An effect of 16S rRNA intercistronic variability on coevolutionary analysis in symbiotic bacteria: molecular phylogeny of Arsenophonus triatominarum

The genes of ribosomal RNA are the most popular and frequently used markers for bacterial phylogeny and reconstruction of insect-symbiont coevolution. In primary symbionts, such as Buchnera and Wigglesworthia, genome economization leads to the establishment of a single copy of these sequences. In phylogenetic studies, they provide sufficient information and yield phylogenetic trees congruent with host evolution. In contrast, other symbiotic lineages (e.g., the genus Arsenophonus) carry a higher number of rRNA copies in their genomes, which may have serious consequences for phylogenetic inference. In this study, we show that in Arsenophonus triatominarum the degree of heterogeneity can affect reconstruction of phylogenetic relationships and mask possible coevolution between the symbiont and its host. Phylogenetic arrangement of individual rRNA copies was used, together with a calculation of their divergence time, to demonstrate that the incongruent 16S rDNA trees and low nucleotide diversity in the secondary symbiont could be reconciled with the coevolutionary scenario.     

The secondary endosymbiotic bacterium of the pea aphid Acyrthosiphon pisum (Insecta: homoptera)

The secondary intracellular symbiotic bacterium (S-symbiont) of the pea aphid Acyrthosiphon pisum was investigated to determine its prevalence among strains, its phylogenetic position, its localization in the host insect, its ultrastructure, and the cytology of the endosymbiotic system. A total of 14 aphid strains were examined, and the S-symbiont was detected in 4 Japanese strains by diagnostic PCR. Two types of eubacterial 16S ribosomal DNA sequences were identified in disymbiotic strains; one of these types was obtained from the primary symbiont Buchnera sp., and the other was obtained from the S-symbiont. In situ hybridization and electron microscopy revealed that the S-symbiont was localized not only in the sheath cells but also in a novel type of cells, the secondary mycetocytes (S-mycetocytes), which have not been found previously in A. pisum. The size and shape of the S-symbiont cells were different when we compared the symbionts in the sheath cells and the symbionts in the S-mycetocytes, indicating that the S-symbiont is pleomorphic under different endosymbiotic conditions. Light microscopy, electron microscopy, and diagnostic PCR revealed unequivocally that the hemocoel is also a normal location for the S-symbiont. Occasional disordered localization of S-symbionts was also observed in adult aphids, suggesting that there has been imperfect host-symbiont coadaptation over the short history of coevolution of these organisms.     

Host-symbiont recognition in the environmentally transmitted sepiolid squid-Vibrio mutualism

Associations between environmentally transmitted symbionts and their hosts provide a unique opportunity to study the evolution of specificity and subsequent radiation of tightly coupled host-symbiont assemblages [3, 8, 24]. The evidence provided here from the environmentally transmitted bacterial symbiont Vibrio fischeri and its sepiolid squid host (Sepiolidae: Euprymna) demonstrates how host-symbiont specificity can still evolve without vertical transmission of the symbiont [1]. Infection by intraspecific V. fischeri symbionts exhibited preferential colonization over interspecific V. fischeri symbionts, indicating a high degree of specificity for the native symbiotic strains. Inoculation with symbiotic bacteria from other taxa (monocentrid fish and loliginid squids) produced little or no colonization in two species of Euprymna, despite their presence in the same or similar habitats as these squids. These findings of host specificity between native Vibrios and sepiolid squids provides evidence that the presence of multiple strains of symbionts does not dictate the composition of bacterial symbionts in the host.     

