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Heritable, facultative symbionts are common in arthropods, often functioning in host defence. Despite moderately reduced genomes, facultative symbionts retain evolutionary potential through mobile genetic elements (MGEs). MGEs form the primary basis of strain-level variation in genome content and architecture, and often correlate with variability in symbiont-mediated phenotypes. In pea aphids (Acyrthosiphon pisum), strain-level variation in the type of toxin-encoding bacteriophages (APSEs) carried by the bacterium Hamiltonella defensa correlates with strength of defence against parasitoids. However, co-inheritance creates difficulties for partitioning their relative contributions to aphid defence. Here we identified isolates of H. defensa that were nearly identical except for APSE type. When holding H. defensa genotype constant, protection levels corresponded to APSE virulence module type. Results further indicated that APSEs move repeatedly within some H. defensa clades providing a mechanism for rapid evolution in anti-parasitoid defences. Strain variation in H. defensa also correlates with the presence of a second symbiont Fukatsuia symbiotica. Predictions that nutritional interactions structured this coinfection were not supported by comparative genomics, but bacteriocin-containing plasmids unique to co-infecting strains may contribute to their common pairing. In conclusion, strain diversity, and joint capacities for horizontal transfer of MGEs and symbionts, are emergent players in the rapid evolution of arthropods., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Insects often harbour heritable symbionts that provide defence against specialized natural enemies, yet little is known about symbiont protection when hosts face simultaneous threats. In pea aphids (Acyrthosiphon pisum), the facultative endosymbiont Hamiltonella defensa confers protection against the parasitoid, Aphidius ervi, and Regiella insecticola protects against aphid-specific fungal pathogens, including Pandora neoaphidis. Here, we investigated whether these two common aphid symbionts protect against a specialized virus A. pisum virus (APV), and whether their antifungal and antiparasitoid services are impacted by APV infection. We found that APV imposed large fitness costs on symbiont-free aphids and these costs were elevated in aphids also housing H. defensa. In contrast, APV titres were significantly reduced and costs to APV infection were largely eliminated in aphids with R. insecticola. To our knowledge, R. insecticola is the first aphid symbiont shown to protect against a viral pathogen, and only the second arthropod symbiont reported to do so. In contrast, APV infection did not impact the protective services of either R. insecticola or H. defensa. To better understand APV biology, we produced five genomes and examined transmission routes. We found that moderate rates of vertical transmission, combined with horizontal transfer through food plants, were the major route of APV spread, although lateral transfer by parasitoids also occurred. Transmission was unaffected by facultative symbionts. In summary, the presence and species identity of facultative symbionts resulted in highly divergent outcomes for aphids infected with APV, while not impacting defensive services that target other enemies. These findings add to the diverse phenotypes conferred by aphid symbionts, and to the growing body of work highlighting extensive variation in symbiont-mediated interactions., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Background: Most phages infect free-living bacteria but a few have been identified that infect heritable symbionts of insects or other eukaryotes. Heritable symbionts are usually specialized and isolated from other bacteria with little known about the origins of associated phages. Hamiltonella defensa is a heritable bacterial symbiont of aphids that is usually infected by a tailed, double-stranded DNA phage named APSE., Methods: We conducted comparative genomic and phylogenetic studies to determine how APSE is related to other phages and prophages., Results: Each APSE genome was organized into four modules and two predicted functional units. Gene content and order were near-fully conserved in modules 1 and 2, which encode predicted DNA metabolism genes, and module 4, which encodes predicted virion assembly genes. Gene content of module 3, which contains predicted toxin, holin and lysozyme genes differed among haplotypes. Comparisons to other sequenced phages suggested APSE genomes are mosaics with modules 1 and 2 sharing similarities with Bordetella-Bcep-Xylostella fastidiosa-like podoviruses, module 4 sharing similarities with P22-like podoviruses, and module 3 sharing no similarities with known phages. Comparisons to other sequenced bacterial genomes identified APSE-like elements in other heritable insect symbionts (Arsenophonus spp.) and enteric bacteria in the family Morganellaceae., Conclusions: APSEs are most closely related to phage elements in the genus Arsenophonus and other bacteria in the Morganellaceae., (© 2021. The Author(s).)

