Intracellular defensive symbiont is culturable and capable of transovarial, vertical transmission

ABSTRACT Insects frequently form heritable associations with beneficial bacteria that are vertically transmitted from parent to offspring. Long-term vertical transmission has repeatedly resulted in genome reduction and gene loss, rendering many such bacteria incapable of establishment in axenic culture. Among aphids, heritable endosymbionts often provide context-specific benefits to their hosts. Although these associations have large impacts on host phenotypes, experimental approaches are often limited by an inability to cultivate these microbes. Here, we report the axenic culture of Candidatus Fukatsuia symbiotica strain WIR, a heritable bacterial endosymbiont of the pea aphid, Acyrthosiphon pisum. Whole-genome sequencing revealed similar genomic features and high sequence similarity to previously described strains, suggesting that the cultivation techniques used here may be applicable to Ca. F. symbiotica strains from distantly related aphids. Microinjection of cultured Ca. F. symbiotica into uninfected aphids revealed that it can reinfect developing embryos and that infections are maintained in subsequent generations via transovarial maternal transmission. Artificially infected aphids exhibit phenotypic and life history traits similar to those observed for native infections. Our results show that Ca. F. symbiotica may be a useful tool for experimentally probing the molecular mechanisms underlying host-symbiont interactions in a heritable symbiosis. IMPORTANCE Diverse eukaryotic organisms form stable, symbiotic relationships with bacteria that provide benefits to their hosts. While these associations are often biologically important, they can be difficult to probe experimentally because intimately host-associated bacteria are difficult to access within host tissues, and most cannot be cultured. This is especially true for the intracellular, maternally inherited bacteria associated with many insects, including aphids. Here, we demonstrate that a pea aphid-associated strain of the heritable endosymbiont, Candidatus Fukatsuia symbiotica, can be grown outside of its host using standard microbiology techniques and can readily re-establish infection that is maintained across host generations. These artificial infections recapitulate the effects of native infections, making this host-symbiont pair a useful experimental system.

present at low abundances.Most tools for forward or reverse genetics in microbes are designed for pure cultures and cannot be performed on bacteria within host tissues.Most studied maternally transmitted endosymbionts cannot be independently cultured using conventional microbiological techniques (14).
Aphids are globally distributed pest insects of the order Hemiptera that have been established as a useful model for understanding heritable symbioses.Nearly all aphids harbor an obligate endosymbiont, Buchnera aphidicola, that supplies essential amino acids lacking in the host diet (5,6,15).The association between Buchnera and aphids is ancient, and over the course of an estimated 200 MY, Buchnera has experienced extreme gene loss and genome reduction (16).Several non-essential or facultative endosymbionts have formed more recent associations and can be found at intermedi ate frequencies in natural populations (17,18).Maternal transmission for Buchnera and facultative endosymbionts is similar, occurring early in embryonic development, and generally results in stable transmission to all offspring of infected individuals (19,20).Very early during embryonic development, Buchnera and any co-infecting faculta tive endosymbionts are endocytosed at the posterior end of the blastula, entering a central, syncytial cell, before packaging into bacteriocytes.These facultative symbioses are potentially more amenable to experimental manipulation.Culture-assisted methods would facilitate our understanding of the molecular mechanisms underlying symbiont transmission in this system.However, to date, no heritable aphid endosymbionts have been successfully reintroduced and stably maintained after axenic culture.
One particularly promising candidate for axenic cultivation is the pea aphid endosymbiont, Candidatus Fukatsuia symbiotica (previously X-type or PAXS).So far, one pea aphid-associated strain, Ca.F. symbiotica strain 5D, has been co-cultured with insect cells, and its genome has been sequenced (21).As compared to other vertically transmitted aphid endosymbionts with sequenced genomes, this strain has relatively intact metabolic capabilities, only moderate gene loss, and intermediate genome-wide GC content, suggesting a more recent transition to a vertically transmitted lifestyle (21).Ca.F. symbiotica strains have variable effects on host phenotypes, with reported benefits ranging from heightened parasitoid resistance, protection against fungal pathogens, and increased tolerance to extreme heat (22)(23)(24).However, high costs to host fitness have also been observed in infected aphids, and some strains provide minimal relief to tested biotic or abiotic stressors (25).Given the lack of easily culturable, vertically transmitted bacterial symbionts, we attempted to axenically cultivate Ca.F. symbiotica from a naturally infected pea aphid, in an effort to establish a tractable system to study its diverse effects on the host.

