cell wall via the ß-N-acetylgucosaminidase activity of FlgJ [ 35 , 36 ]. Like the PGN pathway, loss of both cell division and FBB genes is more extensive in all subfamilies relative to the Aphidinae ( Fig 3 ). However, there is no apparent coincidence in these losses with those of PGN pathway genes that would implicate a dependence of either pathway on the cell wall. For example, though symbionts of both C. viminalis (Chaitophorinae) and Stegophylla sp. (Phyllaphididae) lack virtually all PGN genes, the former maintains many cell division genes, similar to the tick-borne pathogens of the genus Ehrlichia [ 21 ], while the latter lacks all of them ( Fig 3 ), a unique phenomenon among obligately intracellular bacteria [ 21 , 26 ]. Similarly, Buchnera of C. cedri, E. lanigerum (Eriosomatinae), and P. obesinymphae (Pemphiginae) lack the PGN pathway but maintain many FBB genes ( Fig 3 ). Furthermore, cell wall synthesis and remodeling genes that are known components of the E. coli divisome are not preferentially maintained in aphid symbionts that lack the PGN pathway but maintain cell division genes ( Fig 3 ) [ 35 ]. Additionally, all Buchnera FlgJ enzymes lack the PGN hydrolase domain, effectively demonstrating a decoupling of FBB assembly to cell wall metabolism. Thus, the Buchnera cell wall generally does not appear to support cell division or FBB assembly.

In contrast, the incidence of aphid amiD in different lineages strongly suggests a link to symbiont cell wall metabolism. The loss of host amiD from most subfamilies with highly reduced Buchnera genomes (Chaitophorinae, Phyllaphidinae, Eriosomatinae, and Pemphiginae) or Buchnera loss (Fordinae) suggests that the gene is expendable in the absence of Buchnera PGN ( Fig 3 ). The putative ability of AmiD to cleave stem peptides from anhydro-MurNAc suggests a dependence on the prior activity of LTs [ 53 ], which are distinct from lysozymes in their production of cyclic anhydro-MurNAc. As LTs are found only in Buchnera from Aphidinae aphids (emtA and/or mltA) and Skilesia (mltG), the presence of aphid amiD largely correlates with LT incidence, with the exception of C. cedri ( Fig 3 ). In tribe Aphidini, the loss of host amiD coincides with that of murF, murC, and/or murE in symbionts—while the encoded enzymes putatively function in two distinct pathways of PGN metabolism (remodeling and lipid II synthesis, respectively), all four enzyme activities involve PGN stem peptides ( Fig 1 ), implying the possibility of a functional relationship. Alternatively, a lineage-specific functional redundancy of host amiD with symbiont amiB could have rendered amiD obsolete. The presence of both aphid amiD and ldcA in H. cornu coincides with incomplete PGN gene loss in its symbiont ( Fig 3 ), suggesting a stronger reliance on host HTGs for symbiont PGN remodeling. Finally, though Buchnera from Cinara cedri (Lachninae) lacks all PGN pathway genes, the host maintains both amiD and ldcA. Many Lachninae aphids harbor secondary symbionts that have taken on essential roles for host survival [ 54 – 56 ]—aphid PGN genes may regulate PGN metabolism in these secondary symbionts instead of Buchnera.

Aphid ldcA exhibits a mixed distribution among host species harboring symbionts with and without PGN. The uniform conservation of ldcA in the Aphidinae subfamily, where Buchnera maintains most PGN pathway genes ( Fig 3 ), supports a relationship between ldcA and symbiont PGN metabolism, at least in this lineage. Aphid ldcA is also present in H. cornu, whose Buchnera harbors a partial PGN pathway ( Fig 3 ). However, aphid ldcA is present in three aphid subfamilies with symbionts lacking PGN pathway genes (Eriosomatinae, Lachninae, and Pemphiginae) and is absent in two (Chaitophorinae, Phyllaphidinae). While the role of ldcA in the former lineages is unclear, Buchnera from each of the latter species has undergone significantly more gene loss relative to symbionts of other aphids [ 27 ]. Aphid ldcA is also absent in Geopemphigus sp. (Fordinae), where excessive genome reduction in Buchnera is thought to have led to its evolutionary replacement with the Bacteroidetes symbiont Skilesia [ 29 ]. Thus, our data suggest that the two independent losses of ldcA in these clades are associated with extensive loss of Buchnera genes, including most PGN genes (Figs 2 and 3 ).

