Vertical Transmission at the Pathogen-Symbiont Interface: Serratia symbiotica and Aphids

Insects have evolved various mechanisms to reliably transmit their beneficial bacterial symbionts to the next generation. Sap-sucking insects, including aphids, transmit symbionts by endocytosis of the symbiont into cells of the early embryo within the mother’s body.

In this work, we investigated whether cultured S. symbiotica strains are capable of vertical transmission similar to facultative or obligate symbionts. We isolated a new S. symbiotica strain, HB1, that shares many features with CWBI-2.3 T but is notably less pathogenic. We examined the capacity of each of these strains to colonize hemolymph of the pea aphid (Acyrthosiphon pisum) and access embryos through the transovarial route described for B. aphidicola (8). Both strains are endocytosed into embryos, but embryos infected by the transovarial route do not appear to develop properly, and offspring infected by a transovarial route are not observed. Using green fluorescent protein (GFP)-tagged strains, we addressed whether transovarial transmission is open to any bacterial cell that comes into contact with the embryo or whether this process involves specific partner recognition. We found that Escherichia coli cannot colonize embryos, despite achieving a high titer within hosts. Thus, the endocytosis step required for transovarial transmission limits the taxonomic range of bacteria that can readily evolve to become aphid symbionts.

RESULTS
Pathogenic S. symbiotica strains form a distinct group closely related to mutualistic strains. We examined the evolutionary relationships of all S. symbiotica strains with publicly available complete genome sequences. To this list, we added a recently isolated and sequenced strain, designated S. symbiotica HB1, from the melon aphid (Aphis gossypii). Our phylogenetic analysis was based on 176 shared orthologous genes and was rooted with outgroups that included other Serratia and more distant Enterobacterales species ( Fig. 1; see Fig. S1 and Table S1A in the supplemental material). S. symbiotica strains are split into two clades, as previously reported (46). Clade A is composed of strains that act as pathogens, mutualists, or co-obligate symbionts in aphids from across the family Aphididae, while clade B is composed of strains that live only as co-obligate symbionts in aphids of the subfamily Lachninae. Clade A strains possess a range of genome sizes (1.54 to 3.58 Mb), GC content (48.7 to 52.5%), host species, and lifestyles. Within clade A, the cultured, gut pathogen strains (CWBI-2.3 T , HB1, Apa8A1, and 24.1) form a clade closely related to vertically transmitted mutualist  Table S1A in the supplemental material. Bootstrap values are indicated with symbols on nodes. The scale bar represents the expected number of substitutions per site. The complete phylogeny is presented in Fig. S1.
Cultured S. symbiotica strains are pathogenic when injected into pea aphid hemolymph. Based on previous studies, S. symbiotica CWBI-2.3 T acts as a gut pathogen in its original host, the black bean aphid (44). In both black bean aphid and pea aphid hosts, S. symbiotica CWBI-2.3 T appears to be restricted to the gut and is not observed infecting hemolymph (44,47,48). To determine whether S. symbiotica CWBI-2.3 T and HB1 can persist and act as pathogens in the hemolymph of pea aphids, we injected fourth-instar pea aphids with tagged strain CWBI-GFP or HB1-GFP at two doses: low (;80 cells injected, 3 replicate trials per treatment) and high (;800 cells injected, 1 trial per treatment). Then, we tracked aphid survival and bacterial titer over time. For comparison, we simultaneously performed injections of Serratia marcescens Db11, a known insect pathogen (49), injections of hemolymph from pea aphids infected with facultative, vertically transmitted S. symbiotica Tucson (23), and injections of buffer as a negative control. We found that S. symbiotica CWBI-GFP and HB1-GFP act The relative titer was calculated as the copy number of S. symbiotica dnaK normalized by copy number of the single-copy Acyrthosiphon pisum gene ef1a. CWBI and HB1 samples used for qPCR are the same as those used to determine bacterial titer by spot-plating. Letters a and b denote two groups of strains that have significantly different titers from one another and indistinguishable titers within groups at an a of 0.01 by the Kruskal-Wallis test, followed by Dunn's multiple-comparison test. In all box plots, boxes mark upper and lower quartiles, and the central line denotes the median value.
as pathogens in pea aphid hemolymph, regardless of dose injected. The survival rates of aphids injected with S. symbiotica CWBI-GFP, S. symbiotica HB1-GFP, and S. marcescens Db11 were much lower than those of aphids injected with buffer or S. symbiotica Tucson (P , 0.001, Cox proportional-hazards model) ( Fig. 2A).
