Horizontal Transfer of Bacterial Symbionts: Heritability and Fitness Effects in a Novel Aphid Host

Members of several bacterial lineages are known only as symbionts of insects and move among hosts through maternal transmission. Such vertical transfer promotes strong fidelity within these associations, favoring the evolution of microbially mediated effects that improve host fitness. However, phylogenetic evidence indicates occasional horizontal transfer among different insect species, suggesting that some microbial symbionts retain a generalized ability to infect multiple hosts. Here we examine the abilities of three vertically transmitted bacteria from the Gammaproteobacteria to infect and spread within a novel host species, the pea aphid, Acyrthosiphon pisum. Using microinjection, we transferred symbionts from three species of natural aphid hosts into a common host background, comparing transmission efficiencies between novel symbionts and those naturally infecting A. pisum. We also examined the fitness effects of two novel symbionts to determine whether they should persist under natural selection acting at the host level. Our results reveal that these heritable bacteria vary in their capacities to utilize A. pisum as a host. One of three novel symbionts failed to undergo efficient maternal transmission in A. pisum, and one of the two efficiently transmitted bacteria depressed aphid growth rates. Although these findings reveal that negative fitness effects and low transmission efficiency can prevent the establishment of a new infection following horizontal transmission, they also indicate that some symbionts can overcome these obstacles, accounting for their widespread distributions across aphids and related insects.     

Side-stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea

To elucidate the co-evolutionary relationships between phloem-feeding insects and their secondary, or facultative, bacterial symbionts, we explore the distributions of three such microbes--provisionally named the R-type (or PASS, or S-sym), T-type (or PABS), and U-type--across a number of aphid and psyllid hosts through the use of diagnostic molecular screening techniques and DNA sequencing. Although typically maternally transmitted, phylogenetic and pairwise divergence analyses reveal that these bacteria have been independently acquired by a variety of unrelated insect hosts, indicating that horizontal transfer has helped to shape their distributions. Based on the high genetic similarity between symbionts in different hosts, we argue that transfer events have occurred recently on an evolutionary timescale. In several instances, however, closely related symbionts associate with related hosts, suggesting that horizontal transfer between distant relatives may be rarer than transmission between close relatives. Our findings on the prevalence of these symbionts within many aphid taxa, along with published observations concerning their effects on host fitness, imply a significant role of facultative symbiosis in aphid ecology and evolution.     

Facultative symbiont infections affect aphid reproduction

Some bacterial symbionts alter their hosts reproduction through various mechanisms that enhance their transmission in the host population. In addition to its obligatory symbiont Buchnera aphidicola, the pea aphid Acyrthosiphon pisum harbors several facultative symbionts influencing several aspects of host ecology. Aphids reproduce by cyclical parthenogenesis whereby clonal and sexual reproduction alternate within the annual life cycle. Many species, including the pea aphid, also show variation in their reproductive mode at the population level, with some lineages reproducing by cyclical parthenogenesis and others by permanent parthenogenesis. While the role of facultative symbionts has been well studied during the parthenogenetic phase of their aphid hosts, very little is known on their possible influence during the sexual phase. Here we investigated whether facultative symbionts modulate the capacity to produce sexual forms in various genetic backgrounds of the pea aphid with controlled symbiont composition and also in different aphid genotypes from natural populations with previously characterized infection status and reproductive mode. We found that most facultative symbionts exhibited detrimental effects on their hosts fitness under sex-inducing conditions in comparison with the reference lines. We also showed that the loss of sexual phase in permanently parthenogenetic lineages of A. pisum was not explained by facultative symbionts. Finally, we demonstrated that Spiroplasma infection annihilated the production of males in the host progeny by inducing a male-killing phenotype, an unexpected result for organisms such as aphids that reproduce primarily through clonal reproduction.     