Candidatus F. symbiotica strain WIR is capable of axenic culture
We attempted to grow Ca.F. symbiotica axenically from a naturally infected pea aphid using standard microbiology media.Growth is observed on heart infusion agar supplemented with 5% defibrinated sheep's blood, with clear to cloudy colonies forming after 2 weeks of incubation at room temperature under ambient atmosphere.No growth was observable at higher temperatures (25°C, 30°C, or 35°C).Light microscopy revealed long (~5 µm), rod-shaped morphology (Fig. 1A).Scanning electron microscopy (SEM) confirmed long, rod-shaped morphology (Fig. 1B and C).

Genomic features and phylogenetic placement of Ca. F. symbiotica WIR
Ca. F. symbiotica strain WIR was subjected to whole-genome sequencing, using a combination of Illumina short reads and Oxford Nanopore long reads, for a total coverage depth of 463×.The complete genome assembly of Ca.F. symbiotica WIR has a total size (3.065Mbp) and GC content (43.77%) very close to that of strain 5D (Table S1).
We generated a maximum likelihood phylogeny based on 340 shared single-copy orthologs, for phylogenetic placement of strain WIR.Ca.Fukatsuia strains form a well-supported monophyletic clade within the family Yersiniaceae (Fig. 2A).Strains associated with pea aphid, Cinara confinis, and Drepanosiphum platanoidis form a tight clade with the recently described strains associated with the aphid genus Anoecia forming a distinct sister clade consistent with recent work (26).Pairwise comparisons show that average nucleotide identity is also high (>99.8%)among the pea aphid-, Cinara-, and Drepanosiphum-associated strains (Fig. 2B) with a steep decline in compari sons with Anoecia-associated strains.

Cultured Ca. F. symbiotica WIR is vertically transmitted to offspring following injection
Given its overall high genomic similarity to vertically transmitted strains and its stable maintenance in naturally infected aphids in the laboratory, we tested whether our Ca.F. symbiotica strain WIR is capable of stably recolonizing uninfected aphids.Bacterial colonization of aphid embryos is a selective process, only occurring during a specific stage early in embryonic development (19,20).When transferring endosymbionts through hemolymph injection, only aphid embryos at the receptive developmental stage are colonized, resulting in a delay between time of injection and production of infected offspring.Among facultative endosymbionts that persist and proliferate within the insect hemocoel, this results in an increase in infection frequency with time from injection (27)(28)(29).
To determine if our cultivated Ca.F. symbiotica is capable of colonizing developing embryos after axenic culture, uninfected aphids were injected with a suspension of bacterial cells from cultures.Offspring produced as early as 8 days after injection tested positive for Ca.F. symbiotica (Fig. 3A).A general upward trend was observed, with 69% of offspring born 13 days after injection testing positive.To determine if these experimen tally established infections are stably maintained, a second set of injections was per formed, using the same methods.Offspring born 10-12 days post-injection were screened for infection.Of the 24 aphids tested for Ca.F. symbiotica, 15 tested positive for infection (62.5%).These aphids were allowed to mature to adulthood and reproduce before sampling, allowing us to screen subsequent generations (Fig. 3B).By the third generation after injection, 100% infection frequency was observed (Fig. 3B).Six sublines of infected aphids were maintained by transferring three nymphs to new plants, biweekly.Ca.F. symbiotica infection status was re-examined approximately 5 months later in these lines, corresponding to approximately 10 aphid generations after injection, and was detected in 100% (24/24) of the aphids screened.Thus, once established, Ca.F. symbiotica is inherited with very high fidelity.

