High throughput method for analysis of repeat number for 28 phase variable loci of C. jejuni strain NCTC11168

Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method for rapidly determining ON/OFF states of these 28 phase-variable loci in a large number of individual colonies. The method combines a series of multiplex PCR assays with a GeneScan assay and automated extraction of tract length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. An important output of this assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species

Spiroplasma Bacteria Enhance Survival of Drosophila hydei Attacked by the Parasitic Wasp Leptopilina heterotoma

Maternally-transmitted associations between endosymbiotic bacteria and insects are ubiquitous. While many of these associations are obligate and mutually beneficial, many are facultative, and the mechanism(s) by which these microbes persist in their host lineages remain elusive. Inherited microbes with imperfect transmission are expected to be lost from their host lineages if no other mechanisms increase their persistence (i.e., host reproductive manipulation and/or fitness benefits to host). Indeed numerous facultative heritable endosymbionts are reproductive manipulators. Nevertheless, many do not manipulate reproduction, so they are expected to confer fitness benefits to their hosts, as has been shown in several studies that report defense against natural enemies, tolerance to environmental stress, and increased fecundity.We examined whether larval to adult survival of Drosophila hydei against attack by a common parasitoid wasp (Leptopilina heterotoma), differed between uninfected flies and flies that were artificially infected with Spiroplasma, a heritable endosymbiont of Drosophila hydei that does not appear to manipulate host reproduction. Survival was significantly greater for Spiroplasma-infected flies, and the effect of Spiroplasma infection was most evident during the host's pupal stage. We examined whether or not increased survival of Spiroplasma-infected flies was due to reduced oviposition by the wasp (i.e., pre-oviposition mechanism). The number of wasp eggs per fly larva did not differ significantly between Spiroplasma-free and Spiroplasma-infected fly larvae, suggesting that differential fly survival is due to a post-oviposition mechanism.Our results suggest that Spiroplasma confers protection to D. hydei against wasp parasitism. This is to our knowledge the first report of a potential defensive mutualism in the genus Spiroplasma. Whether it explains the persistence and high abundance of this strain in natural populations of D. hydei, as well as the widespread distribution of heritable Spiroplasma in Drosophila and other arthropods, remains to be investigated