Independent origins and horizontal transfer of bacterial symbionts of aphids

Many insect groups have obligate associations with primary endosymbionts: mutualistic bacteria that are maternally transmitted and derived from an ancient infection. Often, the same insects are hosts to 'secondary' bacterial symbionts which are maternally transmitted but relatively labile within host lineages. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. Three types of bacteria besides Buchnera were found repeatedly; all three fall within the Enterobacteriaceae. The R-type has a 16S rDNA less than 0.1% different from that of the secondary symbiont previously reported from Acyrthosiphon pisum and is related to Serratia species. The T-type includes a symbiont previously reported from a whitefly; the U-type comprises a new cluster near the T-type. The T-type was found in every one of 40 Uroleucon ambrosiae clones collected throughout the United States. In contrast, A. pisum individuals were infected by any combination of the three symbiont types. Secondary symbionts were maternally transmitted for 11 months within laboratory-reared A. pisum clones and were present in sexually produced eggs. PCR screens for a bacteriophage, APSE-1, indicated its presence in both A. pisum and U. ambrosiae containing secondary symbionts. Electron microscopy of R-type and T-type bacteria in A. pisum and in U. ambrosiae revealed rod-shaped organisms that attain extremely high densities within a few bacteriocytes.     

Multiple introductions of the Spiroplasma bacterial endosymbiont into Drosophila

Bacterial endosymbionts are common in insects and can have dramatic effects on their host's evolution. So far, the only heritable symbionts found in Drosophila have been Wolbachia and Spiroplasma . While the incidence and effects of Wolbachia have been studied extensively, the prevalence and significance of Spiroplasma infections in Drosophila are less clear. These small, gram-positive, helical bacteria infect a diverse array of plant and arthropod hosts, conferring a variety of fitness effects. Male-killing Spiroplasma are known from certain Drosophila species; however, in others, Spiroplasma appear not to affect sex ratio. Previous studies have identified different Spiroplasma haplotypes in Drosophila populations, although no extensive surveys have yet been reported. We used a multilocus sequence analysis to reconstruct a robust Spiroplasma endosymbiont phylogeny, assess genetic diversity, and look for evidence of recombination. Six loci were sequenced from over 65 Spiroplasma -infected individuals from nine different Drosophila species. Analysis of these sequences reveals at least five separate introductions of four phylogenetically distinct Spiroplasma haplotypes, indicating that more extensive sampling will likely reveal an even greater Spiroplasma endosymbiont diversity. Patterns of variation in Drosophila mitochondrial haplotypes in Spiroplasma -infected and uninfected flies imply imperfect vertical transmission in host populations and possible horizontal transmission.     

Population dynamics of defensive symbionts in aphids

Vertically transmitted micro-organisms can increase in frequency in host populations by providing net benefits to hosts. While laboratory studies have identified diverse beneficial effects conferred by inherited symbionts of insects, they have not explicitly examined the population dynamics of mutualist symbiont infection within populations. In the pea aphid, Acyrthosiphon pisum, the inherited facultative symbiont, Hamiltonella defensa, provides protection against parasitism by the wasp, Aphidius ervi. Despite a high fidelity of vertical transmission and direct benefits of infection accruing to parasitized aphids, Hamiltonella remains only at intermediate frequencies in natural populations. Here, we conducted population cage experiments to monitor the dynamics of Hamiltonella and of another common A. pisum symbiont, Serratia symbiotica, in the presence and absence of parasitism. We also conducted fitness assays of Hamiltonella-infected aphids to search for costs to infection in the absence of parasitism. In the population cages, we found that the frequency of A. pisum infected with Hamiltonella increased dramatically after repeated exposure to parasitism by A. ervi, indicating that selection pressures from natural enemies can lead to the increase of particular inherited symbionts in insect populations. In our laboratory fitness assays, we did not detect a cost to infection with Hamiltonella, but in the population cages not exposed to parasitism, we found a significant decline in the frequency of both Hamiltonella and Serratia. The declining frequencies of Hamiltonella-infected aphids in population cages in the absence of parasitism indicate a probable cost to infection and may explain why Hamiltonella remains at intermediate frequencies in natural populations.     