We hypothesized that the virulence of S. symbiotica CWBI-GFP and HB1-GFP was related to their titer in hemolymph. To test this hypothesis, aphids were sacrificed every 24 h to measure the number of CFU per aphid. Regardless of the injection dose, S. Serratia symbiotica at the Pathogen-Symbiont Interface ® symbiotica CWBI-GFP and HB1-GFP grow exponentially within aphids, plateauing at ;10 9 CFU per aphid by 5 to 7 days postinjection (dpi) ( Fig. 2B; Fig. S2). The difference in growth of CWBI-GFP and HB1-GFP at 3 and 4 dpi may explain the difference in aphid survival rate across these treatment groups (Fig. 2B). As S. symbiotica Tucson is not culturable, quantitative PCR (qPCR) was used to compare titers of CWBI-GFP and HB1-GFP to those of S. symbiotica Tucson, immediately after injection (initial) and several days after injection (5 dpi), and to the titers at which other mutualist S. symbiotica strains persist in pea aphids reared in the laboratory (Fig. 2C). The relative titer was calculated as the copy number of a single-copy Serratia gene (dnaK) normalized by a single-copy pea aphid gene (ef1a). Although similar numbers of S. symbiotica CWBI-GFP, HB1-GFP, and Tucson cells were injected into aphids, both S. symbiotica CWBI-GFP and HB1-GFP reach higher titers than S. symbiotica Tucson at 5 dpi (Fig. 2C). The titer of S. symbiotica Tucson at 5 dpi is comparable to the steady-state titer of S. symbiotica strains maintained in naturally infected, clonal pea aphids established as laboratory lines (Fig. 2C).
Cultured S. symbiotica strains are not vertically transmitted in pea aphids. Typically, S. symbiotica CWBI-2.3 T infects aphids by a fecal-oral route or from plants (44). To determine if S. symbiotica CWBI-GFP and HB1-GFP can be transmitted to offspring via the transovarial route used by mutualistic symbiotic strains, we screened the offspring of surviving aphids for S. symbiotica by looking for GFP fluorescence and by plating for CFU. As transovarial transmission of symbionts occurs early in embryonic development, there is a delay between injection and the birth of infected offspring (38). To determine when after injection we should begin to observe offspring infected by transovarial transmission, we injected mutualistic S. symbiotica Tucson and monitored its transmission by sampling offspring and using PCR to screen for the presence of S. symbiotica. Transmission of S. symbiotica Tucson was identified in newborn offspring starting at 10 dpi (Fig. 3A). The proportion of infected offspring per mother increased over time, reaching 100% for most mothers by 15 dpi (Fig. 3A).
In contrast to aphids injected with S. symbiotica Tucson, most aphids injected with S. symbiotica CWBI-GFP and HB1-GFP did not survive to 10 dpi ( Fig. 2A) or did not produce offspring past 10 dpi. However, 36 of the 144 females injected with a low dose (;80 cells) of S. symbiotica HB1 did survive and produce offspring beyond 10 dpi. Of these, only 20 females (55.6%) produced both infected offspring and offspring that survived for more than 5 days. The number of offspring produced by these females decreased from 10 to 15 dpi, and no females survived past 16 dpi (Fig. 3B). At 15 dpi, all offspring born from 10 to 15 dpi were screened for GFP fluorescence to determine infection status. A large proportion of offspring born from 10 to 15 dpi were fluorescent at 15 dpi (72/117, or 61.5%) and at 20 dpi (58/88, 65.9%) (Fig. 3B). Fluorescence from S. symbiotica HB1-GFP appeared to be limited to the guts of these offspring ( Fig. 3C; Fig. S3). To determine if offspring infected with HB1-GFP could transmit HB1-GFP to the next generation, we waited until they were reproductive adults (9 days old) and dissected out embryos from 12 fluorescent and 12 nonfluorescent offspring. We observed no evidence of HB1-GFP in embryos (Fig. 3D). We plated the remaining offspring (46 fluorescent, 18 not fluorescent) and observed that only fluorescent aphids produced fluorescent colonies. Together, these results suggest that the majority of infected offspring that survive to adulthood are infected by a fecal-oral route and that S. symbiotica HB1-GFP cannot be stably vertically transmitted through the transovarial route across multiple generations.