Cospeciation between bacterial endosymbionts (Buchnera) and a recent radiation of aphids (Uroleucon) and pitfalls of testing for phylogenetic congruence

Abstract.— Previous studies of phylogenetic congruence between aphids and their symbiotic bacteria ( Buchnera ) supported long-term vertical transmission of symbionts. However, those studies were based on distantly related aphids and would not have revealed horizontal transfer of symbionts among closely related hosts. Aphid species of the genus Uroleucon are closely related phylogenetically and overlap in geographic ranges, habitats, and parasitoids. To examine support for congruence of phylogenies of Buchnera and Uroleucon , sequences from four mitochondrial, one nuclear, and one endosymbiont gene ( trpB ) were obtained. Congruence of phylogenies based on pooled aphid genes with phylogenies based on trpB was highly significant: Most nodes resolved by trpB corresponded to nodes resolved by the pooled aphid genes. Furthermore, no nodes were both inconsistent between the trees and strongly supported in both trees. Two kinds of analyses testing the null hypothesis of perfect congruence between pairwise combinations of datasets and tree topologies were performed: the Kishino-Hasegawa test and the likelihood-ratio test. Both tests indicated significant disagreement among most pairwise combinations of mitochondrial, nuclear, and symbiont datasets. Because rampant recombination among mitochondrial genomes of different aphid species is unlikely, inaccurate assumptions in the evolutionary models underlying these tests appear to be causing the hypothesis of a shared history to be incorrectly rejected. Moreover, trpB was more consistent with the aphid genes as a set than any single aphid gene was with the others, suggesting that the symbionts show the same phylogeny as the aphids. Overall, analyses support the interpretation that symbionts and aphids have undergone strict cospeciation, with no horizontal transmission of symbionts even among closely related, ecologically similar aphid hosts.     

Large-scale label-free quantitative proteomics of the pea aphid-Buchnera symbiosis

Many insects are nutritionally dependent on symbiotic microorganisms that have tiny genomes and are housed in specialized host cells called bacteriocytes. The obligate symbiosis between the pea aphid Acyrthosiphon pisum and the γ-proteobacterium Buchnera aphidicola (only 584 predicted proteins) is particularly amenable for molecular analysis because the genomes of both partners have been sequenced. To better define the symbiotic relationship between this aphid and Buchnera, we used large-scale, high accuracy tandem mass spectrometry (nanoLC-LTQ-Orbtrap) to identify aphid and Buchnera proteins in the whole aphid body, purified bacteriocytes, isolated Buchnera cells and the residual bacteriocyte fraction. More than 1900 aphid and 400 Buchnera proteins were identified. All enzymes in amino acid metabolism annotated in the Buchnera genome were detected, reflecting the high (68%) coverage of the proteome and supporting the core function of Buchnera in the aphid symbiosis. Transporters mediating the transport of predicted metabolites were present in the bacteriocyte. Label-free spectral counting combined with hierarchical clustering, allowed to define the quantitative distribution of a subset of these proteins across both symbiotic partners, yielding no evidence for the selective transfer of protein among the partners in either direction. This is the first quantitative proteome analysis of bacteriocyte symbiosis, providing a wealth of information about molecular function of both the host cell and bacterial symbiont.     

An Acanthamoeba sp. containing two phylogenetically different bacterial endosymbionts

Acanthamoebae are ubiquitous free-living amoebae and important predators of microbial communities. They frequently contain obligate intracellular bacterial symbionts, which show a worldwide distribution. All Acanthamoeba spp. described so far harboured no or only a single specific endosymbiont phylotype, and in some cases evidence for coevolution between the symbiotic bacteria and the amoeba host has been reported. In this study we have isolated and characterized an Acanthamoeba sp. (strain OEW1) showing a stable symbiotic relationship with two morphologically different endosymbionts. 16S rRNA sequence analysis assigned these symbionts to the candidate genus Procabacter (Betaproteobacteria) and the genus Parachlamydia (Chlamydiae) respectively. Fluorescence in situ hybridization and transmission electron microscopy confirmed the affiliation of the endosymbionts and showed their co-occurrence in the amoeba host cells and their intracellular location within separate compartments enclosed by host-derived membranes. Further analysis of this stable relationship should provide novel insights into the complex interactions of intracellular multiple-partner associations.     

Tracing the evolution of gene loss in obligate bacterial symbionts

The gamma-proteobacterial symbionts of insects are a model group for comparative studies of genome reduction. The phylogenetic proximity of these reduced genomes to the larger genomes of well-studied free-living bacteria has enabled reconstructions of the process by which genes and DNA are lost. Three genome sequences are now available for Buchnera aphidicola. Analyses of Buchnera genomes in comparison with those of related enteric bacteria suggest that extensive changes including large deletions, repetitive element proliferation and chromosomal rearrangements occurred initially, followed by extreme stasis in gene order and slow decay of additional genes. This pattern appears to be characteristic of symbiont evolution.     

