Complex host/symbiont integration of a multi-partner symbiotic system in the eusocial aphid Ceratovacuna japonica

Summary Some hemipteran insects rely on multiple endosymbionts for essential nutrients. However, the evolution of multi-partner symbiotic systems is not well-established. Here, we report a co-obligate symbiosis in the eusocial aphid, Ceratovacuna japonica. 16S rRNA amplicon sequencing unveiled co-infection with a novel Arsenophonus sp. symbiont and Buchnera aphidicola, a common obligate endosymbiont in aphids. Both symbionts were housed within distinct bacteriocytes and were maternally transmitted. The Buchnera and Arsenophonus symbionts had streamlined genomes of 432,286 bp and 853,149 bp, respectively, and exhibited metabolic complementarity in riboflavin and peptidoglycan synthesis pathways. These anatomical and genomic properties were similar to those of independently evolved multi-partner symbiotic systems, such as Buchnera–Serratia in Lachninae and Periphyllus aphids, representing remarkable parallelism. Furthermore, symbiont populations and bacteriome morphology differed between reproductive and soldier castes. Our study provides the first example of co-obligate symbiosis in Hormaphidinae and gives insight into the evolutionary genetics of this complex system.


INTRODUCTION
Endosymbionts, microorganisms that live inside the body or cells of host organisms, are widespread in eukaryotes, serving as important resources for evolutionary innovation in hosts (Moran and Baumann, 2000;Nowack and Melkonian, 2010;Pawlowska et al., 2018;Rosenberg et al., 2010). Endosymbionts have been shown to confer beneficial functions to hosts, such as provisioning essential nutrients that are not synthesized by hosts (Akman Gü ndü z and Douglas, 2012;Douglas et al., 2001;Shigenobu et al., 2000;The International Aphid Genomics Consortium, 2010;Wilson et al., 2010), providing resistance to heat stress (Chen et al., 2000;Russell and Moran, 2006) and protection against natural enemies via toxin production (Degnan et al., 2009;Nakabachi et al., 2013;Oliver et al., 2003). The acquisition of novel traits carried by endosymbionts contributes to niche divergence and lineage diversification of hosts (Janson et al., 2008;Sudakaran et al., 2017).
Aphids are good models for studying endosymbiosis. Almost all aphid species harbor the obligate mutualistic symbiont Buchnera aphidicola (Gammaproteobacteria) in specialized host cells known as bacteriocytes, and these symbionts are vertically transmitted to offspring (Braendle et al., 2003;Miura et al., 2003). Buchnera provides essential amino acids and riboflavin (vitamin B 2 ), which aphids cannot synthesize and are deficient in their sole diet of phloem sap (Akman Gü ndü z and Douglas, 2012;Douglas et al., 2001;Nakabachi and Ishikawa, 1999;Shigenobu and Wilson, 2011). In addition to the primary symbiont Buchnera, aphids are often associated with facultative symbionts, which are not essential but can contribute to host fitness and phenotypes (Oliver et al., 2010). Buchnera genomes have experienced significant reductions down to $0.6 Mb with $600 protein-coding genes. Despite the drastic genome reduction, Buchnera genomes retain genes involved in the biosynthesis of amino acids and vitamins essential for the host aphids (Chong et al., 2019;Van Ham et al., 2003;Shigenobu et al., 2000). Facultative symbionts exhibit moderate genome reductions. For example, Serratia symbiotica, Hamiltonella defensa and Regiella insecticola of pea aphids have $2.0 Mb genomes encoding $2,000 genes (Burke and Moran, 2011;Degnan et al., 2009Degnan et al., , 2010. Yorimoto, 2022). Novel symbionts, such as Sphingopyxis, Pectobacterium, Sodalis-related, and Erwiniarelated bacteria, have been found by 16S rRNA amplicon sequencing in a wide variety of aphid lineages (McLean et al., 2019;Xu et al., 2020Xu et al., , 2021. In addition, co-obligate symbioses, where multiple species of symbionts are essential for host survival, have been found in several aphid lineages. Co-obligate symbiosis involving Buchnera and Serratia evolved repeatedly in multiple aphid lineages, including the subfamily Lachninae, genus Periphyllus, Microlophium carnosum, and Aphis urticata (Manzano-Marín et al., 2017;Monnin et al., 2020). Co-obligate Buchnera of Lachninae and Periphyllus (Chaitophorinae) aphids have $0.4 Mb genomes encoding $400 genes and have lost genes involved in the biosynthesis of some essential amino acids and vitamins that the co-obligate Serratia is presumed to compensate for (Lamelas et al., 2011a;Manzano-Marín et al., 2016;Monnin et al., 2020;Pé rez-Brocal et al., 2006).
The tribe Cerataphidini (Hemiptera: Aphididae: Hormaphidinae) is an aphid clade that possesses a number of biologically interesting characteristics, including a complex life history, gall formation, and eusociality. They are able to alternate between primary host plants, where sexual reproduction occurs, and secondary host plants, where parthenogenesis occurs, seasonally (Aoki and Kurosu, 2010;Fukatsu et al., 1994). The fundatrix of Cerataphidini aphids hatches from an overwintering fertilized egg and induces morphologically diverse galls on the Styrax trees, their primary host plant (Aoki and Kurosu, 2010;Fukatsu et al., 1994). Cerataphidini aphids are eusocial; they produce sterile individuals known as a soldier caste to protect the colony from natural enemies (Stern and Foster, 1996). Fukatsu et al. (1994) reported that several species of Cerataphidini have secondary symbionts in addition to Buchnera (Fukatsu et al., 1994), although their identities and roles remain elusive. In several Cerataphidini aphids, the primary symbiont Buchnera has been replaced with extracellular yeast-like symbionts (Fukatsu et al., 1994).
In this study, we investigated bacterial symbionts of Ceratovacuna japonica in the Cerataphidini tribe (Figure 1A), taking an advantage of a recently established laboratory rearing system for this species (Hattori et al., 2013). We uncovered two endosymbionts of Ce. japonica by deep sequencing of 16S rRNA amplicons, Buchnera and an Arsenophonus-related bacterium, and these were consistently detected together. We sequenced the whole genomes of the two symbionts to assess the roles of both symbionts. We conducted microscopic observations to investigate their localization and dynamics in the host. We also compared symbiont populations between social castes. Our results suggest that the newly detected Arsenophonus-related symbiont is a novel species that has established an obligate relationship with the host Ce. japonica through collaboration with Buchnera.