Cultured S. symbiotica strains are capable of colonizing pea aphid embryos. Stable intergenerational transmission of S. symbiotica CWBI-2.3 T and HB1 was not observed, apparently due to the pathogenicity of these strains and/or their limited ability to colonize offspring. In order to determine whether these strains were, nevertheless, capable of transovarial transmission, we injected aphids with S. symbiotica CWBI-2.3 T and HB1, dissected ovarioles at 7 dpi, and used fluorescence in situ hybridization (FISH) to visualize its infection pattern relative to that of the primary symbiont B. aphidicola during embryonic development. To compare these results to the transmission pattern of a mutualist strain, we injected aphids with hemolymph from pea aphids infected with S. symbiotica Tucson (Fig. 4A to D).
Embryos injected with S. symbiotica Tucson display regular growth and development, reaching 400 mm in length by the time of katatrepsis (41) (Fig. 4A). As previously described, B. aphidicola and mutualistic S. symbiotica are transmitted to the syncytium of stage 7 blastula from hemolymph via an endocytic process (8,41,50). S. symbiotica Tucson appears to be unable to infect embryos at later developmental stages, as embryos that were beyond stage 7 at the time of injection lack S. symbiotica (Fig. 4B). This observation is supported by the absence of S. symbiotica in offspring born the first 10 days after injection (Fig. 3A) (38). Those embryos exposed to S. symbiotica Tucson at stage 7 possess S. symbiotica in sheath cells, but strain Tucson does not invade primary bacteriocytes that contain B. aphidicola (Fig. 4C). The endocytosis of S. symbiotica Tucson into stage 7 embryos occurs at the posterior end of the embryo (Fig. 4D), as previously described for the closely related strain S. symbiotica IS (8).
Despite its pathogenicity, the dominant infection pattern of CWBI-2.3 T is similar to that of the nonpathogenic Tucson strain (Fig. 4E and F). Cells of CWBI-2.3 T are attached to the embryonic surface but do not infect embryos that are beyond stage 7 (Fig. 4G). Following infection, CWBI-2.3 T is packaged similarly to the Tucson strain; both are Serratia symbiotica at the Pathogen-Symbiont Interface ® sorted into sheath cells and cannot colonize the primary bacteriocytes that house B. aphidicola ( Fig. 4C and H). CWBI-2.3 T is endocytosed into early embryos with B. aphidicola (Fig. 4I), as is S. symbiotica HB1 (Movie S1). Remarkably, at 7 dpi, CWBI-2.3 T greatly outnumbers B. aphidicola in the syncytial cell (Fig. 4I). In contrast to S. symbiotica Tucson, infection with cultured S. symbiotica CWBI-2.3 T stunts embryonic growth, though it does not prevent progression through characteristic early developmental stages ( Fig. 4E and F).
Transovarial transmission is a specific capability of S. symbiotica strains. To determine if endocytosis is selective at the level of bacterial species, we tested the transmission capability of E. coli K-12 strain BW25113. E. coli is related to B. aphidicola, S. symbiotica, and several other mutualistic symbionts of aphids, which are all within Enterobacterales (20). We chose strain BW25113 because it can infect the gut and hemolymph of pea aphids and kills aphids a few days postinfection (51). We created the tagged E. coli strain BW25113-GFP and injected it into fourth-instar aphids as described above. For comparison, we injected S. symbiotica CWBI-GFP into a separate set of fourth-instar aphids. E. coli BW25113-GFP forms a robust infection in pea aphid hemolymph, reaching titers comparable to those of S. symbiotica CWBI-GFP at 5 dpi (Fig. 5A). We dissected single ovarioles from 10 aphids in each treatment group at 5 dpi and observed early embryos to determine infection status. Using this approach, S. symbiotica CWBI-GFP could be seen infecting early embryos ( Fig. 5B and C; Movies S2 and S3). In contrast, E. coli BW25113-GFP attaches to the embryonic surface, sometimes coating the entire exterior of the embryo, but was never observed endocytosing into embryos ( Fig. 5D and E; Movies S4 and S5).