Transmission,relatedness, and the evolution of cooperative symbionts

Cooperative interactions between species, termed mutualisms, play a key role in shaping natural ecosystems, economically important agricultural systems, and in influencing human health. Across different mutualisms, there is significant variation in the benefit that hosts receive from their symbionts. Empirical data suggest that transmission mode can help explain this variation: vertical transmission, where symbionts infect their host's offspring, leads to symbionts that provide greater benefits to their hosts than horizontal transmission, where symbionts leave their host and infect other hosts in the population. However, two different theoretical explanations have been given for this pattern: firstly, vertical transmission aligns the fitness interests of hosts and their symbionts; secondly, vertical transmission leads to increased relatedness between symbionts sharing a host, favouring cooperation between symbionts. We used a combination of analytical models and dynamic simulations to tease these factors apart, in order to compare their separate influences and see how they interact. We found that relatedness between symbionts sharing a host, rather than transmission mode per se, was the most important factor driving symbiont cooperation. Transmission mode mattered mainly because it determined relatedness. We also found evolutionary branching throughout much of our simulation, suggesting that a combination of transmission mode and multiplicity of infections could lead to the stable coexistence of different symbiont strategies.     

Horizontal transfer of facultative endosymbionts is limited by host relatedness

Heritable microbial symbionts can have important effects on many aspects of their hosts’ biology. Acquisition of a novel symbiont strain can provide fitness benefits to the host, with significant ecological and evolutionary consequences. We measured barriers to horizontal transmission by artificially transferring facultative symbionts from the grain aphid, Sitobion avenae, and five other aphid species into two clonal genotypes of S. avenae. We found the symbiont Hamiltonella defensa establishes infections more easily following a transfer from the same host species and that such infections are more stable. Infection success was also higher when the introduced symbiont strain was more closely related to the strain that was originally present in the host (but which had previously been removed). There were no differences among successfully established symbiont strains in their effect on aphid fecundity. Hamiltonella defensa did not confer protection against parasitoids in our S. avenae clones, although it often does in other aphid hosts. However, strains of the symbiont Regiella insecticola originating from two host species protected grain aphids against the pathogenic fungus Pandora neoaphidis. This study helps describe the extent to which facultative symbionts can act as a pool of adaptations that can be sampled by their eukaryote hosts.     

Evolutionary genetics of a defensive facultative symbiont of insects: exchange of toxin-encoding bacteriophage

The facultative endosymbiont of aphids, Hamiltonella defensa , kills parasitoid wasp larvae, allowing aphid hosts to survive and reproduce. This protection may depend on toxins that are encoded by the genomes of H. defensa and of its bacteriophage (APSE). Strains of H. defensa vary in degree of protection conferred upon Acyrthosiphon pisum (pea aphid). Although H. defensa is known to undergo some horizontal transmission among aphid maternal lineages, divergence, recombination, and population structure in H. defensa and APSE have not been characterized. We performed a multilocus sequence analysis of 10 bacterial and five phage loci for strains isolated from A. pisum and other aphid species. The H. defensa chromosome was found to be largely clonal, allowing us to generate a well-resolved H. defensa strain phylogeny. In contrast, APSE chromosomes undergo recombination and numerous H. defensa strains have probably lost the phage. Within a set of H. defensa strains that are indistinguishable on the basis of chromosomal genes or restriction digests of chromosomal fragments, loss of APSE is associated with decreased protection, strongly suggesting that APSE-encoded genes contribute to the defensive phenotype. Thus, homologous recombination of APSE genes and sexual transmission of symbionts and phage are likely factors influencing the exchange of ecologically important genes among symbionts. Although H. defensa has been lost, transferred and gained within A. pisum , one subclade of H. defensa appears to be universal within a subclade of the aphid genus Uroleucon , suggesting a transition from facultative, horizontal transmission to strictly vertical inheritance.     

