The Tripartite Associations between Bacteriophage, Wolbachia, and Arthropods

Abstract

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.

Synopsis

Symbiotic bacteria that are maternally inherited are widespread in terrestrial invertebrates. Such bacteria infect the cells of reproductive tissues and can have important evolutionary and developmental effects on the host. Often these inherited symbionts develop beneficial relationships with their hosts, but some species can also selfishly alter invertebrate reproduction to increase the numbers of infected females (the transmitting sex of the bacteria) in the population. Bacterial-mediated distortions such as male-killing, feminization, parthenogenesis induction, and cytoplasmic incompatibility are collectively known as “reproductive parasitism.” In this article, the investigators show that the associations between the most common reproductive parasite in the biosphere (Wolbachia) and a parasitic wasp host are affected by a mobile element—a temperate bacteriophage of Wolbachia. In contrast to recent reports that suggest bacteriophage WO-B may induce reproductive parasitism, the authors' quantitative and ultrastructural analyses indicate that lytic phage WO-B are lethal and therefore associate with a reduction in both Wolbachia densities and reproductive parasitism. Based on these data, the authors propose a phage density model in which lytic phage development specifically leads to a reduction, rather than induction, of reproductive parisitism. The study is among the first investigations to show that lytic bacteriophage inversely associate with the densities and phenotype of an obligate intracellular bacterium.