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.     

Phylogenetic congruence of mealybugs and their primary endosymbionts

Tight interactions between unrelated organisms such as is seen in plant-insect, host-parasite, or host-symbiont associations may lead to speciation of the smaller partners when their hosts speciate. Totally congruent phylogenies of interacting taxa have not been observed often but a number of studies have provided evidence that various hemipteran insect taxa and their primary bacterial endosymbionts share phylogenetic histories. Like other hemipterans, mealybugs (Pseudococcidae) harbour multiple intracellular bacterial symbionts, which are thought to be strictly vertically inherited, implying codivergence of hosts and symbionts. Here, robust estimates of phylogeny were generated from four fragments of three nuclear genes for mealybugs of the subfamily Pseudococcinae, and a substantial fragment of the 16S-23S rDNA of their P-endosymbionts. Phylogenetic congruence was highly significant, with 75% of nodes on the two trees identical, and significant correlation of branch lengths indicated coincident timing of cladogenesis. It is suggested that the low level of observed incongruence was influenced by uncertainty in phylogenetic estimation, but evolutionary outcomes other than congruence, including host shifts, could not be rejected.     

Decay of mutualistic potential in aphid endosymbionts through silencing of biosynthetic loci: Buchnera of Diuraphis

Buchnera, the primary bacterial endosymbiont of aphids, is known to provision essential amino acids lacking in the hosts' diet of plant sap. The recent discovery of silenced copies of genes for tryptophan biosynthesis (trpEG) in certain Buchnera lineages suggests a decay in symbiotic functions in some aphid species. However, neither the distribution of pseudogenes among lineages nor the impact of this gene silencing on amino-acid availability in hosts has been assessed. In Buchnera of the aphid Diuraphis noxia, tandem repeats of these pseudogenes have persisted in diverse lineages, and thpEG pseudogenes have originated at least twice within this aphid genus. Measures of amino-acid concentrations in Diuraphis species have shown that the presence of the pseudogene is associated with a decreased availability of tryptophan, indicating that gene silencing decreases nutrient provisioning by symbionts. In Buchnera of Diuraphis, rates of nonsynonymous substitutions are elevated in functional trpE copies, supporting the hypothesis that pseudogene origin and persistence reflect a reduced selection for symbiont biosynthetic contributions. The parallel evolution of trpEG pseudogenes in Buchnera of Diuraphis and certain other aphid hosts suggests that either selection at the host level is not effective or that fitness in these aphids is not limited by tryptophan availability.     

Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses. Correspondence to: D.L. Distel     

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.     

Characterization of sponge-associated Verrucomicrobia: microcompartment-based sugar utilization and enhanced toxin–antitoxin modules as features of host-associated Opitutales

Bacteria of the phylum Verrucomicrobia are ubiquitous in marine environments and can be found as free-living organisms or as symbionts of eukaryotic hosts. Little is known about host-associated Verrucomicrobia in the marine environment. Here we reconstructed two genomes of symbiotic Verrucomicrobia from bacterial metagenomes derived from the Atlanto-Mediterranean sponge Petrosia ficiformis and three genomes from strains that we isolated from offshore seawater of the Eastern Mediterranean Sea. Phylogenomic analysis of these five strains indicated that they are all members of Verrucomicrobia subdivision 4, order Opitutales. We compared these novel sponge-associated and seawater-isolated genomes to closely related Verrucomicrobia. Genomic analysis revealed that Planctomycetes-Verrucomicrobia microcompartment gene clusters are enriched in the genomes of symbiotic Opitutales including sponge symbionts but not in free-living ones. We hypothesize that in sponge symbionts these microcompartments are used for degradation of l -fucose and l -rhamnose, which are components of algal and bacterial cell walls and therefore may be found at high concentrations in the sponge tissue. Furthermore, we observed an enrichment of toxin–antitoxin modules in symbiotic Opitutales. We suggest that, in sponges, verrucomicrobial symbionts utilize these modules as a defence mechanism against antimicrobial activity deriving from the abundant microbial community co-inhabiting the host.     