Next, we considered whether the presence or absence of host PGN genes coincides with the degree of symbiont PGN gene loss. The ubiquity of aphid i-type lys and bLys genes in host genomes suggests no involvement of these genes with symbiont PGN metabolism (Figs 2 and 3 ). While the incidence of rlpA paralog types varies among aphid lineages, all species contain multiple rlpA genes, implying no relationship with symbiont gene repertoire. This is less surprising when the functional basis of RlpA’s role in bacterial cell division is considered. Bacterial RlpA proteins participate in cell division largely via the N-terminal sporulation-related repeat (SPOR) domain (Pfam 05036) that binds “denuded” (stemless) PGN glycan chains that accumulate at the septum [ 49 , 50 ]. In contrast, aphid RlpA proteins contain only the catalytic double-ψ ß-barrel (DPBB) domain (Pfam 03330) of RlpA [ 12 , 13 ]. While the RlpA DPBB domain is unique among LTs for its ability to cleave denuded PGN [ 51 , 52 ], the absence of SPOR domains from aphid RlpA proteins suggests that these enzymes likely do not participate in symbiont cell division. Furthermore, aphid RlpA proteins contain one or more eukaryotic inhibitor cysteine-knot (ICK)/knottin domains that are unrelated to PGN metabolism [ 12 , 13 ], further distancing RlpA from a role in this pathway. Collectively, these observations suggest that the DPBB domain may serve an alternative function in aphid RlpA proteins.

Filled cells indicate gene presence, while empty cells indicate gene absence. For aphid genes, numbers indicate the number of gene paralogs identified per genome. For symbiont genes, filled cells all indicate a single copy per genome. All symbionts are Buchnera except those of Geopemphigus sp, indicated by an asterisk, which contains a novel Bacteroidetes symbiont. Host species containing known co-obligate or uncharacterized facultative symbionts are indicated with an obelisk (†) or a double obelisk (‡), respectively. Each species code represents the combined first two letters of the genus and species ( S1 Table ). Colored circles indicate genes with dual-membership in the pathway of the same color, while black indicates dual-membership in amino acid metabolism.

We anticipated that the symbionts of host species lacking amiD and/or ldcA genes might display greater loss of genes from PGN-related pathways due to a functional link between these host genes and symbiont cell wall metabolism. To test this idea, we looked for patterns of coincidence between host and symbiont PGN gene repertoires among all available sequenced aphid species. First, we measured the incidence of 138 bacterial genes, collectively involved in PGN metabolism or two potentially related pathways, within the primary symbiont genome of each aphid species ( Fig 3 and S8 Table ). As noted previously, Buchnera from aphids of tribe Macrosiphini (subfamily Aphidinae) harbor the most complete set of PGN metabolism genes among Buchnera species, maintaining a nearly complete lipid II synthesis pathway and the minimal enzyme functions necessary for cell wall synthesis (glycosyltransferase and d,d-transpeptidase) ( Fig 3 ) [ 21 , 27 ]. Though Macrosiphini Buchnera generally maintain genes encoding two of the three essential activities for cell wall remodeling, an amidase (amiB) and at least one lytic transglycosylase (LT; mltA, emtA) gene, they often lack the gene typically encoding the third, endopeptidase activity (mepM) ( Fig 1 ). In comparison, Buchnera of the tribe Aphidini (subfamily Aphidinae) display more frequent loss of murF, murC, and/or murE of the lipid II synthesis pathway. Most other subfamilies (Chaitophorinae, Phyllaphidinae, Lachninae, Eriosomatinae, and Pemphiginae) exhibit near-complete loss of all PGN pathway genes, while Buchnera from Hormaphis cornu (subfamily Hormaphidinae) represents an intermediate, maintaining a single cell wall synthesis gene (mrcB, encoding the bifunctional glycosyltransferase/d,d-transpeptidase PBP1B) and partial lipid II synthesis pathway ( Fig 3 ). Notably, no aphid primary symbionts, including Buchnera of most aphids and Candidatus Skilesia alterna (referred to hereafter as Skilesia) of Geopemphigus, maintain any of the PGN recycling genes used in our searches ( S8 Table ).