DISCUSSION
Transovarial transmission is a key feature of many insect-bacterium symbioses wherein bacteria provide a benefit to their host. This transmission route is linked to irreversible bacterial transitions, from pathogenicity to mutualism (2). However, many relationships that rely on transovarial transmission are ancient, and their early stages cannot be experimentally recapitulated, leaving unanswered if and how pathogenic bacteria access this transmission route. Focusing on secondary symbionts may be  more useful for understanding these early transitions, as some secondary symbionts or their close relatives are culturable, genetically tractable, and can be removed from or introduced to hosts without dramatically compromising host fitness (52)(53)(54). For example, Sodalis praecaptivus, a close relative to Sodalis species found as host-restricted symbionts across diverse insects, uses quorum sensing to attenuate virulence and gain access to vertical transmission in a nonnative host, the tsetse fly (55). The bacterial species S. symbiotica was first known as a vertically transmitted mutualist, but pathogenic strains were subsequently cultured from aphids collected in Europe and Africa (29,(33)(34)(35) and, in this study, in North America. These strains have provided a new opportunity to dissect the early steps involved in the transition to a host-restricted lifestyle. Knowing that vertical transmission is a key to this transition, we aimed to determine whether culturable, pathogenic S. symbiotica could access this pathway and what, if any, limitations are faced by S. symbiotica in this transition.
Cultured S. symbiotica strains are close relatives to nonculturable, vertically transmitted mutualists. However, several lines of evidence suggest that these strains lack a history of maternal transmission in aphids. For one, persistent vertical transmission generally leads to the irreversible loss of genes such that symbionts can no longer access a free-living or pathogenic lifestyle (56). In comparison to vertically transmitted strains, CWBI-2.3 T , HB1, Apa8A1, and 24.1 are culturable, maintain larger genomes, and possess more ancestral genes common to free-living Serratia (Fig. 1). Second, these strains do not appear to undergo vertical transmission in natural infections. Following ingestion by black bean or pea aphids, S. symbiotica CWBI-2.3 T is not subsequently detected in hemolymph or embryos but is present in the gut and in honeydew, suggesting that the dominant route of transmission for this strain is fecal-oral (33,43,47,48). We injected CWBI-2.3 T and HB1 into hemolymph to determine if they are nonetheless capable of transovarial transmission in pea aphids. Vertical transmission is theorized to be the primary force driving permanent bacterial transitions from pathogenicity to mutualism, but to do so, vertical transmission must precede mutualism (2). That pathogenic S. symbiotica strains CWBI-2.3 T and HB1 are endocytosed into the syncytial cell of early embryos along with B. aphidicola provides empirical evidence for the precedence of vertical transmission in this system. Together, these results suggest that the aphid gut has served as an access point for environmental or plant-associated Serratia to infect aphids and that gut pathogenicity was an ancestral lifestyle for strains that are now intracellular and mutualistic (33).
S. symbiotica is common to natural populations of pea aphids (38,57), and previous 16S rRNA gene surveys have identified S. symbiotica in aphid tribes from across the Aphidoidea (33,58). However, pathogenic and mutualistic strains have near-identical 16S rRNA sequences, so it is unclear how many cases represent S. symbiotica pathogens. To date, pathogenic strains have been cultured only from Aphis species. However, S. symbiotica CWBI-2.3 T can horizontally transmit across aphids feeding on the same plant (43) and can infect the guts of alternative aphid species, including the pea aphid (47), suggesting that pathogenic S. symbiotica may be more widespread across aphid genera in nature. The global distribution of Aphis-associated strains, along with their ability to transmit using the same transovarial route as B. aphidicola, suggests that related gut-associated strains may serve as a source pool for the evolution of commensal or mutualistic strains. Mutualism may have arisen several times independently in S. symbiotica and, along with the subsequent horizontal transfer, would contribute to the phylogenetic discordance between facultative strains and their aphid hosts (58). The acquisition and replacement of secondary symbionts have occurred during the evolution of many ancient insect-microbe symbioses and may help hosts to escape the "evolutionary rabbit hole" of dependence on a primary symbiont that has become an ineffective mutualist due to genome decay (59)(60)(61).
Transovarial transmission in aphids generally occurs by bacterial endocytosis into the syncytial cell of early embryos (8). What host and bacterial factors are involved in this pathway are unclear, but insights may be gained from other systems in which hosts are genetically tractable. In Drosophila, knockout of yolk proteins or the Yolkless receptor results in reduced localization to and/or endocytosis of Spiroplasma in embryos, suggesting that the vitellogenin pathway is involved in transovarial transmission (62). While parthenogenetic aphids do not undergo vitellogenesis or produce visible yolk, it is possible that similar receptor-mediated processes are used for B. aphidicola and S. symbiotica transmission and that specific bacterial ligands are required. If this is the case, S. symbiotica strains that normally live in the aphid gut appear to possess the requisite molecular determinants, as they display an innate potential for endocytosis into embryos. Furthermore, the inability of E. coli BW25113 to endocytose into the syncytial cell of embryos suggests that the endocytic step of transovarial transmission contributes to selectivity in this system. While many bacterial taxa occasionally infect pea aphids (34,63), few are found as long-term mutualists. The primary symbiont, B. aphidicola, is stably maintained in most aphid lineages, though rare replacements exist (e.g., see reference 64). Additionally, few species are found as secondary symbionts, and most are members of the Enterobacterales, including S. symbiotica, "Ca. H. defensa," "Ca. R. insecticola," "Ca. F. symbiotica," "Ca. Erwinia haradaeae," and Arsenophonus; and, less commonly, other bacterial groups, including Wolbachia, Rickettsia, and Spiroplasma (21,22,61).