Ca. F. symbiotica WIR is maintained via transovarial transmission
To determine if the artificially generated Ca.F. symbiotica infections are maintained via the route of transovarial transmission known for other aphid symbionts (20,28), fluorescent in situ hybridization (FISH) microscopy was performed.Three generations after injection, developing embryos were dissected out of adult aphids.These embryos were fixed and stained using 4′,6-diamidino-2-phenylindole (DAPI), and fluorescent probes targeting Ca.F. symbiotica 16S rRNA and Buchnera 16S rRNA.Prior to Buchnera colonization, the syncytium of developing embryos is also devoid of Ca.F. symbiotica (Fig. 4A; Fig. S1).The earliest observed entry into developing embryos was concurrent with Buchnera colonization (Fig. 4B; Fig. S1).Older embryos contain Ca.F. symbiotica, where it is housed within bacteriocytes and sheath cells (Fig. 4C and D).Control images of uninfected aphids treated with the same probes and imaged with the same methods are included in Fig. S2.

Ca. F. symbiotica phenotypes are recapitulated in artificial infections
High costs to host fitness have been associated with Ca.F. symbiotica infection under benign laboratory conditions (23,25).To determine if our artificially infected lines exhibit similar effects, we measured the number of offspring produced within the first 5 days of reproduction and adult body weight.Artificially infected (LSR1X) and naturally infected (WIRX) lines were compared to uninfected aphids of the same host genetic backgrounds (LSR1 and WIRC, respectively).In both native or artificially generated infections, we observed a reduction in the total number of offspring produced (Fig. 5A).This reduction was statistically significant both for natural infections (t = 5.04, df = 33.0,P < 0.001) and artificially generated infections (t = 7.66, df = 40.2,P < 0.001).Naturally infected aphids averaged 45.1% fewer offspring than cured aphids of the same genotype.In artificial infections, the average was 37.9%.A significant reduction in adult body weight was also observed for aphids with native (t = 6.84, df = 178.2,P < 0.001) and artificial infections (t = 6.39,df = 165.9,P < 0.001).This reduction in weight was similar for both natural (19.2%) and artificially generated infections (18.6%), compared to their uninfected counterparts (Fig. 5B).
To determine if Ca. F. symbiotica infection protects against non-specialist fungal pathogens, we performed challenges using a strain of Fusarium (Ascomycota), isolated from an aphid collected in Austin, TX, USA.The same lineages of aphids described above were exposed to conidial suspensions or mock treatments of vehicle solution, and mortality was monitored until aphids were up to 18 days old, an age at which most reproduction is completed.Natively infected aphids experienced reduced mortality compared to uninfected aphids, although this difference was not statistically significant at α = 0.05 (Cox proportional hazard model, P = 0.061) (Fig. S3A and B).In the artificially infected line (LSR1X), this difference was significant (P = 0.027).Mock treatments experienced low mortality and did not differ by symbiont infection status (Fig. S3C and  D).