Aphid lineages vary in whether HTGs have been lost or duplicated ( Fig 2 ). RlpA subfamilies are maintained to different extents among aphid lineages, with each genome containing at least one type ( Fig 2 ). We examined the phylogenetic relationships of aphid RlpA proteins and found that each sequence falls within one of four paralogous subfamilies: RlpA4-like, RlpA3-like, RlpA1-like, and RlpA2/5-like ( S2 Fig ). RlpA4-like proteins are the most basally branching subfamily and RlpA2/5 is the most recent branch, supporting the initial analysis of A. pisum rlpA genes [ 13 ]. Ancestral reconstruction indicates that the most recent aphid common ancestor had all rlpA subfamilies, implying that the underlying gene duplications occurred in a common ancestor of the Aphidoidea ( Fig 2 ). In contrast, amiD and ldcA have been lost independently five and two times, respectively ( Fig 2 ). The repeated loss of these genes provides an opportunity to test our hypothesis—if aphid HTGs contribute to symbiont PGN metabolism, the loss of aphid HTGs should correlate with the absence of interacting symbiont genes.

Unique to superfamily Aphidoidea are the HTGs rlpA, ldcA, and amiD, all of which were acquired in a shared ancestor of this superfamily ( Fig 2 ). With the exception of an amiD homolog within the D. citri genome, none of the bacteriocyte-expressed HTGs found in aphids were detected outside of the Aphidoidea ( Fig 2 ). In the case of AmiD, phylogenetic analysis revealed that the D. citri homolog clusters separately from the aphid copies, indicating that D. citri amiD was acquired from a bacterial source independently of aphid amiD ( S1 Fig ).

We first evaluated the distribution of genes encoding enzymes that target PGN and genes that represent conserved components of insect innate immunity ( S7 Table ). PGRPs sense bacterial infection by binding and/or cleaving PGN and activating downstream pathways that stimulate the humoral immune response, which includes increased production of bacteriostatic lysozymes [ 40 ]. Lysozymes, characterized by their N-acetylmuramidase activity, include members from four different glycoside hydrolase families: chicken- and invertebrate-type (c- and i-types, respectively; GH22), goose-type (g-type; GH23), viral-type (v-type; GH24), and Chalaropsis-type (ch-type; GH25) [ 40 ]. Both PGRPs and c-type lysozymes are evolutionarily conserved in insects [ 40 , 41 ]. We found no genes encoding PGRPs or c-type lysozymes among aphid or phylloxerid genomes, but their presence in the scale insect, whitefly [ 42 ], and psyllid [ 43 , 44 ] genomes support their loss in a shared ancestor of the Aphidomorpha infraorder ( Fig 2 ). The absence of these key immune genes may have facilitated the establishment of endosymbioses in aphids [ 45 ], though their absence from phylloxerids, which lack symbionts [ 39 ] and presence in other outgroups that harbor obligate symbionts suggest that alternative forces underlie their loss from the Aphidomorpha. We observed little variation in the number of invertebrate-type (i-type) lysozymes among most insects investigated ( Fig 2 ), indicating a functional difference between these and c-type lysozymes. Major exceptions are Cinara cedri (Lachninae) and Eriosoma lanigerum (Eriosomatinae) which harbor eight to nine i-lysozyme paralogs compared with three or four paralogs in other species. Finally, we found bLys in all Aphidomorpha genomes, indicating that this gene family was acquired concomitantly with the loss of PGRPs and c-type lysozymes ( Fig 2 ). The ch-type lysozyme domain of bLys is most closely related to domains in prophages of Wolbachia-like bacteria [ 13 , 46 ] and is minimally expressed within bacteriocytes, implying a role outside of symbiosis [ 13 ]. The multiple independent HGTs of ch-type lysozymes to all domains of life [ 46 ] and the unique N,O-diacetylmuramidase activity described for some ch-type lysozymes [ 47 , 48 ] suggest that aphid bLys is more likely involved in host defense against bacterial pathogens than in symbiont PGN metabolism.