Co-infection of Buchnera and Arsenophonus in natural populations of Ceratovacuna japonica
To assess the symbiont communities in Ce. japonica, we collected 26 colonies at nine geographically distinct populations from three different host plant species across Japan for high-throughput 16S rRNA amplicon sequencing ( Figures 1B, 1C, Tables S1, and S2). The sequencing of 16S rRNA hypervariable regions, V1-V2 and V3-V4, yielded 332,656 and 432,594 reads after quality filtering. V1-V2 and V3-V4 reads were classified into 13 and 25 ASVs (amplicon sequence variants), respectively. The prevalent bacterial genera were identified as Buchnera (mean relative abundance GSE of V1-V2: 81.48 G 2.63%, V3-V4: 92.15 G 2.22%), Arsenophonus (V1-V2: 14.89 G 2.41%, V3-V4: 2.55 G 0.76%), and Hamiltonella (V1-V2: 3.60 G 1.58%, V3-V4: 5.25 G 1.88%) ( Figure 1C, Table S1, and S2). Buchnera, the nearly ubiquitous endosymbiont of aphids, was detected in all populations at an extremely high relative abundance (86.92 G 1.86%) ( Figure 1C). In addition, Arsenophonus was detected in all populations of Ce. japonica, regardless of host plant and geographical location ( Figures 1B and 1C, Table S1 and S2). Hamiltonella was detected in specimens from 10 populations in the Nagano area (#8-17 in Figure 1) feeding on Sasa senanensis; however, it was not detected in the Hokkaido population (#1 in Figure 1) on the same host plant (Figures 1B, 1C, Table S1, and S2). Serratia was detected in only a single colony at a low level ( Figures 1B and 1C, Table S2). We then performed PCR analyses to confirm the presence or absence of Buchnera, Arsenophonus, and Hamiltonella in the Ce. japonica populations with specific primers targeting a single-copy gene, dnaK, for each bacterium. Consistent with the result of 16S rRNA amplicon sequencing, PCR analyses showed that both Buchnera and Arsenophonus were detected in all populations, whereas Hamiltonella was detected only in populations in the central Japan area (Figures 1D and S1A). Next, we investigated the infection status at the individual level. Diagnostic PCR analyses of each of the 12 individuals in four geographically distinct populations detected both Buchnera and Arsenophonus in all cases ( Figure S1B). Taken  iScience Article always co-infected with Buchnera and Arsenophonus, forming a multi-partner symbiosis. In addition, Ce. japonica could be infected with Hamiltonella as a facultative symbiont.
We established an isofemale line of the aphid Ce. japonica, named NOSY1, from a single individual collected at the foot of Mt. Norikura (location #14 in Figures 1, S1, Tables S1, and S2). It was cultured on bamboo shoots in the laboratory to undergo parthenogenetic reproduction for detailed molecular and microscopic analyses, as described below.
We also built a phylogenetic tree of Arsenophonus of Ce. japonica (hereafter referred to as Arsenophonus CJ) and related bacteria based on the 16S rRNA sequences ( Figure S2A). The tree indicated that Arsenophonus CJ was related to endosymbionts of hematophagous Diptera (the superfamily Hippoboscoidea), such as Lipoptena and Trichobius, and lice species (Group A in Figure S2A), whereas Arsenophonus CJ was not included in the clade of symbionts of Hemiptera and Hymenoptera (Group B in Figure S2A). This unusual clustering that does not reflect the phylogenetic relationship of the host insect might be an artifact caused by long-branch attraction and base-pair shift, which are often reported in the phylogenetic analyses of insect endosymbionts . To overcome this problem, we constructed the phylogeny of Arsenophonus using 204 single-copy orthologous protein sequences composed of 54,777 amino acid residues that are conserved among Arsenophonus and Riesia. The phylogenetic tree strongly supported the monophyly of the obligate endosymbionts of hematophagous insects and plant sap-feeding insects, all of which possess streamlined small genomes ( Figure 2B). This phylogenetic position of Arsenophonus CJ was also supported by the phylogenetic trees based on Dayhoff6 recoded protein sequences, whose method minimized the heterogeneous composition and long-branch attraction ( Figure S2B). The 16S rRNA sequence of Arsenophonus CJ showed the highest similarity (93.0%) to that of Candidatus Arsenophonus lipoptenae ( Figure S3A), an obligate endosymbiont of the blood-sucking deer fly Lipoptena cervi with a role as a B vitamin provider (Nová ková et al., 2016). This 93.0% identity was far lower than typical thresholds for assignment to the same species (e.g., %97%) (Stackebrandt and Goebel, 1994), indicating that the symbiont discovered in this study was a novel species in the genus Arsenophonus.
The 16S rRNA sequence of Hamiltonella of Ce. japonica (hereafter referred to as Hamiltonella CJ) was almost identical (99.8%) to that of Candidatus Hamiltonella defensa, a facultative symbiont of the pea aphid Acyrthosiphon pisum ( Figure S3B).