Host-based divergence in populations of the pea aphid: insights from nuclear markers and the prevalence of facultative symbionts

In North America, the pea aphid Acyrthosiphon pisum encompasses ecologically and genetically distinct host races that offer an ideal biological system for studies on sympatric speciation. In addition to its obligate symbiont Buchnera, pea aphids harbour several facultative and phylogenetically distant symbionts. We explored the relationships between host races of A. pisum and their symbiotic microbiota to gain insights into the historical process of ecological specialization and symbiotic acquisition in this aphid. We used allozyme and microsatellite markers to analyse the extent of genetic differentiation between populations of A. pisum on pea, alfalfa and clover in France. In parallel, we examined: (i) the distribution of four facultative symbionts; and (ii) the genetic variation in the Buchnera genome across host-associated populations of A. pisum. Our study clearly demonstrates that populations of A. pisum on pea, clover and alfalfa in France are genetically divergent, which indicates that they constitute distinct host races. We also found a very strong association between host races of A. pisum and their symbiotic microbiota. We stress the need for phylogeographic studies to shed light on the process of host-race formation and acquisition of facultative symbionts in A. pisum. We also question the effects of these symbionts on aphid host fitness, including their role in adaptation to a host plant.     

Can maternally inherited endosymbionts adapt to a novel host? Direct costs of Spiroplasma infection,but not vertical transmission efficiency,evolve rapidly after horizontal transfer into D. melanogaster

Maternally inherited symbionts are common in arthropods and many have important roles in host adaptation. The observation that specific symbiont lineages infect distantly related host species implies new interactions are commonly established by lateral transfer events. However, studies have shown that symbionts often perform poorly in novel hosts. We hypothesized selection on the symbiont may be sufficiently rapid that poor performance in a novel host environment is rapidly ameliorated, permitting symbiont maintenance. Here, we test this prediction for a Spiroplasma strain transinfected into the novel host Drosophila melanogaster. In the generations immediately following transinfection, the symbiont had low transmission efficiency to offspring and imposed severe fitness costs on its host. We observed that effects on host fitness evolved rapidly, being undetectable after 17 generations in the novel host, whereas vertical transmission efficiency was poorly responsive over this period. Our results suggest that long-term symbiosis may more readily be established in cases where symbionts perform poorly in just one aspect of symbiosis.     

Protection against a fungal pathogen conferred by the aphid facultative endosymbionts Rickettsia and Spiroplasma is expressed in multiple host genotypes and species and is not influenced by co‐infection with another symbiont

Many insects harbour facultative endosymbiotic bacteria, often more than one type at a time. These symbionts can have major effects on their hosts' biology, which may be modulated by the presence of other symbiont species and by the host's genetic background. We investigated these effects by transferring two sets of facultative endosymbionts (one Hamiltonella and Rickettsia, the other Hamiltonella and Spiroplasma) from naturally double‐infected pea aphid hosts into five novel host genotypes of two aphid species. The symbionts were transferred either together or separately. We then measured aphid fecundity and susceptibility to an entomopathogenic fungus. The pathogen‐protective phenotype conferred by the symbionts Rickettsia and Spiroplasma varied among host genotypes, but was not influenced by co‐infection with Hamiltonella. Fecundity varied across single and double infections and between symbiont types, aphid genotypes and species. Some host genotypes benefit from harbouring more than one symbiont type.     

Cohabitation and roommate bias of symbiotic bacteria in insect hosts

Symbiotic interactions between insects and bacteria have long fascinated ecologists. Aphids have emerged as the model system on which to study the effect of endosymbiotic bacteria on their hosts. Aphid‐symbiont interactions are ecologically interesting as aphids host multiple secondary symbionts that can provide broad benefits, such as protection against heat stress or specialist natural enemies (parasitic wasps and entomopathogenic fungi). There are nine common aphid secondary symbionts and individual aphids host on average 1–2 symbionts. A cost‐benefit trade‐off for hosting symbionts is thought to explain why not all aphids host every possible symbiont in a population. Both positive and negative associations between various symbionts occur, and this could happen due to increased costs when cohosting certain combinations or as a consequence of competitive interactions between the symbionts within a host. In this issue of Molecular Ecology, Mathé‐Hubert, Kaech, Hertaeg, Jaenike, and Vorburger (2019) use data on the symbiont status of field‐collected aphids to inform a model on the evolution of symbiont co‐occurrence. They vary the effective female population size as well as the rate of horizontal and maternal transmission to infer the relative impact of symbiont‐symbiont interactions versus random drift. Additional data analysis revisits an association between two symbionts in a fruit fly species using a long‐term data set to highlight that such interactions are not limited to aphids.     