DISCUSSION
Ca. Fukatsuia symbiotica, which is maternally transmitted but remains amenable to axenic culture, presents a rare opportunity to experimentally probe questions that are usually not feasible with vertically transmitted bacteria.Most vertically transmitted endosymbionts cannot live independently from their hosts, reflecting the gene loss that accompanies long-term vertical transmission (30).One major advantage of axenically cultivating symbiotic bacteria is the ability to efficiently sequence genomes without large portions of reads mapping to host genomes.Whole-genome sequencing of Ca.F. symbiotica WIR places it among other Ca.Fukatsuia symbionts of aphids.Ca.F. symbio tica strains share average nucleotide identities >99.8%, even among distantly related Cinara and Drepanosiphum hosts, suggesting relatively recent interspecific horizontal transmission, as previously noted (21).Some evidence supports intraspecific, horizontal transmission among pea aphids (18).Given their high sequence similarity, additional Ca.F. symbiotica strains from other aphid lineages may also be capable of independent cultivation.
Close relatives of the vertically transmitted aphid endosymbiont Serratia symbiotica also have been axenically cultured.However, they differ in fundamental features.These culturable strains of S. symbiotica comprise a clade of gut-associated pathogens that exhibit major differences in their gene content, localization, transmission, and effects on host fitness and that differ from the vertically transmitted strains (28,(31)(32)(33)(34).We found that Ca.F. symbiotica transmission occurs in a similar manner to that observed for both culturable and non-culturable strains of S. symbiotica (28).In each case, bacterial cells in the hemocoel enter embryos at an early stage of development when the syncytial cell that takes up Buchnera is exposed (Fig. 4B).Limited pathogenicity, as observed for non-culturable S. symbiotica and culturable Ca.F. symbiotica WIR, is a prerequisite for stable long-term vertical transmission, which requires hosts to survive to become reproductive adults.
Ca. F. symbiotica strains have been shown to confer a variety of effects on hosts (23).A strain collected in North America conferred protection against parasitoid wasps, when coinfecting with Candidatus Hamiltonella defensa (22).Strains of Ca.F. symbiotica from aphids collected in Europe are associated with many reported benefits, ranging from improved parasitoid resistance, defense against fungal pathogens, and improved recovery following heat stress (23,24).However, at least one North American strain has failed to reproduce any of the same defensive phenotypes (25).Possibly, methodological differences, including rearing temperatures, contribute to this variation in observed outcomes.
Culture-assisted methods may help clarify the impact of strain-level variation on the complex effects on hosts.Comparative genomics using protective and non-protective strains from pea aphids could provide insights into the genetic basis for these differences and into potential mechanisms of protective effects.
We demonstrated that Ca.F. symbiotica strain WIR imposes substantial costs to host fitness in the absence of natural enemies but also appears to confer protection against an entomopathogenic Fusarium isolate (Fig. S3A).Several other aphid endosym bionts, including Candidatus Regiella insecticola, have been reported to protect against the distantly related aphid specialist Pandora neoaphidis (35).However, to our knowl edge, no studies have shown pea aphid-associated heritable endosymbionts protecting against generalist entomopathogens, within Entomophthorales or Ascomycota (36,37).Although Ca.F. symbiotica imposes relatively high fitness costs under benign conditions (Fig. 5A and B), we speculate that the breadth of its protective effects helps explain its persistence in natural populations.In field collections, Ca.F. symbiotica commonly coinfects with Ca. H. defensa, and superinfection has been shown to reduce host fitness cost, which also helps explain Ca.F. symbiotica's persistence in natural populations (25,38).
Axenic culture may enable other approaches, including genetic manipulation.For example, the culturing of Sodalis glossinidius has made genetic methods possible for identifying genes involved in host colonization (39).Similar approaches may be applied in this system.Strain WIR grows slowly (~14 days to observe colonies), as is consistent with intimate host association.Supplementation with additional B vitamins, amino acids, or sterilized aphid extracts failed to noticeably improve growth, and we saw no appreciable growth in the range of liquid media tested.Other groups have successfully used co-cultivation with insect cell lines for growing Ca.F. symbiotica, as well as Ca.Regiella insecticola and Ca.Hamiltonella in liquid media (21,40,41).A combination of these approaches may be useful for the study and manipulation of these endosymbionts outside of their hosts.

Insect rearing
Pea aphid (Acyrthosiphon pisum) lines used for experiments were reared on fava seedlings (Vicia fava) either at 15°C for long-term maintenance or at 20°C for microin jection experiments.Aphids were kept under long day conditions (18:6, L:D) to ensure clonal, asexual reproduction.The aphid line WIRX was collected in Madison Wisconsin, in 2017, and harbors a natural Ca.F. symbiotica infection.This line was cured of a natural Candidatus H. defensa infection in 2021.A subline was also cured of Ca.F. symbiotica infection as well, resulting in an aphid line cured of all secondary symbiont infection (WIRC).All experiments were performed more than 20 generations after antibiotic treatments.For recolonization and fitness experiments, the aphid line LSR1 was used and was previously cured of its natural Candidatus Regiella insecticola infection in 2009.

