Cryptic community structure, transmission dynamics, and within-host ecology of heritable pea aphid microbiomes
Authors:   , Peng Linyao , Russell Jacob A 
Published: July 2022

Maternally transmitted microbes are prevalent among insects. Existing frequently within multi-species communities, symbiont fates within insect populations should stem, largely, from their maternal transmission efficiency and host-level fitness effects. Despite the prevalence of multi-species symbiont communities, I have seen only modest efforts to differentiate the impacts of single vs. multi-species consortia on host fitness or symbiont transmission. Furthermore, the frequencies of non-maternal transfer have gone underexplored in many systems. With the capacities of horizontal or paternal transmission to create new communities, heritable symbionts could be selected for their competitive and dispersive abilities - properties less expected under strict maternal transfer. To investigate these possibilities I performed a range of molecular and experimental studies on the pea aphid, Acyrthosiphon pisum. Pea aphids are sap-feeding insects with a variety of maternally transmitted bacteria, including the well-studied obligate nutritional symbiont, Buchnera. Beyond this ubiquitous bacterium, pea aphids can harbor variable combinations of seven defensive symbiont species. While useful through their protection against parasitoids, pathogens, or heat, these symbionts are not ubiquitous within pea aphid populations. Previous screening for these more labile, "facultative" symbionts identified consistent community structure at the species level, with varying symbionts tending to co-infect with (or to co-exclude), regular symbiont partners (or non-partners). Recent work, however, suggests hidden diversity, with different "strains" of facultative symbiont species conferring distinct effects on host fitness. To precisely define community structure at the strain level, I sequenced protein-coding loci for all known pea aphid symbiont species across broad temporal and spatial scales within North America (Chapter 2). Defining boundaries through single nucleotide differences among conspecific symbiont isolates, I identified strain variation for all symbiont species except for Rickettsiella viridis, detecting variability in the relative abundance of strains, for most symbionts, over space and time. Most diverse was the anti-parasitoid defender Hamiltonella defensa, whose five common strains (or haplotypes) differed strongly in community structure. Hamiltonella strain B showed strong tendencies to co-infect with other symbionts, primarily Fukatsuia symbiotica and, separately, one strain (B) of a Spiroplasma species (Chapter 2). To understand the stability of these communities I studied their maternal transmission efficiencies (Chapter 3). Detecting no transmission failure for any of these symbionts in the lab - except for Spiroplasma B under hot conditions - I observed regular failure for each in the field. Occurring in the absence of other facultative symbionts, this suggested the likelihood of eventual loss of Hamiltonella B, and its co-infecting symbionts, from pea aphid populations in the absence of: 1) semi-regular non-maternal transfer, or 2) strong fitness benefits. Through the genotyping of obligate Buchnera symbionts across three loci, I examined the former possibility (Chapter 2). Given their exclusive reliance on maternal transmission, Buchnera genotypes track aphid matrilineage history. Distributions of facultative symbiont strains across unrelated Buchnera would, hence, imply their non-maternal transfer. Through a 3-locus genotyping methodology, I uncovered seven Buchnera genotypes across four genotyped pea aphid populations. Intriguingly, Hamiltonella B and Fukatsuia were found across six of these matrilineages. In contrast, Hamiltonella strain C, with its common Regiella symbiont co-infectors, was almost exclusively found in one matrilineage, as was Hamiltonella strain A - a variant commonly found to live alone. With the expectation that Hamiltonella strains, and their associated communities, might differ in their capacities to undergo non-maternal transfer, I created rare symbiont communities, using microinjection to mimic horizontal transfer (Chapter 3). When combined with stable, common symbiont communities (i.e. Spiroplasma strain A single infection; or Serratia-Rickettsiella), jointly injected Hamiltonella B and Fukatsuia established with high efficiency, frequently evicting the original symbionts. Establishment and eviction rates were lower for Hamiltonella C-Regiella and for Hamiltonella A after microinjection, supporting the hypothesis of their inferior horizontal transfer/competitive capacities. Working with a series of field-collected aphids used previously for studies of maternal transmission efficiency, my strain-level genotyping (Chapter 3) revealed that much of the common maternal transmission failure in nature occurred in the context of rare communities. For Hamiltonella B, maternal transmission was considerably higher in the presence of Fukatsuia, while under some contexts it was also boosted by Rickettsia - another common partner, typically through triple infection. Communities involving these symbionts exhibited high transmission efficiency when living alongside Serratia and/or its common partner Rickettsiella, which were often, themselves, poorly transmitted when living alongside these Hamiltonella B-associated communities To summarize, four discoveries add up to suggest a new paradigm for understanding the pea aphid's heritable microbiome: First, was the somewhat regular detection of rare, unstable communities in nature. Second, was my discovery that some common, communities are found across multiple aphid matrilineages. Building off of this was the third discovery, made here, that symbionts with such a suggested history of non-maternal transfer exhibit higher capacities for horizontal transfer and competition under lab assays. Fourth was the prior finding, re-defined here through strain genotyping, that common community partners improve each others' maternal transmission efficiency - in some cases even when living alongside other, uncommon partner symbionts. Combined, these findings suggest that symbiont-level selection may act on competitive, and dispersive capacities of some facultative symbiont species. Furthermore, facultative symbionts may coevolve in response to evolving variation within several pools of genomes: the host (aphid) genome, the Buchnera genome, genomes of the natural enemies they defend against, and genotypes of other - competing or cooperating - facultative symbionts. I discuss these possibilities and their implications for biological control.