Diversity of bacteria associated with natural aphid populations

The bacterial communities of aphids were investigated by terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments generated by PCR with general eubacterial primers. By both methods, the gamma-proteobacterium Buchnera was detected in laboratory cultures of six parthenogenetic lines of the pea aphid Acyrthosiphon pisum and one line of the black bean aphid Aphis fabae, and one or more of four previously described bacterial taxa were also detected in all aphid lines except one of A. pisum. These latter bacteria, collectively known as secondary symbionts or accessory bacteria, comprised three taxa of gamma-proteobacteria (R-type [PASS], T-type [PABS], and U-type [PAUS]) and a rickettsia (S-type [PAR]). Complementary analysis of aphids from natural populations of four aphid species (A. pisum [n = 74], Amphorophora rubi [n = 109], Aphis sarothamni [n = 42], and Microlophium carnosum [n = 101]) from a single geographical location revealed Buchnera and up to three taxa of accessory bacteria, but no other bacterial taxa, in each aphid. The prevalence of accessory bacterial taxa varied significantly among aphid species but not with the sampling month (between June and August 2000). These results indicate that the accessory bacterial taxa are distributed across multiple aphid species, although with variable prevalence, and that laboratory culture does not generally result in a shift in the bacterial community in aphids. Both the transmission patterns of the accessory bacteria between individual aphids and their impact on aphid fitness are suggested to influence the prevalence of accessory bacterial taxa in natural aphid populations.     

The natural occurrence of secondary bacterial symbionts in aphids

1. Many insects host secondary bacterial symbionts that are known to have wide‐ranging effects on their hosts, from host‐plant use to resistance against natural enemies. This has been most widely studied in aphids, which have become a model system to study insect–bacteria interactions. 2. While there is an increasing understanding of the role of symbionts in aphids from controlled laboratory studies, we are only beginning to explore the impact of hosting these symbionts on eco‐evolutionary dynamics in natural systems. To date, many research groups have identified bacterial symbionts from various aphid species, providing us with a bank of literature on aphid–symbiont associations in natural populations. 3. The role of secondary symbionts in aphids is discussed, and the taxonomic and geographical distribution of symbionts among aphids are summarised, and the potential reasons for the patterns observed. The need to test for multiple symbiont species (and co‐infections) across many individuals and the whole distribution range of an aphid is highlighted, including sampling on all known host‐plant species. 4. It is further important also to consider variation within the symbiont, the aphid‐host and the surrounding community, e.g. host‐plants or the natural enemies, to understand how these have the potential to mediate aphid–symbiont interactions. 5. Finally, the knowledge gained from experimental work should now be used to understand the role of aphid secondary symbionts in field systems, to fully understand the potentially far‐reaching consequences of aphid endosymbionts on community and ecosystem processes.     

Does host outcrossing disrupt compatibility with heritable symbionts?

Vertically transmitted microbes are common in macro‐organisms and can enhance host defense against environmental stress. Because vertical transmission couples host and symbiont lineages, symbionts may become specialized to host species or genotypes. Specialization and contrasting reproductive modes of symbiotic partners could create incompatibilities between inherited symbionts and novel host genotypes when hosts outcross or hybridize. Such incompatibilities could manifest as failed colonization or poor symbiont growth in host offspring that are genetically dissimilar from their maternal host. Moreover, outcrossing between host species could influence both host and symbiont reproductive performance. We tested these hypotheses by manipulating outcrossing between populations and species of two grasses, Elymus virginicus and E. canadensis, that host vertically transmitted fungal endophytes (genus Epichloё). In both greenhouse and field settings, we found that host–symbiont compatibility was robust to variation in host genetic background, spanning within‐population, between‐population and between‐species crosses. Symbiont transmission into the F₁ generation was generally high and weakly affected by host outcrossing. Furthermore, endophytes grew equally well in planta regardless of host genetic background and transmitted at high frequencies into the F₂ generation. However, outcrossing, especially inter‐specific hybridization, reduced reproductive fitness of the host, and thereby the symbiont. Our results challenge the hypothesis that host genetic recombination, which typically exceeds that of symbionts, is a disruptive force in heritable symbioses. Instead, symbionts may be sufficiently generalized to tolerate ecologically realistic variation in host outcrossing.     