Infection status
To screen for secondary symbiont infection, DNA was extracted from individual aphids using an ethanol precipitation protocol (42).Diagnostic PCRs were run using the primers 10F (AGTTTGATCATGGCTCAGATTG) and X420R (GCAACACTCTTTGCATTGCT) and performed using the cycling conditions described (17).The PCR conditions were the following: 94°C for 2 min, 10 cycles of 94°C for 1 min, 65°C-55°C for 1 min lowered 1°C each cycle, and 72°C for 2 min; 25 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min; and a final extension at 72°C for 6 min, with products held at 4°C.PCR products were run on a 2% agarose gel and visually assessed for amplification.

Isolation of Candidatus Fukatsuia symbiotica
To grow Ca.F. symbiotica outside of its aphid host, single WIRX adults were briefly surface sterilized in 10% bleach with 0.5% TWEEN80, then rinsed in sterile water, before homogenization in 150 µL of phosphate-buffered saline (PBS).This suspension was serially diluted in PBS and spot plated onto heart infusion agar supplemented with 5% sheep's blood, and Columbia agar with 5% sheep's blood.Plates were incubated under ambient atmosphere at room temperature (approximately 20°C) and wrapped in parafilm to prevent drying.Colony PCR was performed using the same primers used for screening for Ca.F. symbiotica in aphids, and amplicons were submitted for Sanger sequencing at Eton Biosciences (San Diego, CA, USA).

Light and electron microscopy
Imaging was performed 14-21 days after inoculation onto fresh plates.For light microscopy, confluent Ca.F. symbiotica culture was suspended in PBS and imaged on a NIKON Eclipse TE2000-U epifluorescence microscope.For scanning electron microscopy, growth was fixed in 2.5% glutaraldehyde and stained with 1.0% osmium in sodium cacodylate.Samples were incubated with 1.0% thiocarbohydrazide, before an addi tional treatment of 1.0% osmium in DI water.Graded ethanol washes were performed from 15% ethanol to absolute ethanol, and drying was performed after treatment in hexamethyldisilazane. Dehydrated samples were sputter coated in 5 nm gold/palladium, and imaging was performed using a Zeiss Supra 40V Scanning Electron Microscope.SEM was performed at the Center for Biomedical Research Support Microscopy and Imaging Facility at UT Austin (RRID:SCR_021756).

Host recolonization
To examine if Ca. F. symbiotica can recolonize its aphid host, confluent growth was scraped into PBS and diluted to an optical density at 600 nm (OD 600 ) of 1.0.Approxi mately 0.1 μL of this bacterial suspension was injected into uninfected, 7-day-old aphids of the LSR1 genotype.Injected aphids were transferred to fava leaves in petri dishes for 24 hours for recovery, before being transferred to plants.Once reproducing, aphids were transferred onto leaves in 1.5% agar daily.Offspring produced between 8 and 13 days after injection were sampled at 7-8 days old.Injections were repeated to assess stability of infections across generations.Here, groups of 7-10 injected aphids were transferred every 2 days.Mature offspring produced 10-12 days post-injection were placed on individual leaves in petri dishes in 1.5% agar and allowed to reproduce overnight.These aphids were screened for Ca.F. symbiotica infection as described above.Offspring from mothers testing positive were transferred to new plants.This process was repeated for another generation, and after the third-generation post-injection, six sublines of infected aphids were maintained by transferring three adults to fresh plants.After 5 months, these lines were screened again for infection.