Aphids that are coinfected with B. aphidicola and mutualistic secondary symbionts possess several known mechanisms that limit the competition between these bacteria. For one, hosts can sort symbionts into distinct cell types, with B. aphidicola in primary bacteriocytes and S. symbiotica relegated to secondary bacteriocytes and sheath cells. Despite its pathogenicity, we observed that CWBI-2.3 T is not able to invade primary bacteriocytes with B. aphidicola and is compartmentalized into sheath cells in a manner similar to that of mutualistic strains (Fig. 3). Pea aphid genotypes may vary in their ability to associate with secondary symbionts. In contrast to the results obtained with Acyrthosiphon pisum LSR1 in our study, when the facultative, host-restricted strain S. symbiotica IS was transferred to Acyrthosiphon pisum AIST, it showed a disordered localization, invading primary bacteriocytes with B. aphidicola (8,65,80). In these cases, S. symbiotica IS was trapped in primary bacteriocytes, unable to exocytose during transmission (8). The specific exocytosis of B. aphidicola also likely plays a role in limiting competition between B. aphidicola and secondary symbionts across multiple generations.
The vertical transmission of CWBI-2.3 T and HB1 in pea aphids is limited by their virulence in hemolymph. Both CWBI-2.3 T and HB1 are more pathogenic in hemolymph than facultative S. symbiotica Tucson but also notably far less pathogenic than S. marcescens Db11. Possibly, adaptation to the gut selects for reduced Serratia virulence by allowing Serratia the time to form a robust gut infection and transmit to other aphids, including offspring, via honeydew (44). The genome of CWBI-2.3 T appears to already reflect some transition to symbiont status, having lost some genes common to free-living Serratia, such as those underlying chemotaxis (66). However, this strain also retains factors that promote host cell invasion and may continue to contribute to pathogenicity, including a complete type III secretion system. The virulence of CWBI-2.3 T and HB1 coincides with unregulated titer, as both of these strains attain 100-to 1,000-fold higher titers than mutualistic S. symbiotica when injected into hemolymph. This enormous difference in titer, and the constancy of the low titers observed for the symbiotic strains, suggests that mutualistic S. symbiotica growth is regulated. The regulation of virulence and titer is important in the establishment of vertical transmission. Self-regulation of both titer and virulence through quorum sensing has been demonstrated in Sodalis praecaptivus and allows this species to establish vertically transmitted infections in weevil and tsetse fly hosts (55,67). Whether mutualistic S. symbiotica strains have relied on similar mechanisms to establish persistent vertical transmission in aphids is unclear. Alternatively, S. symbiotica virulence may be attenuated by the loss of one or several key virulence factors before the establishment of vertical transmission. The experimental tractability of these strains will allow for future investigations focused on these attenuation mechanisms and the role of vertical transmission in the transition to a host-restricted lifestyle in aphids.

MATERIALS AND METHODS
Isolation and culture of S. symbiotica. S. symbiotica strain CWBI-2.3 T (DSM 23270) was obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures and grown on tryptic soy agar (TSA) plates at 27°C (29). S. symbiotica strain HB1 was isolated from the melon aphid (Aphis gossypii), collected in August 2018 from HausBar Farms in Austin, Texas. Details are provided in Text S1 in the supplemental material.
S. symbiotica HB1 genome sequencing. S. symbiotica HB1 was grown in tryptic soy broth (TSB) at room temperature and harvested at an optical density at 600 nm (OD 600 ) of ;1.0, and DNA was extracted with the DNeasy blood and tissue kit (Qiagen). A paired-end sequencing library with dual barcodes was prepared using the Illumina Nextera XT DNA kit, and sequencing was performed on an Illumina iSeq 100. Raw reads were trimmed using Trimmomatic (68) and assembled using the SPAdes algorithm (69) via Unicycler (70). Genome contamination and completeness were assessed using CheckM (71).