Buchnera and Arsenophonus are intracellular symbionts housed inside distinct bacteriocytes
We inspected the internal morphology of Ce. japonica to uncover the localization of the three bacteria identified by 16S rRNA amplicon sequencing. Our histological observation identified a pair of symbiotic organs (bacteriomes) in the thorax of the adult parthenogenetic viviparous female ( Figures 3A and 3C). Each bacteriome exhibited an oval-shaped structure with a major axis of approximately 191 mm composed of several large presumably polyploid nuclei surrounded by the cytoplasm, which was filled with bacterial cells ( Figure 3B), a typical characteristic of aphid bacteriocytes. Of interest, HE staining and DAPI staining patterns allowed us to distinguish between two types of bacteriocytes. There were several uninucleate bacteriocytes on the surface of the bacteriome and a single double-nucleate bacteriocyte at the center of the bacteriome; the latter type of bacteriocytes showed less intense HE signals and more intense DAPI signals than those of the former type ( Figures 3D and S4).
We next conducted fluorescence in situ hybridization (FISH) using probes specifically targeting each bacterium, Buchnera CJ, Arsenophonus CJ, and Hamiltonella CJ. The FISH analysis revealed that Buchnera and Arsenophonus infected the same bacteriome but were localized in different bacteriocytes, consistent with the differential staining pattern of HE and DAPI. Buchnera was localized in the uninucleate bacteriocytes in the outer layer of the bacteriome, whereas Arsenophonus was localized in the single double-nucleate bacteriocyte at the center of the bacteriome ( Figures 3E and 3G). Both Buchnera and Arsenophonus were observed exclusively in these specialized bacteriocytes and we had no evidence for the extracellular localization (e.g. hemolymph) of these endosymbionts ( Figure 3E). In addition, ovarioles in the abdomen contained many embryos infected with both Buchnera and Arsenophonus ( Figure 3E), suggesting that both symbionts are maternally and vertically transmitted to offspring. In contrast to the systematic localization of Buchnera and Arsenophonus in the bacteriocytes, sporadic Hamiltonella were found in the hemocoel and some were detected probably in the sheath cells located between bacteriocytes, where they often coexisted with Arsenophonus ( Figures 3F, 3H, and 3I).    iScience Article Electron microscopy also revealed three types of symbionts that differed in morphology and electron density ( Figure 3I). Buchnera and Arsenophonus were round and resided within their specialized bacteriocytes, whereas Hamiltonella was rod-shaped and resided within sheath cells (Figures 3I-3K and 3O). Buchnera and Arsenophonus had a three-layered membrane structure ( Figures 3M and 3N), indicating that both symbionts are packed inside the host-derived membranes called symbiosomal membranes, as reported in Buchnera of the pea aphid (Munson et al., 1991). In contrast, Hamiltonella had only a two-layered membrane, i.e., a bacterial inner membrane and outer membrane ( Figure 3O).