Host fitness assays
Reproductive adults of each aphid line were placed on fava seedlings for 24 hours to generate age-controlled cohorts.Aphids were weighed on the first day of adulthood, transferred to individual fava seedlings, and monitored daily for the production of offspring.Reproductive output was measured as the number of offspring produced over the first 5 days of reproduction.Data for both reproductive output and weight are pooled from two trials initiated on separate days.

Fungal isolation and pathogen challenge
To determine if Ca. F. symbiotica protects against ecologically relevant fungal pathogens beyond those previously reported, we attempted to isolate fungal pathogens from naturally infected aphids.Pea aphids were collected in Austin, TX, in April 2023, and individual adults were placed on fava leaves in 1.5% agar.An individual aphid exhibiting fungal infection was ground in PBS and plated on Sabouraud agar.A single colony was passaged and maintained on yeast peptone, dextrose agar.For species identification, genomic DNA was extracted using a DNeasy DNA Extraction Kit (Qiagen), and a diagnostic PCR was performed, amplifying the internal transcribed spacer, using the primers ITS1 and ITS4 (52).Cleaned amplicons were submitted to ACGT (Germantown, MD) for Sanger sequencing.The consensus sequence for forward and reverse reactions was used as a query against the NCBI non-redundant nucleotide database to identify the fungal strain to genus.
For susceptibility assays, conidia were scraped from 14-day-old plate cultures and suspended in 0.05% TWEEN80.Dosage was quantified using a hemocytometer and normalized to approximately 10 7 conidia/mL.Aphids were each submerged in the conidial suspension and transferred to fava seedlings.Mock treatments in which aphids were submerged in 0.05% TWEEN80 were also performed, and survival for each treatment was monitored daily.

Statistical analyses
Statistical analyses were performed in RStudio (R version 4.2.2).Comparisons of host fitness between infected and uninfected aphids were performed using Welch's t-tests.Kaplan-Meier survival curves were generated using the survfit and ggsurvplot functions, in the R package survminer.Statistical significance was assigned at α = 0.05.

FIG 1
FIG 1 Morphology of Ca.F. symbiotica WIR cultivated outside of its aphid host.(A) Light microscopy of Ca.F. symbiotica WIR resuspended after growth on heart infusion agar with 5% sheep's blood.Scale bar indicates 5 µm.(B, C) Scanning electron microscopy performed on plate-grown Ca.F. symbiotica WIR.Scale bars indicate 2 µm.

FIG 2
FIG 2 Phylogenetic placement and sequence similarity of Ca.F. symbiotica WIR relative to other strains of interest.(A) Maximum likelihood phylogeny of Ca.Fukatsuia strains and other gram-negative bacteria, based on the concatenated amino acid alignments of 340 single-copy orthologs.Scale bar indicates number of substitutions per site.Genome accessions and related information are included in Table S2.(B) Pairwise comparisons of average nucleotide identity of Ca.Fukatsuia strains.

FIG 3
FIG 3 Colonization pattern of Ca.F. symbiotica WIR following injection into uninfected aphids.(A) Bar chart indicating the portion of individuals testing positive for Ca.F. symbiotica infection, in the generation immediately following injection, sampled daily.(B) Bar chart showing the portion of infected individuals in subsequent generations.The number of individuals testing positive over number sampled is shown above each bar.

FIG 4
FIG 4 Localization of Ca.F. symbiotica in embryos three generations after injection.(A) Prior to Buchnera colonization, the syncytium of developing embryos does not contain Ca.F. symbiotica.(B) Entry of Ca.F. symbiotica into the syncytial space of stage 7 embryos is concurrent with Buchnera colonization.(C, D) Ca.F. symbiotica is visible in bacteriocytes and sheath cells of older embryos.Ca.F. symbiotica cells are labeled in green, Buchnera in red, and aphid DNA in blue.Linear adjustment for brightness was applied across all parts of each image to improve clarity.Two-channel images for panels A and B are provided in Fig. S1.Control images of aphid embryos not infected with Ca.F. symbiotica are shown in Fig. S2.