The aphid phylogeny was constructed using maximum likelihood from a concatentated nucleotide alignment of 829 core genes shared by all 23 insect species. Bootstrap supports after 1,000 replicates are shown, with red branches indicating supports < 70%. All symbionts are Buchnera except those of Geopemphigus sp, indicated by an asterisk, which contains a novel Bacteroidetes symbiont. Host species containing known co-obligate or uncharacterized facultative symbionts are indicated with an obelisk (†) or a double obelisk (‡), respectively. Each species code represents the combined first two letters of the genus and species. The ancestral state (gray) and placement of gene gains (black), duplications (black), and losses (white) were determined using Count (38). Numbers in shapes indicate the number of gene copies involved in the event. Shapes indicate distinct gene families, and colored borders denote the four RlpA paralog types, named after the A. pisum rlpA genes.

In order to understand 1) the incidence of A. pisum HTG homologs among different aphid lineages and 2) the evolutionary history of host-encoded PGN genes, we reconstructed the aphid phylogeny using the four newly sequenced aphid genomes, 13 available aphid genomes, and six related insects in suborder Sternorrhyncha included as outgroups ( S1 Table ). The phylogeny was based on 829 single-copy genes found in at least 80% of the species ( Fig 2 ). Genes with putative activities pertaining to PGN metabolism, including peptidoglycan recognition proteins (PGRPs), lysozymes, and aphid HTGs (bLys, ldcA, amiD, and rlpA), were identified by BLAST search using A. pisum protein sequences as queries ( S7 Table ). With the exception of Daktulosphaira vitifoliae [ 39 ], the outgroup species contain bacterial symbionts, none closely related to Buchnera.

To understand the evolution of genes related to PGN pathways, we sequenced the genomes of four divergent aphid lineages, including the first genome sequences for subfamilies Fordinae (Geopemphigus sp.), Phyllaphidinae (Stegophylla sp.) and Pemphiginae (Pemphigus sp.), and the second genome sequence for subfamily Chaitophorinae (Chaitophorus sp.) after Sipha flava (NCBI BioProject PRJNA472250). The Geopemphigus genome also represents the first aphid genome for which Buchnera has been lost and replaced with an unrelated endosymbiont [ 29 ]. For each genome, we generated 38–58 Gb of genomic data from one short insert size Illumina library ( S2 Table ) and 5–7 Gb RNA-seq data ( S3 Table ). After assembly, estimated genome sizes range from 245 Mb—658 Mb ( S4 Table ). The sequenced genomes show high completeness based on the presence of single-copy Benchmarking Universal Single-Copy Orthologs (BUSCOs) for Hemiptera (n = 2,510) [ 38 ], ranging from 97%-99% for both the assemblies ( S5 Table ) and our gene annotation results ( S6 Table ). These values are similar to those for other aphid genome sequences [ 33 ]. All sequencing data can be found at NCBI under the accession number PRJNA758084.

Discussion

The HTGs rlpA, ldcA, and amiD of A. pisum encode putative PGN hydrolases that are more highly expressed in bacteriocytes than other host tissues, suggesting that they might play a role in Buchnera cell wall metabolism [13]. The putative functions of these HTGs collectively represent two of the three (LT and amidase) enzyme activities required for cell wall remodeling [60]. Furthermore, some LdcA homologs from intracellular bacteria are known to exhibit the third (endopeptidase) activity [61–63], implying that aphid HTGs might function cooperatively. Indeed, we recently showed that the A. pisum LdcA enzyme exhibits enhanced endopeptidase activity relative to its E. coli homolog, cleaving both a greater range and amount of PGN substrates [22]. Further, we found evidence of this activity in the composition of Buchnera PGN from pea aphids, demonstrating its biological relevance.

We examined the idea that aphid HTGs and Buchnera PGN metabolism might be functionally linked by identifying correlations between host and symbiont PGN gene repertoires based on newly sequenced genomes from basally branching aphid lineages. Aphid HTGs vary in their incidence and copy number among different aphid species, with each gene family displaying a unique distribution. While aphid rlpA genes are ubiquitous in aphids, the absence of aphid ldcA and amiD genes in some lineages coincides with greater loss of symbiont PGN pathway genes, suggesting functional linkage of ldcA and amiD with symbiont PGN metabolism. These results demonstrate that each aphid HTG family has an independent evolutionary trajectory, implying that they are unlikely to function cooperatively as mealybug HTGs do for symbiont PGN synthesis [17].