A shift to parasitism in the jellyfish symbiont Symbiodinium microadriaticum

One of the outstanding and poorly understood examples of cooperation between species is found in corals, hydras and jellyfish that form symbioses with algae. These mutualistic algae are mostly acquired infectiously from the seawater and, according to models of virulence evolution, should be selected to parasitize their hosts. We altered algal transmission between jellyfish hosts in the laboratory to examine the potential for virulence evolution in this widespread symbiosis. In one experimental treatment, vertical transmission of algae (parent to offspring) selected for symbiont cooperation, because symbiont fitness was tied to host reproduction. In the other treatment, horizontal transmission (infectious spread) decoupled symbiont fitness from the host, potentially allowing parasitic symbionts to spread. Fitness estimates revealed a striking shift to parasitism in the horizontal treatment. The horizontally transmitted algae proliferated faster within hosts and had higher dispersal rates from hosts compared to the vertical treatment, while reducing host reproduction and growth. However, a trade-off was detected between harm caused to hosts and symbiont fitness. Virulence trade-offs have been modelled for pathogens and may be critical in stabilising 'infectious' symbioses. Our results demonstrate the dynamic nature of this symbiosis and illustrate the potential ease with which beneficial symbionts can evolve into parasites.     

Symbiont modifies host life-history traits that affect gene flow

The evolution of herbivore-host plant specialization requires low levels of gene flow between populations on alternate plant species. Accordingly, selection for host plant specialization is most effective when genotypes have minimal exposure to, and few mating opportunities with individuals from, alternate habitats. Maternally transmitted bacterial symbionts are common in insect herbivores and can influence host fecundity under a variety of conditions. Symbiont-mediated effects on host life-history strategies, however, are largely unknown. Here, we show that the facultative bacterial symbiont Candidatus Regiella insecticola strikingly alters both dispersal and mating in the pea aphid, Acyrthosiphon pisum. Pea aphids containing Regiella produced only half the number of winged offspring in response to crowding and, for two out of three aphid lineages, altered the timing of sexual reproduction in response to conditions mimicking seasonal changes, than did aphids lacking Regiella. These symbiont-associated changes in dispersal and mating are likely to have played a key role in the initiation of genetic differentiation and in the evolution of pea aphid-host plant specialization. As symbionts are widespread in insects, symbiont-induced life history changes may have promoted specialization, and potentially speciation, in many organisms.     

Costs and benefits of symbiont infection in aphids: variation among symbionts and across temperatures

Symbiosis is prevalent throughout the tree of life and has had a significant impact on the ecology and evolution of many bacteria and eukaryotes. The benevolence of symbiotic interactions often varies with the environment, and such variation is expected to play an important role in shaping the prevalence and distributions of symbiosis throughout nature. In this study, we examine how the fitness of aphids is influenced by infection with one of three maternally transmitted bacteria, 'Candidatus Serratia symbiotica', 'Candidatus Hamiltonella defensa' and 'Candidatus Regiella insecticola', addressing how symbiont benevolence varies with temperature. We find that the effects of these 'secondary' symbionts on Acyrthosiphon pisum depend on when and whether aphids are exposed to a brief period of heat shock. We also demonstrate that symbionts--even closely related isolates--vary in their effects on hosts. Our results indicate similar effects of S. symbiotica and H. defensa in conferring tolerance to high temperatures and a liability of R. insecticola under these same conditions. These findings reveal a role for heritable symbionts in the adaptation of aphids to their abiotic environments and add to an expanding body of knowledge on the adaptive significance of symbiosis.