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.

Keywords: antifungal defense; endosymbionts; insect; vertical transmission.

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

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

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

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.

Fig 5

Phenotypic effects of Ca. F. symbiotica in native or artificial infections. (A) Boxplots indicating the number of offspring produced by aphids varying in infection status. ***, P < 0.001, n = 22–27 aphids per treatment. (B) Adult mass measured in milligrams. ***, P < 0.001, n = 82–96 aphids per treatment. Welch’s t-tests performed within each host genotypic background.