Phylogenetic analysis. S. symbiotica and outgroup genomes used for phylogenetic analysis are listed in Table S1A in the supplemental material. Genomes were downloaded from the NCBI Assembly Database on 2 March 2020. All outgroup genomes were filtered for .95% completeness and ,5% contamination using CheckM (71). Annotations were obtained using Prokka (72), and 176 single-copy orthologs were identified by OrthoFinder (73). These single-copy orthologs were aligned with MAFFT (74), trimmed using a BLOSUM62 matrix in BMGE (75), and concatenated using an in-house script, producing an alignment with 56,881 total amino acid positions. A tree was constructed by maximum likelihood with a JTT1R10 model and 100 bootstraps, using IQ-Tree (76). The complete phylogeny is available in Fig. S1. The presence of Serratia marker genes was determined using CheckM with the Serratia marker gene set provided with CheckM. The average nucleotide identity for S. symbiotica genomes was calculated using FastANI (77).
Tracking aphid survival, fecundity, and transmission after injection with S. marcescens, S. symbiotica, and injection buffer. Fourth-instar pea aphids were injected with S. marcescens Db11, recombinant S. symbiotica CWBI-GFP, recombinant S. symbiotica HB1-GFP, hemolymph from pea aphids infected with S. symbiotica Tucson, or injection buffer, as described in Text S1. Every 24 h, survival was recorded, offspring were collected, and surviving adults were moved to a fresh dish. Adults were collected at death or at the end of the experiment at 15 dpi and screened for the presence or absence of S. symbiotica. Details are provided in Text S1.
Bacterial titer by spot-plating and qPCR. Fourth-instar pea aphids were injected with recombinant S. symbiotica CWBI-GFP, recombinant S. symbiotica HB1-GFP, or hemolymph from pea aphids infected with S. symbiotica Tucson, as described in Text S1. At 24 h, aphids were transferred in sets of 15 to seedlings of Vicia faba and stored under long-day conditions (16-h light, 8-h dark) in incubators held at a constant 20°C. At each time point, aphids were collected in separate tubes, surface sterilized in 10% bleach for 1 min, rinsed in deionized water for 1 min, and then crushed and resuspended in 100 ml phosphate-buffered saline (PBS). For aphids injected with culturable S. symbiotica CWBI-GFP or HB1-GFP, 50 ml of this homogenate was used for spot-plating and 50 ml was frozen for DNA extraction and quantitative PCR (qPCR). For aphids injected with S. symbiotica Tucson, all 100 ml of homogenate was frozen and used for DNA extraction and qPCR. Details are provided in Text S1.
Statistical analyses. All statistical analyses and graphing were performed in the R programming language (version 3.6.3) (79). Survival rates for each treatment group were visualized as Kaplan-Meier survival curves, and comparisons of rates across treatment groups were performed using the Cox proportional-hazards model. Bacterial titers across treatment groups were compared using the Kruskal-Wallis analysis of variance, followed by Dunn's multiple-comparison test.
FISH microscopy. Fourth-instar pea aphids were injected with wild-type S. symbiotica CWBI-2.3 T , wild-type S. symbiotica HB1, or hemolymph from pea aphids infected with S. symbiotica Tucson, as described in Text S1. Embryos were dissected at 4 dpi (Movie S1) or 7 dpi (Fig. 4) in 70% ethanol. Fluorescence in situ hybridization (FISH) was performed as described by Koga et al. (8) with slight modifications. Details are provided in Text S1.
Live imaging of E. coli and S. symbiotica in pea aphids. For live imaging, fourth-instar Acyrthosiphon pisum LSR1 aphids were injected with recombinant S. symbiotica CWBI-GFP or recombinant E. coli BW25113-GFP, as described in Text S1. At 24 h, the aphids were transferred in sets of 15 to seedlings of V. faba and stored under long-day conditions (16-h light, 8-h dark) in incubators held at a constant 20°C. At 5 dpi, a subset of aphids from each treatment group were used to obtain titer counts via spot-plating, as described above, and the remaining aphids were dissected in TC-100 insect medium. Single ovarioles from 10 infected aphids per treatment group were observed under a Zeiss LSM 710 confocal microscope.
Data availability. This whole-genome shotgun project for S. symbiotica HB1 has been deposited at DDBJ/ENA/GenBank under accession no. JACBGK000000000. The version described in this paper is version JACBGK010000000.