Streamlined small genomes of Buchnera CJ and Arsenophonus CJ exhibit complementary metabolic capacity
We conducted a shotgun sequencing of the hologenome of the isofemale line of Ce. japonica NOSY1. A metagenomic assembly approach (see STAR Methods for details) yielded assemblies of the complete genomes of three bacterial symbionts, Buchnera CJ, Arsenophonus CJ, and Hamiltonella CJ ( Figure S5). The Buchnera CJ genome consisted of one circular chromosome and two plasmids, pLeu and pTrp. (Figures 4A and 4D and Table 1). The Buchnera CJ chromosome had a length of 414,725 bp, G + C content of 20.0%, and coding density of 87.3%. The genome size of Buchnera CJ was significantly smaller than the typical size (approximately 600 kb) of Buchnera of many aphid species (Chong et al., 2019;Van Ham et al., 2003;Shigenobu and Yorimoto, 2022;Shigenobu et al., 2000;Tamas et al., 2002). It was similar to the sizes of genomes of Buchnera of aphids belonging to the genera Cinara, Tuberolachnus, and Periphyllus (Lamelas et al., 2011a;Manzano-Marín et al., 2016;Monnin et al., 2020;Pé rez-Brocal et al., 2006), all of which coexist with another obligate symbiont, S. symbiotica. The chromosome of Buchnera CJ encoded 370 protein-coding genes (CDSs), 3 rRNAs (16, 5, and 23S), 30 tRNAs, and 6 pseudogenes. The plasmid pLeu consisted of at least two tandem repeats, the 7,001 bp units of six genes ( Figure 4D). The plasmid pTrp consisted of at least two tandem repeats, the 10,560 bp units of one trpE, one pseudogenized trpG, and 10 trpG genes ( Figure 4D).
The Arsenophonus CJ genome consisted of one circular chromosome (Figures 4B and Table 1). The Arsenophonus CJ chromosome had a length of 853,149 bp and G + C content of 18.5%. The genome of Arsenophonus CJ was considerably smaller than the genome of Arsenophonus nasoniae (5.0 Mb), a son-killer bacterium of Nasonia vitripennis (Darby et al., 2010), and similar in size to the genome of Candidatus Arsenophonus lipoptenae (0.84 Mb), an obligate intracellular symbiont of the blood-feeding deer ked L. cervi (Nová ková et al., 2016). Note that Arsenophonus lipoptenae was most closely related to Arsenophonus CJ based on the 16S rRNA sequences, as mentioned above ( Figure S2A). In total, the Arsenophonus CJ genome encoded 512 proteins, 3 rRNAs (16, 5, and 23S), 32 tRNAs, and 11 pseudogenes. Notably, the Arsenophonus CJ genome exhibited a low coding density (59.9%), which may be a sign of ongoing genomic erosion.
The Hamiltonella CJ genome had a length of 2,258,324 bp, although we could not produce a circular contig probably because of the long repeats over 10,000 bp at both ends of the contig (Figures 4C and Table 1). The genome size of Hamiltonella CJ was similar to that of Hamiltonella of A. pisum (Degnan et al., 2009), which is a facultative symbiont of aphids, and was larger than that of Hamiltonella of Bemisia tabaci (Rao et al., 2015), which is a co-obligate endosymbiont of whiteflies. The Hamiltonella CJ genome encoded 2,097 proteins, 9 rRNAs (3 of 16S, 3 of 5S, and 3 of 23S), 42 tRNAs, and 72 pseudogenes.
Based on two observations (1) the systematic co-infection of two endosymbionts, Buchnera and Arsenophonus in Ce. japonica, and (2) the drastic genome reduction of these two bacteria, we hypothesized  iScience Article that the Ce. japonica aphid depends on Buchnera CJ and Arsenophonus CJ forming a co-obligate symbiosis. In several hemipteran insects, metabolic complementarity between co-symbionts has been observed (Husnik et al., 2013;Lamelas et al., 2011b;McCutcheon et al., 2009;Rao et al., 2015;Szabó et al., 2020;Wu et al., 2006). To determine if Buchnera CJ and Arsenophonus CJ form co-obligate associations with the host, we examined the genomic signature of metabolic complementarity by comparing the gene repertoires of these bacteria. We detected a complementarity gene repertoire in the metabolic pathways of riboflavin and peptidoglycan between Buchnera CJ and Arsenophonus CJ. Although all genes responsible for riboflavin biosynthesis were missing from the Buchnera CJ genome, these genes were retained in the Arsenophonus CJ genome ( Figure 4F and Table S5), indicating a complementary riboflavin-related gene set between the two symbionts. Although Buchnera CJ lost all genes related to peptidoglycan synthesis, the Arsenophonus CJ genome retained genes to synthesize dap-type peptidoglycan (glmMSU, murABCDEFG, mraY, mrcB, ftsI, and dacA) (Table S6). This complementary gene repertoire related to nutrition and cell wall components represent a genomic signature of co-obligate symbiosis.
