A Veritable Menagerie of Heritable Bacteria from Ants, Butterflies, and Beyond: Broad Molecular Surveys and a Systematic Review

Maternally transmitted bacteria have been important players in the evolution of insects and other arthropods, affecting their nutrition, defense, development, and reproduction. Wolbachia are the best studied among these and typically the most prevalent. While several other bacteria have independently evolved a heritable lifestyle, less is known about their host ranges. Moreover, most groups of insects have not had their heritable microflora systematically surveyed across a broad range of their taxonomic diversity. To help remedy these shortcomings we used diagnostic PCR to screen for five groups of heritable symbionts—Arsenophonus spp., Cardinium hertigii, Hamiltonella defensa, Spiroplasma spp., and Wolbachia spp.—across the ants and lepidopterans (focusing, in the latter case, on two butterfly families—the Lycaenidae and Nymphalidae). We did not detect Cardinium or Hamiltonella in any host. Wolbachia were the most widespread, while Spiroplasma (ants and lepidopterans) and Arsenophonus (ants only) were present at low levels. Co-infections with different Wolbachia strains appeared especially common in ants and less so in lepidopterans. While no additional facultative heritable symbionts were found among ants using universal bacterial primers, microbes related to heritable enteric bacteria were detected in several hosts. In summary, our findings show that Wolbachia are the dominant heritable symbionts of ants and at least some lepidopterans. However, a systematic review of symbiont frequencies across host taxa revealed that this is not always the case across other arthropods. Furthermore, comparisons of symbiont frequencies revealed that the prevalence of Wolbachia and other heritable symbionts varies substantially across lower-level arthropod taxa. We discuss the correlates, potential causes, and implications of these patterns, providing hypotheses on host attributes that may shape the distributions of these influential bacteria.Organismic and Evolutionary Biolog

Army Ants Harbor a Host-Specific Clade of Entomoplasmatales Bacteria ▿ †

In this article, we describe the distributions of Entomoplasmatales bacteria across the ants, identifying a novel lineage of gut bacteria that is unique to the army ants. While our findings indicate that the Entomoplasmatales are not essential for growth or development, molecular analyses suggest that this relationship is host specific and potentially ancient. The documented trends add to a growing body of literature that hints at a diversity of undiscovered associations between ants and bacterial symbionts

Specialization and Geographic Isolation among Wolbachia Symbionts from Ants and Lycaenid Butterflies

Wolbachia are the most prevalent and influential bacteria described among the insects to date. But despite their significance, we lack an understanding of their evolutionary histories. To describe the evolution of symbioses between Wolbachia and their hosts, we surveyed global collections of two diverse families of insects, the ants and lycaenid butterflies. In total, 54 Wolbachia isolates were typed using a Multi Locus Sequence Typing (MLST) approach, in which five unlinked loci were sequenced and analyzed to decipher evolutionary patterns. AMOVA and phylogenetic analyses demonstrated that related Wolbachia commonly infect related hosts, revealing a pattern of host association that was strongest among strains from the ants. A review of the literature indicated that horizontal transfer is most successful when Wolbachia move between related hosts, suggesting that patterns of host association are driven by specialization on a common physiological background. Aside from providing the broadest and strongest evidence to date for Wolbachia specialization, our findings also reveal that strains from New World ants differ markedly from those in ants from other locations. We, therefore, conclude that both geographic and phylogenetic barriers have promoted evolutionary divergence among these influential symbionts.Organismic and Evolutionary Biolog

Bacterial gut symbionts are tightly linked with the evolution of herbivory in ants

Ants are a dominant feature of terrestrial ecosystems, yet we know little about the forces that drive their evolution. Recent findings illustrate that their diets range from herbivorous to predaceous, with “herbivores” feeding primarily on exudates from plants and sap-feeding insects. Persistence on these nitrogen-poor food sources raises the question of how ants obtain sufficient nutrition. To investigate the potential role of symbiotic microbes, we have surveyed 283 species from 18 of the 21 ant subfamilies using molecular techniques. Our findings uncovered a wealth of bacteria from across the ants. Notable among the surveyed hosts were herbivorous “turtle ants” from the related genera Cephalotes and Procryptocerus (tribe Cephalotini). These commonly harbored bacteria from ant-specific clades within the Burkholderiales, Pseudomonadales, Rhizobiales, Verrucomicrobiales, and Xanthomonadales, and studies of lab-reared Cephalotes varians characterized these microbes as symbiotic residents of ant guts. Although most of these symbionts were confined to turtle ants, bacteria from an ant-specific clade of Rhizobiales were more broadly distributed. Statistical analyses revealed a strong relationship between herbivory and the prevalence of Rhizobiales gut symbionts within ant genera. Furthermore, a consideration of the ant phylogeny identified at least five independent origins of symbioses between herbivorous ants and related Rhizobiales. Combined with previous findings and the potential for symbiotic nitrogen fixation, our results strongly support the hypothesis that bacteria have facilitated convergent evolution of herbivory across the ants, further implicating symbiosis as a major force in ant evolution