Heritable symbionts are common in terrestrial arthropods and often provide beneficial services to hosts. Unlike obligate, nutritional symbionts that largely persist under strict host control within specialized host cells, heritable facultative symbionts exhibit large variation in within-host lifestyles and services rendered with many retaining the capacity to transition among roles. One enigmatic symbiont, Candidatus Fukatsuia symbiotica, frequently infects aphids with reported roles ranging from pathogen, defensive symbiont, mutualism exploiter, and nutritional co-obligate symbiont. Here, we used an in vitro culture-assisted protocol to sequence the genome of a facultative strain of Fukatsuia from pea aphids (Acyrthosiphon pisum). Phylogenetic and genomic comparisons indicate that Fukatsuia is an aerobic heterotroph, which together with Regiella insecticola and Hamiltonella defensa form a clade of heritable facultative symbionts within the Yersiniaceae (Enterobacteriales). These three heritable facultative symbionts largely share overlapping inventories of genes associated with housekeeping functions, metabolism, and nutrient acquisition, while varying in complements of mobile DNA. One unusual feature of Fukatsuia is its strong tendency to occur as a coinfection with H. defensa. However, the overall similarity of gene inventories among aphid heritable facultative symbionts suggests that metabolic complementarity is not the basis for coinfection, unless playing out on a H. defensa strain-specific basis. We also compared the pea aphid Fukatsuia with a strain from the aphid Cinara confinis (Lachninae) where it is reported to have transitioned to co-obligate status to support decaying Buchnera function. Overall, the two genomes are very similar with no clear genomic signatures consistent with such a transition, which suggests co-obligate status in C. confinis was a recent event., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Many insects host facultative, bacterial symbionts that confer conditional fitness benefits to their hosts. Hamiltonella defensa is a common facultative symbiont of aphids that provides protection against parasitoid wasps. Protection levels vary among strains of H. defensa that are also differentially infected by bacteriophages named APSEs. However, little is known about trait variation among strains because only one isolate has been fully sequenced. Generating complete genomes for facultative symbionts is hindered by relatively large genome sizes but low abundances in hosts like aphids that are very small. Here, we took advantage of methods for culturing H. defensa outside of aphids to generate complete genomes and transcriptome data for four strains of H. defensa from the pea aphid Acyrthosiphon pisum. Chosen strains also spanned the breadth of the H. defensa phylogeny and differed in strength of protection conferred against parasitoids. Results indicated that strains shared most genes with roles in nutrient acquisition, metabolism, and essential housekeeping functions. In contrast, the inventory of mobile genetic elements varied substantially, which generated strain specific differences in gene content and genome architecture. In some cases, specific traits correlated with differences in protection against parasitoids, but in others high variation between strains obscured identification of traits with likely roles in defense. Transcriptome data generated continuous distributions to genome assemblies with some genes that were highly expressed and others that were not. Single molecule real-time sequencing further identified differences in DNA methylation patterns and restriction modification systems that provide defense against phage infection.

Heritable symbionts are common in insects with many contributing to host defence. Hamiltonella defensa is a facultative, bacterial symbiont of the pea aphid, Acyrthosiphon pisum that provides protection against the endoparasitoid wasp Aphidius ervi Protection levels vary among strains of H. defensa that are differentially infected by bacteriophages named APSEs. By contrast, little is known about mechanism(s) of resistance owing to the intractability of host-restricted microbes for functional study. Here, we developed methods for culturing strains of H. defensa that varied in the presence and type of APSE. Most H. defensa strains proliferated at 27°C in co-cultures with the TN5 cell line or as pure cultures with no insect cells. The strain infected by APSE3, which provides high levels of protection in vivo , produced a soluble factor(s) that disabled development of A. ervi embryos independent of any aphid factors. Experimental transfer of APSE3 also conferred the ability to disable A. ervi development to a phage-free strain of H. defensa Altogether, these results provide a critical foundation for characterizing symbiont-derived factor(s) involved in host protection and other functions. Our results also demonstrate that phage-mediated transfer of traits provides a mechanism for innovation in host restricted symbionts., (© 2017 The Author(s).)