Although Arsenophonus-related bacteria infect some aphids as facultative symbionts, the genus has not been reported as obligate symbionts in aphids, to the best of our knowledge. As such, we further inspected the genome of Arsenophonus CJ to understand the evolution of the obligate symbiosis. Arsenophonus CJ had a streamlined 853 kb genome with an extremely low GC content (18.5%), a typical feature of obligatory endosymbionts. Notably, the Arsenophonus CJ genome exhibited a very low coding density (59.9%), suggesting ongoing genomic erosion. A COG analysis demonstrated that Arsenophonus CJ is missing many genes with broad functions, including essential housekeeping functions, such as ''Replication, recombination and repair'' ( Figure 4E). For example, the Arsenophonus CJ genome lacked the SOS system genes recA, lexA, umuCD, and uvrABC, involved in DNA repair, as observed in Buchnera. The Arsenophonus CJ genome lacked a majority of genes involved in amino acid biosynthesis pathways; however, it retained all genes responsible for riboflavin biosynthesis ( Figure 4F, Tables S4, and S5). Of interest, genes for lipid A biosynthesis were missing or pseudogenized in the Arsenophonus CJ genome. The lipid A biosynthesis pathway is highly conserved in free-living bacteria and facultative bacterial symbionts; however, obligate intracellular symbionts often lack all or some components of this pathway, which is interpreted as an adaptation to the host immune responses (Wu et al., 2006). Among nine key genes involved in lipid A biosynthesis, five genes were lost and four were found to be pseudogenes in the Arsenophonus CJ genome (Table S7). The analysis of lipid A-related gene repertoire of the Arsenophonus CJ provided two insights: (1) Arsenophonus CJ lost the ability to produce functional lipid A, as observed in other obligatory symbionts, and (2) the degenerative process occurred relatively recently, because four pseudogenes were detected. Taken together, the Arsenphonus CJ genome showed the features of obligatory symbionts and the signature of genomic erosion because of the ongoing obligate interaction.  (F) Comparison of gene repertoires responsible for nutrient synthesis by Buchnera and Arsenophonus. E. coli is shown as an example of a free-living bacterium. Color blocks indicate the completeness of the minimal gene set for metabolic pathways: red, orange, yellow, light blue, and blue mean 0-24%, 25-49%, 50-74%, 75%-99%, and 100% of the completeness, respectively. In the aphid-Buchnera symbiosis, a metabolic collaboration is known for amino acid syntheses, where the host complements the steps missing from Buchnera as observed in the pathways of leucine, isoleucine, valine, and methionine syntheses (Shigenobu and Wilson, 2011;The International Aphid Genomics Consortium, 2010 iScience Article multi-partner symbiosis in Ce. japonica, we analyzed the formation of the bacteriome and dynamics of symbionts during parthenogenetic embryogenesis. We dissected ovarioles from the third or fourth instar nymphs of viviparous aphids, within which a series of developing embryos can be observed. Until anatrepsis (stage 8), when the germband is invaginating from both the dorsal and ventral sides, no symbionts were detected ( Figure 5A). Around the time of germband elongation and folding into an S shape (stage 11), we found a mixed population of Buchnera and Arsenophonus incorporated into the embryo from the posterior part ( Figure 5B). At the later stage of the germband elongation, when it twisted with more abdominal segments (stage 12), we observed the first formation of bacteriocytes, where huge host nuclei and symbiont masses were cellularized into bacteriocytes ( Figure 5C). At this stage, both Buchnera and Arsenophonus coexisted in the central bacteriocyte; however, only Buchnera cells were found in peripheral bacteriocytes ( Figures 5C, 5D, and 5E). By the time of the initiation of limb bud formation (stage 13), several bacteriocytes aggregated into an organ-like structure (i.e., a bacteriome) located at the ventral part of the embryo. Within the bacteriome, Buchnera and Arsenophonus were segregated into different types of bacteriocytes: Arsenophonus resided in a single central syncytial bacteriocyte containing four host nuclei, whereas Buchnera resided in multiple peripheral uninucleate bacteriocytes surrounding the Arsenophonus-containing bacteriocyte ( Figure 5F). After katatrepsis, the bacteriome was located in the dorsal abdomen of the embryo ( Figures 5G and 5H). At this stage, the morphology of the bacteriome changed from a round shape to a U-shape, maintaining the orientation of the two types of bacteriocytes-outer Buchnera-containing bacteriocytes and inner Arsenophonus-containing bacteriocytes. In the late embryo before larviposition, the bacteriome divided laterally to form a pair of bacteriomes at the anterior abdomen ( Figure 5I). After larviposition, the pair of bacteriomes was located at the anterior abdomen, maintaining the orientation of the two types of bacteriocytes ( Figure 6E). The morphology and localization of the bacteriomes in first instar larvae were comparable to those observed in adults ( Figure 3A).

