The tripartite associations between bacteriophage, Wolbachia, and arthropods

© 2006 Bordenstein et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The definitive version was published in PLoS Pathogens 2(2006): e43, doi:10.1371/journal.ppat.0020043.By manipulating arthropod reproduction worldwide, the heritable endosymbiont Wolbachia has spread to pandemic levels. Little is known about the microbial basis of cytoplasmic incompatibility (CI) except that bacterial densities and percentages of infected sperm cysts associate with incompatibility strength. The recent discovery of a temperate bacteriophage (WO-B) of Wolbachia containing ankyrin-encoding genes and virulence factors has led to intensifying debate that bacteriophage WO-B induces CI. However, current hypotheses have not considered the separate roles that lytic and lysogenic phage might have on bacterial fitness and phenotype. Here we describe a set of quantitative approaches to characterize phage densities and its associations with bacterial densities and CI. We enumerated genome copy number of phage WO-B and Wolbachia and CI penetrance in supergroup A- and B-infected males of the parasitoid wasp Nasonia vitripennis. We report several findings: (1) variability in CI strength for A-infected males is positively associated with bacterial densities, as expected under the bacterial density model of CI, (2) phage and bacterial densities have a significant inverse association, as expected for an active lytic infection, and (3) CI strength and phage densities are inversely related in A-infected males; similarly, males expressing incomplete CI have significantly higher phage densities than males expressing complete CI. Ultrastructural analyses indicate that approximately 12% of the A Wolbachia have phage particles, and aggregations of these particles can putatively occur outside the Wolbachia cell. Physical interactions were observed between approximately 16% of the Wolbachia cells and spermatid tails. The results support a low to moderate frequency of lytic development in Wolbachia and an overall negative density relationship between bacteriophage and Wolbachia. The findings motivate a novel phage density model of CI in which lytic phage repress Wolbachia densities and therefore reproductive parasitism. We conclude that phage, Wolbachia, and arthropods form a tripartite symbiotic association in which all three are integral to understanding the biology of this widespread endosymbiosis. Clarifying the roles of lytic and lysogenic phage development in Wolbachia biology will effectively structure inquiries into this research topic.This work was supported by grants from the NASA Astrobiology Institute (NNA04CC04A) and National Institutes of Health (R01 GM62626-01) to JJW, and by the Marine Biological Laboratory's Program in Global Infectious Diseases, funded by the Ellison Medical Foundation, to SRB

The Associations of Temperate Phage WO-B and <i>Wolbachia</i> Abundance

<p>Points on the charts denote the absolute copy numbers of a single-copy gene for phage WO-B and <i>Wolbachia</i> in individual adults of (A) A-infected males and (B) B-infected males of N. vitripennis. Correlation coefficients and significances are calculated according to the nonparametric method of Spearman's rho. In (A), the gray rectangle denotes the relative range of copy number variation for B-infected males, as depicted in larger scale in (B).</p

<i>Wolbachia</i> Infecting the Testes of N. vitripennis

<div><p>(A) Negatively stained virus particles of uniform size from PEG-precipitated preparations of A-infected N. vitripennis adults. No tail-like structures were apparent, perhaps due to disruption during the purification process. Bar = 100 nm.</p><p>(B) Low-resolution transmission electron micrograph of individualized spermatids and <i>Wolbachia</i> in N. vitripennis pupal testes. Ax and Md denote spermatid axonemes and mitochondrial derivatives. Several of the noted <i>Wolbachia</i> cells (W) are shown in higher resolution in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020043#ppat-0020043-g005" target="_blank">Figure 5</a>C–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020043#ppat-0020043-g005" target="_blank">5</a>E. Solid arrowheads denote phage particles. Bar = 200 nm.</p><p>(C) A high density of phage particles within <i>Wolbachia</i> is shown; phage tails are occasionally visible and noted by white arrowheads in the inset. Bar = 200 nm; inset bar = 100 nm.</p><p>(D) Virion-free (lower right) and virion-containing <i>Wolbachia</i> (upper right) localized near two N. vitripennis spermatids are shown. Solid arrowheads denote phage particles inside <i>Wolbachia</i>, and Ax and Md denote spermatid axonemes and mitochondrial derivatives, respectively. Bar = 200 nm.</p><p>(E) Solid arrowheads denote phage particles dispersing from within putatively lysed <i>Wolbachia</i> into the extracellular matrix. Bar = 200 nm.</p></div

A Phage Density Model of CI

<p>This flowchart depicts the predicted associations, i.e., (−) negative and (+) positive, between phage densities, <i>Wolbachia</i> densities, and CI levels with arrows connecting the associated variables. The flowchart is based on the following hypothesis: if lytic development of temperate phage WO-B leads to bacterial lysis or slowed cell divisions, the relative copy number of phages per bacterium may negatively associate with the relative copy number of <i>Wolbachia</i> per host, which in turn is well established to positively associate with CI levels. Under this scenario, phage densities can have a secondary negative association with CI levels.</p

Aggregations of Putative Extracellular Virions

<div><p>Solid arrowheads denote phage particles with no detectable <i>Wolbachia</i> membrane surrounding them. In some cases, a single membrane structure can be seen either completely or partially surrounding the phage particle patches.</p><p>(A) A patch of five virions near an individualized spermatid, for size reference.</p><p>(B) A patch of nine virions to the left of an individualized spermatid.</p><p>(C) A patch of 20 virions near a flagellar axoneme with potentially a single membrane structure surrounding the patch.</p></div

The Relationships between Phage Density, <i>Wolbachia</i> Density, and CI Level

<p>Points on the charts denote the relative phage WO-B density, relative <i>Wolbachia</i> density, and the number of female (diploid) offspring produced from cytoplasmically incompatible crosses using A-infected males. CI in haplodiploid species is expressed as a reduced number of female offspring. In (A), the dashed line denotes the estimated number of different lysogenic WO-B prophages per A-<i>Wolbachia</i> genome as determined by OFR7 amplification, cloning, and sequencing of heterogeneous copies [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020043#ppat-0020043-b044" target="_blank">44</a>].</p