A Veritable Menagerie of Heritable Bacteria from Ants, Butterflies, and Beyond: Broad Molecular Surveys and a Systematic Review

<div><p>Maternally transmitted bacteria have been important players in the evolution of insects and other arthropods, affecting their nutrition, defense, development, and reproduction. <em>Wolbachia</em> are the best studied among these and typically the most prevalent. While several other bacteria have independently evolved a heritable lifestyle, less is known about their host ranges. Moreover, most groups of insects have not had their heritable microflora systematically surveyed across a broad range of their taxonomic diversity. To help remedy these shortcomings we used diagnostic PCR to screen for five groups of heritable symbionts—<em>Arsenophonus</em> spp., <em>Cardinium hertigii</em>, <em>Hamiltonella defensa</em>, <em>Spiroplasma</em> spp., and <em>Wolbachia</em> spp.—across the ants and lepidopterans (focusing, in the latter case, on two butterfly families—the Lycaenidae and Nymphalidae). We did not detect <em>Cardinium</em> or <em>Hamiltonella</em> in any host. <em>Wolbachia</em> were the most widespread, while <em>Spiroplasma</em> (ants and lepidopterans) and <em>Arsenophonus</em> (ants only) were present at low levels. Co-infections with different <em>Wolbachia</em> strains appeared especially common in ants and less so in lepidopterans. While no additional facultative heritable symbionts were found among ants using universal bacterial primers, microbes related to heritable enteric bacteria were detected in several hosts. In summary, our findings show that <em>Wolbachia</em> are the dominant heritable symbionts of ants and at least some lepidopterans. However, a systematic review of symbiont frequencies across host taxa revealed that this is not always the case across other arthropods. Furthermore, comparisons of symbiont frequencies revealed that the prevalence of <em>Wolbachia</em> and other heritable symbionts varies substantially across lower-level arthropod taxa. We discuss the correlates, potential causes, and implications of these patterns, providing hypotheses on host attributes that may shape the distributions of these influential bacteria.</p> </div

Proportions of ants and lepidopterans testing positive for heritable symbionts in this study.

†<p><i>Spiroplasma</i> tallies were inferred in this study but were mostly derived from molecular screening in Funaro et al. 2011.</p>††<p>Numbers of surveyed species are indicated in parentheses.</p

The diversity of <i>Wolbachia</i> strains from single insect hosts as inferred through sequencing of <i>wsp</i> libraries.

†<p>Phylotypes described based on 99% sequence similarity, as described in the methods.</p

16S rRNA phylogeny of known heritable symbionts and microbes from ants.

<p>Maximum phylogeny showing relatedness between ant associates and known maternally transmitted bacteria, thus illustrating the range of candidate heritable symbionts across the ants. Lineages of heritable symbionts are labeled and color-coded to indicate the presence of ant-associates. Color strip circles are used to indicate bacterial taxonomy (inner circle) and ant-association (outer circle). For ease of viewing, several clades on the original phylogeny were collapsed (i.e. those without heritable symbionts). The full tree is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051027#pone.0051027.s002" target="_blank">Figure S1</a>. Note that lifestyle heterogeneity within <i>Spiroplasma</i> and <i>Arsenophonus</i> lineages means that identified ant-associates are not certain to be heritable. Also note that the two “possible” symbionts of ants (a <i>Staphylococcus</i> sp. and a <i>Bacillus</i> sp.) were both detected in hemolymph and in eggs laid by queens, suggesting heritability.</p

Frequencies of <i>Wolbachia</i> across arthropod orders.

<p>Proportions of infected species for all orders with n>10 species surveyed for <i>Arsenophonus</i> (A), <i>Cardinium</i> (C), <i>Hamiltonella</i> (H), <i>Spiroplasma</i> (S), and/or <i>Wolbachia</i> (W). “−” symbols indicate cases where data were not illustrated due to sample sizes of less than 10 surveyed species. Numbers of surveyed species are indicated below each respective bar. Data used for this figure were generated with diagnostic PCR surveys and are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051027#pone.0051027.s011" target="_blank">Table S5</a>. The phylogeny was drawn from information on the Tree of Life Website (tolweb.org). Only those orders with at least two symbionts meeting the above criteria are illustrated.</p

16S rRNA phylogeny of <i>Spiroplasma</i> from ants, lepidopterans, and other hosts.

<p>Maximum likelihood phylogeny illustrating relatedness between ant associates and various <i>Spiroplasma</i> symbionts. The inner-most color strip illustrates known lifestyle, with gut associates being presented in light gray, pathogens in dark gray, and heritable bacteria in black. In the outer strip, the phenotype of male-killing is illustrated. Microbes are named after their hosts. Those from ants are in bold and italics; those from lepidopterans are in bold font; and those reported from <i>Polyrhachis</i> in this study are indicated with an asterisk. Non-<i>Spiroplasma</i> clades are collapsed for brevity. Bootstrap values greater than 60 are indicated above their respective nodes.</p