Infection of
Hamiltonella CJ was also detected in the infecting bacterial mass together with Buchnera and Arsenophonus at the S-shape embryo stage ( Figure S7A). However, unlike Buchnera and Arsenophonus, at later stages, Hamiltonella was scattered around the periphery of the bacteriome ( Figure S7B) and was sometimes detected in or around unknown cells or organs, different from bacteriomes. Figure S7B shows an example of the localization near the tip of the germband.

Symbiosis status differs between soldier and reproductive castes
Ce. japonica is eusocial and produces sterile individuals known as a soldier caste specialized for colony protection ( Figures 1A, 6A, and 6B). With our laboratory rearing system, asexual viviparous females cultured on the bamboo S. senanensis produce the normal (reproductive) caste and the soldier caste. Soldiers remain first-instar nymphs throughout their life on the bamboo. We compared symbiosis status between castes. First, we evaluated Buchnera and Arsenophonus titers in first-instar individuals of the  Figures 6C and 6D). Buchnera was detected from both castes in Ce. japonica; however, the Buchnera titer was approximately 44% lower in the soldier caste than in the reproductive caste and the difference was statistically significant ( Figure 6C; Welch's t-test, p < 0.001). Arsenophonus was also detected in both castes in Ce. japonica; however, the Arsenophonus titer was approximately 25% lower in the soldier caste than in the reproductive caste and this difference was statistically significant ( Figure 6D; Welch's t-test, p < 0.05). Second, in an analysis of morphological differences, found that soldiers possess a pair of bacteriomes with a different morphology from that in the reproductive caste ( Figures 6E and 6F). Taken together, soldiers exhibit a different symbiotic condition distinct from that of the reproductive caste, including fewer symbionts in apparently malformed bacteriomes. Arsenophonus is an obligate symbiont in Ce. japonica forming a dual symbiosis with Buchnera We investigated bacterial symbionts of the eusocial aphid Ce. japonica by an integrative approach using molecular, genomic, and microscopic techniques. We found that two bacterial species, Buchnera CJ and Arsenophonus CJ, coexisted in all natural populations and individuals surveyed ( Figures 1C and 1D, S1, Tables S1, and S2). The persistent presence of Buchnera in Ce. japonica is not surprising because the species is a common obligatory endosymbiont in aphids (Baumann et al., 1995;Chong et al., 2019;Shigenobu and Wilson, 2011;Shigenobu et al., 2000). Buchnera CJ showed typical characteristics of Buchnera of other aphids, i.e., a small genome, transovarial inheritance, intracellular localization in bacteriocytes, and a subcellular morphology exhibiting a round-shaped bacterial cell encapsulated by the host-derived membrane (symbiosomal membrane). On the other hand, the systematic infection of Arsenophonus CJ was an unexpected and novel finding in this study. Although Arsenophonus-related bacteria infect some aphids as facultative symbionts (Ayoubi et al., 2020;Wagner et al., 2015;Wulff et al., 2013), the consistent infection of Arsenophonus in Ce. japonica suggests that the symbiosis is obligatory for the host. Microscopic observation showed that Arsenophonus cells were localized inside bacteriocytes, similar to the intracellular symbiosis observed in Buchnera (Figures 3E and 3G). Each Arsenophonus cell was surrounded by the host-derived membrane (symbiosomal membrane), as observed in Buchnera ( Figure 3N). Furthermore, Arsenophonus symbionts were maternally and vertically inherited in the host offspring ( Figures 5B-5I). Arsenophonus CJ had a small, streamlined genome (Figures 3B and Table 1), typical of obligate bacterial symbionts. Taken together, we detected a dual obligate symbiosis involving Arsenophonus sp. and Buchnera in Ce. japonica.

The paraphyly of Arsenophonus genus and the origin of Arsenophonus CJ
The genus Arsenophonus is a cluster of bacterial symbionts found in taxonomically widespread insects (Nová ková et al., 2009) and Arsenophonus-related bacteria have been reported to infect some aphids as facultative symbionts (Ayoubi et al., 2020;Jousselin et al., 2013;Wagner et al., 2015;Wulff et al., 2013). Our molecular phylogenetic analysis indicated that Arsenophonus is a paraphyletic genus sharing a

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common ancestor with Aschnera and Riesia. Based on the phylogenetic tree, obligate endosymbionts of hematophagous insects and plant sap-feeding insects were clustered into a monophyletic group ( Figures 2B and S2), despite the distant host lineage. A. lipoptenae and Riesia bacteria are obligate endosymbionts of blood-feeding insects, Hippoboscoidea flies and Anoplura sucking lice, respectively (Boyd et al., 2017;Nová ková et al., 2016). Arsenophonus of a whitefly Aleurodics dispersus is a co-obligate endosymbiont collaborating with the ancient obligate symbiont, Portiera aleyrodidarum (Santos-Garcia et al., 2018). Of interest, regardless of host lineages, the group of obligate endosymbionts including Arsenophonus CJ shares similar characteristics. These obligate endosymbionts have small genomes ranging from 0.53 to 0.84 Mb with AT-biased nucleotide composition and supply hosts with essential B vitamins that are deficient in the hosts' diet (Boyd et al., 2017;Hosokawa et al., 2012;Nová ková et al., 2016;Santos-Garcia et al., 2018). These features contrast facultative Arsenophonus symbionts that have relatively large genomes, e.g., the son-killer bacterium A. nasoniae (4.99 Mb) of N. vitripennis (Darby et al., 2010), Arsenophonus triatominarum (3.86 Mb) of Triatoma infestans, and Arsenophonus sp. (2.42 Mb) of Aphis craccivora. In sum, Arsenophonus CJ belonged to the group of obligatory endosymbionts, supporting the obligate nature of the observed symbiosis. Also, it should be noted that this is the first report of an obligate type of Arsenophonus endosymbiont found in aphid species.
The phylogenetic position of Arsenophonus CJ raises a question about its origin. Assuming the closest relative is the endosymbiont of hematophagous insects with currently available datasets, Arsenophonus CJ might be originated from symbionts of hematophagous insects by horizontal transfer. Alternatively, because Arsenophonus is widespread in Hormaphidinae aphids (Xu et al., 2021), the ancient acquisition of this symbiont by Ce. japonica is also possible. Extensive phylogenetic analyses of Arsenophonus in Hormaphidinae aphids with more extensive taxon sampling are needed to understand the origin of co-obligate Arsenophonus symbionts.