Despite the differences in the incidence of HTGs among aphids, the distribution of rlpA, amiD, and ldcA throughout the Aphidioidea superfamily and their absence from closely related Sternorrhynchan insects (Fig 2) suggests that their acquisition was a milestone in aphid evolution. The Buchnera symbiont of the most recent aphid ancestor is predicted to have had functional lipid II, cell wall synthesis, and cell wall remodeling pathways, despite evidence for significant genome reduction [27]. Thus, host HTGs could have provided an immediate benefit following their acquisition by influencing symbiont PGN metabolism (Fig 1). Aphid ldcA and amiD may continue to serve this purpose in species for which symbiont PGN pathways are maintained (Aphidinae, Hormaphidinae). Outside of these subfamilies, most symbiont PGN genes were lost, along with many other non-essential genes, due to the elevated genetic drift characteristic of endosymbionts (Fig 3) [64]. Following the loss of PGN metabolism in symbionts, HTGs were not needed and therefore lost (amiD in Chaitophorinae, Phyllaphidinae, Eriosomatinae, and Pemphiginae, and ldcA in the first two), repurposed for interaction with non-Buchnera symbionts (Lachninae, Fordinae), or maintained by purifying selection (ldcA in Eriosomatinae and Pemphiginae). These cases are discussed further below.

In the Lachninae aphids and in Geopemphigus sp., aphid ldcA and amiD may contribute to PGN metabolism in symbionts other than Buchnera. In Geopemphigus sp., Buchnera has been completely replaced with Skilesia [29]. It is possible that the Geopemphigus amiD gene contributes to PGN metabolism in Skilesia, while the ldcA gene could have been lost concomitantly with loss of Buchnera by the Geopemphigus ancestor. In contrast, C. cedri harbors both Buchnera and Serratia [54]. The Buchnera symbionts of Lachninae aphids exhibit significant genome reduction relative to other subfamilies [54–56], including the loss of PGN pathway genes [31]. The gain of a co-obligate symbiont before the complete loss of Buchnera peptidoglycan may have prevented the loss of either aphid ldcA or amiD. Host HTGs may contribute to unconfirmed secondary or co-obligate symbionts in other species harboring Buchnera strains lacking the PGN pathway (Eriosomatinae and Pemphiginae). Reads from Serratia and Pantoea bacteria were found among the E. lanigerum and P. obesinymphae sequencing data, respectively [33]. Though both genera include many insect-associated bacteria [65,66], a mutualistic relationship between host and bacteria cannot be inferred from sequencing alone. Alternatively, host HTGs in lineages that lack symbiont PGN may have evolved functions that differ from their putative original roles in symbiont PGN metabolism (Eriosomatinae and Pemphiginae). Although we did not find evidence of diversifying selection for ldcA in these lineages (Table 1), their presence in some species lacking symbiont PGN and absence in others suggests that they remain functional.

Gene duplication is often accompanied by selection for divergence in function, although we did not detect evidence for positive selection associated with gene duplication in ldcA (Table 1). Phylogenetic analysis of aphid RlpA proteins reveals four groups of paralogs, each distributed throughout all aphid lineages (S2 Fig). Each paralog group exhibits a distinct phylogenetic profile and none coincides with the loss of symbiont PGN pathway genes, implying 1) distinctive functions of each paralog group, and 2) a lack of functional interaction between RlpAs and PGN metabolism. Because each aphid rlpA gene has eukaryotic domains with putative functions unrelated to PGN metabolism, the aphid rlpA gene family may have never been involved in symbiont cell wall metabolism. Knottin domains, characterized by interwoven ß-sheets stabilized by disulfide bonds, represent the conserved scaffold of a diverse range of target-selective proteins found in multiple eukaryotic kingdoms, many of which are components of venom [67]. Most knottins, including Btk1-4 in whiteflies, are encoded as small, single-domain proteins [68]. Aphids do not encode any knottin domains outside of RlpA, suggesting that the RlpA DPBB domain could have been horizontally transferred into the coding sequence of an ancestral, single-domain knottin gene. The role of knottin-RlpA proteins in the aphid-Buchnera symbiosis may derive from the function of their knottin domains, which are involved in immunity in whiteflies [69–71], instead of from the DPBB domain.