Host-bacterial coevolution and the search for new drug targets

Understanding the coevolution between humans and our microbial symbionts and pathogens requires complementary approaches, ranging from community analysis to in-depth analysis of individual genomes. Here we review the evidence for coevolution between symbionts and their hosts, the role of horizontal gene transfer in coevolution, and genomic and metagenomic approaches to identify drug targets. Recent studies have shown that our symbiotic microbes confer many metabolic capabilities that our mammalian genomes lack, and that targeting mechanisms of horizontal gene transfer is a promising new direction for drug discovery. Gnotobiotic ('germ-free') mice are an especially exciting new tool for unraveling the function of microbes, whether individually or in the context of complex communities.     

Diversity in symbiont consortia in the pea aphid complex is associated with large phenotypic variation in the insect host

Virtually all eukaryotes host microbial symbionts that influence their phenotype in many ways. In a host population, individuals may differ in their symbiotic complement in terms of symbiont species and strains. Hence, the combined expression of symbiont and host genotypes may generate a range of phenotypic diversity on which selection can operate and influence host population ecology and evolution. Here, we used the pea aphid to examine how the infection with various symbiotic complements contributes to phenotypic diversity of this insect species. The pea aphid hosts an obligate symbiont (Buchnera aphidicola) and several secondary symbionts among which is Hamiltonella defensa. This secondary symbiont confers a protection against parasitoids but can also reduce the host’s longevity and fecundity. These phenotypic effects of H. defensa infection have been described for a small fraction of the pea aphid complex which encompasses multiple plant-specialized biotypes. In this study, we examined phenotypic differences in four pea aphid biotypes where H. defensa occurs at high frequency and sometimes associated with other secondary symbionts. For each biotype, we measured the fecundity, lifespan and level of parasitoid protection in several aphid lineages differing in their symbiotic complement. Our results showed little variation in longevity and fecundity among lineages but strong differences in their protection level. These differences in protective levels largely resulted from the strain type of H. defensa and the symbiotic consortium in the host. This study highlights the important role of symbiotic complement in the emergence of phenotypic divergence among host populations of the same species.     

毛蚜属Chaitophorus蚜虫(半翅目:蚜科:毛蚜亚科)与初级内共生菌Buchnera的演化关系

【目的】蚜虫体内共生菌种类丰富,二者关系十分密切。几乎所有蚜虫都具有一类专性的初级内共生菌Buchnera aphidicola,二者的专性共生关系使蚜虫-Buchnera成为研究共生关系演化的理想模型。本研究对蚜虫-Buchnera在低级阶元水平上的"平行演化假说"进行了验证。【方法】选取在杨属Populus或柳属Salix植物上营同寄主全周期生活的毛蚜属Chaitophorus蚜虫作为研究对象,基于不同来源的分子标记(蚜虫线粒体基因、核基因和内共生菌基因),运用最大似然法和贝叶斯法重建蚜虫和Buchnera的系统树,并利用Tree Map、Jane和Para Fit检验二者是否具有协同系统发生关系。【结果】Tree Map和Jane分析检测到毛蚜属蚜虫与Buchnera具有显著的共成种信号,Para Fit分析结果表明二者的总体关联极为显著。【结论】毛蚜属蚜虫与其初级内共生菌Buchnera在种级及以下水平上符合"平行演化假说",并且二者的演化关系不会受到寄主植物差异的影响。     

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.     

Evolution of the secondary symbiont "Candidatus serratia symbiotica" in aphid species of the subfamily lachninae

Buchnera aphidicola BCc, the primary endosymbiont of the aphid Cinara cedri (subfamily Lachninae), is losing its symbiotic capacity and might be replaced by the coresident "Candidatus Serratia symbiotica." Phylogenetic and morphological analyses within the subfamily Lachninae indicate two different "Ca. Serratia symbiotica" lineages and support the longtime coevolution of both symbionts in C. cedri.