Buchnera and Arsenophonus as co-obligate symbionts
Our genome analysis revealed that the genome size of Buchnera CJ (Table 1) was significantly smaller than the majority of Buchnera genomes (Chong et al., 2019;Shigenobu and Yorimoto, 2022). It is possible that a loss of some functions in this Buchnera lineage was compensated by the other obligate symbiont Arsenophonus CJ. We examined the genomic signature of metabolic complementarity. All genes responsible for riboflavin biosynthesis were missing from the Buchnera CJ genome but present in the Arsenophonus CJ genome ( Figure 4F and Table S5), indicating a complementary riboflavin-related gene set between two symbionts. Arsenophonus CJ may provide riboflavin to the host, instead of Buchnera, which provides riboflavin in the Aphid-Buchnera symbiosis (Nakabachi and Ishikawa, 1999). Similarly, peptidoglycan synthesis genes were lost in Buchnera CJ and present in the Arsenophonus CJ genome (particularly glmMSU, murABCDEFG, mraY, mrcB, ftsI, and dacA) (Table S6). These results provide evidence for complementary repertoires of genes involved in nutrition and cell wall production in Buchnera CJ and Arsenophonus CJ, further indicating that they represent a co-obligate symbiosis. Similar genomic complementarity was reported in the Buchnera-Serratia co-obligate symbioses found in Lachninae and Periphyllus aphids, as well as in the more recently evolved associations found in A. urticata and M. carnosum  (See below for further discussion).
Our histological observation revealed a characteristic arrangement of the two symbionts within the same bacteriome. Both symbionts resided within the same bacteriome but in separate bacteriocytes; the central syncytial bacteriocytes harboring Arsenophonus CJ were morphologically different from the peripheral uninucleate bacteriocytes harboring Buchnera within the bacteriome of Ce. japonica (Figure 3). In bacteriome formation during embryogenesis, although both Buchnera CJ and Arsenophonus CJ were maternally transmitted simultaneously to the embryos, the symbionts were segregated into different types of bacteriocytes ( Figures 5B-5F). These observations indicate a high level of anatomical and developmental integration of the two symbionts and the host.

Parallel evolution of co-obligate symbiosis in aphids
The co-obligate symbiosis of Buchnera and Serratia has occurred in aphid lineages, such as Cinara and Tuberolachnus in the subfamily Lachninae, Periphyllus in the subfamily Chaitophorinae, and A. urticata and M. carnosum in the subfamily Aphidinae (Lamelas et al., 2011a(Lamelas et al., ,2011bManzano-Marín and Latorre, 2014;Manzano-Marín et al., 2016;Monnin et al., 2020;Pé rez-Brocal et al., 2006). In several Cinara species, co-obligate Serratia has been replaced by Erwinia, Fukatsuia,  Manzano-Marín et al., 2019;Meseguer et al., 2017). Our study is the first report of co-obligate symbiosis in the subfamily Hormaphidinae and revealed a novel partner combination (i.e., Buchnera and Arsenophonus). Regardless of the aphid lineage or co-obligate symbiont species, there are amazing similarities among co-obligate symbioses ranging from genomic features to the morphology of bacteriocytes. Although typical Buchnera genome sizes are $0.6 Mb, those of co-obligate Buchnera in Ce. japonica, Lachninae, and Periphyllus aphids have $0.4 Mb genomes, regardless of phylogenetic positions (Table 1) (Chong et al., 2019;Shigenobu and Yorimoto, 2022). All of these $0.4 Mb Buchnera genomes lost genes in the riboflavin, ornithine, and peptidoglycan biosynthesis pathways, although the ornithine and peptidoglycan biosynthesis pathways were also lost in several Buchnera genomes of other subfamilies ( Figure 4F, Tables S4, S5, and S6) (Chong et al., 2019;Lamelas et al., 2011a;Manzano-Marín et al., 2016Monnin et al., 2020;Pé rez-Brocal et al., 2006). The loss of nutritional functions in Buchnera to provide riboflavin to the host is presumably compensated by the partner obligate symbiont, as evidenced by the complementary gene repertoire. The loss of peptidoglycan pathway genes might be accounted for by the environment Buchnera resides where the obligate endosymbiont may not require cell walls inside the specialized host cells. In dual symbiosis, Buchnera and co-infected symbionts, such as Serratia, Erwinia, Fukatsuia, or Sodalis, are found in the same bacteriome but sorted separately into the distinct bacteriocytes in Lachninae aphids (Manzano-Marín et al., 2017. Mitochondrial density differs between Buchnera-containing bacteriocytes and co-obligate symbiont-containing bacteriocytes: Mitochondrial abundance was lower in bacteriocytes harboring Serratia in Ci. cedri (Gó mez-Valero et al., 2004) and it seems to be the case with Arsenophonus-containing bacteriocytes in Ce. japonica ( Figures 3J and 3K). These common characteristics are consistent with parallel evolution of the co-obligate symbiosis in aphid lineages, implying that there are evolutionary constraints on the aphid-Buchnera symbiosis.
In addition to the parallelism in co-obligate symbioses, there are significant variations across aphid lineages in the localization patterns of new symbionts. As we reported in this study, co-obligate Arsenophonus CJ resided inside the central syncytial bacteriocytes distinct from Buchnera's one and exhibited no extracellular localization in Ce. japonica (Hormaphidinae) (Figure 3). In Lachninae aphids, co-obligate symbionts reside inside the distinct bacteriocytes, whereas the distribution of the bacteriocytes harboring co-obligate symbionts is diversified (Manzano-Marín et al., 2017. The co-obligate Serratia of Pe. lyropictus (Chaitophorinae) exhibits intracellular and extracellular localizations, such as in central syncytium bacteriocytes, sheath cells, hemolymph, and gut of the host (Renoz et al., 2022). The variations in anatomical integration in multi-partner symbioses seem to reflect the degree of the symbiotic association among the host and symbionts.