The question remains as to why the Buchnera cell wall is maintained in some species and not others. We show that cell division and FBB assembly genes, both of which interact with the cell wall in E. coli [35,37], are not coincident with the PGN pathway in Buchnera and are thus unlikely to be functionally co-dependent (Fig 3). Additionally, many intracellular environments are isotonic with the symbiont cytoplasm, eliminating the dependence on the cell wall to maintain turgor pressure [21]. Furthermore, eukaryotes typically recognize PGN via PGRPs [40], which all aphids lack (Fig 2) [45]. Our results suggest that the retention of the Buchnera cell wall is related to the presence of aphid amiD and ldcA—species that are able influence symbiont PGN metabolism are likely better able to control or support their symbionts, maximizing the benefits and reducing the costs associated with symbiosis. In many aphid lineages, genomic degradation in Buchnera has resulted in the loss of the cell wall, and this loss may eliminate the ability of hosts to regulate symbionts through the actions of AmiD and LdcA. Under this scenario, the selective forces maintaining aphid amiD and ldcA are dependent on the presence of a cell wall in symbionts, and the function of the symbiont cell wall is to enable hosts to control and/or support symbionts.

In further support of this hypothesis, we observed cell walls that fully envelope Buchnera cells in R. maidis (Fig 5) despite predictions based on gene repertoires that cell wall synthesis is not possible (Fig 3) [26]. Our experiments demonstrate the functionality of the remaining PGN pathway genes. Thus, the concomitant loss of Buchnera murF, murC, and murE and host amiD, implies these genes are functionally related. As Buchnera genes underwent elimination due to drift, AmiD became useless and was subsequently lost. In Aphidini, ldcA genes may have enhanced importance for regulating symbiosis, as Aphidini ldcA could represent the sole host modulator of Buchnera PGN. The preservation of the cell wall despite loss of multiple genes related to the PGN stem peptide also implies the existence of compensatory mechanisms for stem peptide synthesis. The stem peptide composition of Aphidini Buchnera PGN may differ from that of typical Gram-negative bacteria, prompting evolutionary changes in LdcA substrate selectivity (Table 1). Furthermore, the only amino acid ligase remaining to Aphidini species is MurF, suggesting that this enzyme may have expanded its role to ligate a wide range of amino acids together to construct the stem peptide. Multifunctional enzymes likely represent a widespread adaption to genome reduction in bacteria [72,73], and two such enzymes have been described in pea aphid Buchnera [22,74]. However, Aphidini Buchnera species maintain most genes necessary to synthesize or incorporate each of the canonical stem peptide amino acids: murI (missing only in Pentalonia nigonervosa) produces d-Glu, ddlB synthesizes d-Ala-d-Ala, ftsI encodes PBP3, which forms crosslinkages involving mDap, and mepM is an endopeptidase that cleaves those crosslinkages (Fig 3). Thus, it is unclear whether PGN structure differs between Aphidini Buchnera and other Gram-negative bacteria. Experimentation is needed to test conclusions drawn from genetics alone.

To date, hypotheses about aphid HTG function are informed by studies pertaining to a single aphid species, A. pisum (tribe Macrosiphini). In this system, HTGs are expressed more highly in bacteriocytes than in other tissues [13]. Further, RNAi knockdown [75] and correlations of host-symbiont gene expression [76] both suggest that amiD and ldcA expression positively influence Buchnera abundance. Finally, RlpA4 is localized to Buchnera [77]. Studies utilizing different aphid species, especially from tribe Aphidini and subfamily Hormaphidinae, which harbor symbionts with partial PGN pathways, are needed to establish whether the host-symbiont interactions observed for A. pisum are conserved in other species, especially when the potential for species-specific adaptations is high. Furthermore, sequencing of aphid-symbiont genomes from additional species within each subfamily would enable the use of statistical tools for detecting patterns in gene repertoire, whereas our analysis was limited because only a single representative species is available for most subfamilies. For example, our conclusions about the differences between the Macrosiphini and Aphidini, for which genomes are available from multiple species, are more robust than those pertaining to more basally branching but less deeply sampled lineages.

In conclusion, cell wall structure and HTG function appear to vary even among closely related aphid species, such that the relationship between aphid HTGs and symbiont PGN metabolism appears to be unique for each species. Furthermore, we emphasize the importance of experimental validation of genomics-based hypotheses, as comparative genomics alone is insufficient to predict the functional capabilities of aphids and their symbionts.

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