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

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 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

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 <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

Prevalence, ranges of infected hosts, and correlates of symbiont prevalence as inferred from our systematic review.

†<p>Focusing on traits found in two or more unrelated groups enriched for the given symbiont.</p

Study selection (PRISMA) flow diagram for our systematic review.

<p>Details on identification and selection of diagnostic PCR studies that surveyed for <i>Arsenophonus</i>, <i>Cardinium</i>, <i>Hamiltonella</i>, <i>Spiroplasma</i>, and <i>Wolbachia</i>.</p

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

<p>Proportions of infected species for all families with n>10 species surveyed for Arsenophonus (A), Cardinium (C), Hamiltonella (H), Spiroplasma (S), and/or Wolbachia (W). “−” symbols indicate cases where results were not illustrated due to sample sizes of less than 10 surveyed species. Families of butterflies and moths were heavily sampled in this study and are highlighted in blue. Infection frequencies within the ant family Formicidae are highlighted in red. Names of arthropod orders are listed in capital letters above the data for their respective families. Numbers of surveyed species are indicated above each respective bar. Data used for this figure were generated with diagnostic PCR 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 families with at least two symbionts meeting the above criteria are illustrated.</p