Roles of Arsenophonus CJ
Based on the genomic analysis, Arsenophonus CJ is likely involved in riboflavin provisioning to the host ( Figure 4F, Tables S4, and S5). A similar function in B-vitamin provisioning has been reported for whiteflies, lice, and Hippoboscoidea flies (Nová ková et al., 2015(Nová ková et al., ,2016Santos-Garcia et al., 2018). An experimental study has demonstrated that a facultative Arsenophonus symbiont expands the dietary breadth in the aphid A. craccivora (Wagner et al., 2015). Arsenophonus CJ may also contribute to the usage of host plants, the snowbell tree Styrax japonicus and bamboo grasses, such as Pleioblastus chino, P. simonii and S. senanensis. However, the acquisition of new co-obligate symbiont species is not associated with adaptation to novel ecological niches in the evolution of co-obligate symbiosis of Cinara aphids (Meseguer et al., 2017). Further studies are needed to clarify the roles of the Arsenophonus symbiont in Ce. japonica.
Ongoing reductive evolution of the Arsenophonus CJ genome Synteny analysis showed that Arsenophonus genomes have experienced many rearrangements and deletions ( Figure S6C and S6D). In addition, the Arsenophonus CJ genome showed a low coding density and multiple pseudogenes (Table 1). Recently evolved symbionts are characterized by a low coding density, pseudogene formation, and many genome rearrangements and deletions (Burke and Moran, 2011;Ochman and Davalos, 2006). Accordingly, Arsenophonus CJ is a recently evolved co-obligate symbiont and gene inactivation is ongoing. An interesting example of ongoing genomic erosion in Arsenophonus CJ was the gene repertoire for lipid A biosynthesis. Lipid A is the hydrophobic anchor of lipopolysaccharide in the outer membrane of gram-negative bacteria (Raetz and Whitfield, 2002;Raetz et al., 2009), and elicits a strong immune response in host animals (Raetz and Whitfield, 2002 iScience Article biosynthesis were either missing or pseudogenes in the Arsenophonus CJ genome (Table S7). Losing the ability to produce lipid A in Arsenophonus CJ may be beneficial for the symbiotic association with host animals by accommodating the host immune response. The observed pseudogenization suggests an ongoing genomic erosion of Arsenophonus CJ, probably leading to a more streamlined obligatory endosymbiont specialized for the Arsenophonus-Buchnera dual symbiosis.

Symbiosis in the sterile soldier in eusocial aphids
Eusocial aphid species have been found in only two subfamilies, Hormaphidinae and Eriosomatinae (Pike and Foster, 2008;Stern and Foster, 1996). The eusocial aphid Ce. japonica provides a unique opportunity to study how symbiosis operates in a caste system. In a comparison of normal (reproductive) nymphs and sterile soldiers, we detected significantly fewer symbionts in soldiers than in normal nymphs ( Figures 6C and  6D) as well as a distorted bacteriome shape ( Figures 6E and 6F), indicating that the symbiotic condition differs between social castes in Ce. japonica. Soldiers of eusocial aphids are sterile and do not grow after birth (Chung and Shigenobu, 2022;Stern and Foster, 1996). Such differences in nutritional conditions between castes may be related to the difference in symbiotic status. In the eusocial aphid Colophina arma of the subfamily Eriosomatinae, the soldier caste lacks endosymbionts and bacteriomes entirely (Fukatsu and Ishikawa, 1992). In the eusocial ant tribe Camponotini, the obligate symbiont Blochmannia enhances nutrition, thereby influencing the size of worker ants (Feldhaar et al., 2007;Sinotte et al., 2018;Zientz et al., 2006). The mechanisms and biological significance of caste-dependent symbiosis in social insects are intriguing subjects for future exploration.

Limitations of the study
This study found a dual obligate symbiosis involving Arsenophonus sp. and Buchnera in Ce. japonica with molecular, genomic, and microscopic approaches. First, the role of Arsenophnus CJ remains elusive. Our genomic analysis predicted that Arsenophonus CJ likely has a role involved in riboflavin provisioning to the host, but it should be further validated experimentally. We do not exclude the possibility of other functions the symbionts exert. For the experimental assessment, a technique of selective and specific elimination of each symbiont, which is currently unfeasible for this system, by using antibiotics or some chemicals should be developed. Second, the origin of Arsenophonus CJ is unclear. Our molecular phylogenetic analysis indicated that Arsenophonus CJ was positioned in a clade that includes obligate endosymbionts of diverse host insects in origin such as hematophagous Diptera and sap-sacking Hemiptera. To understand the origin of Arsenophonus CJ and the evolutionary trajectory of Arsenophonus-Buchnera dual symbiosis, further genomic analysis of symbionts with more extensive taxon sampling in the subfamily Hormaphidinae is needed.

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Shuji Shigenobu (shige@nibb.ac.jp).

Materials availability
The isofemale NOSY1 strain of Ce. japonica established in this study has been maintained at the Laboratory of Evolutionary Genomics, National Institute of Basic Biology. iScience 25, 105478, December 22, 2022 iScience Article