Quality over quantity: unraveling the contributions to cytoplasmic incompatibility caused by two coinfecting Cardinium symbionts

Cardinium hertigii is a common maternally-inherited bacterial endosymbiont of arthropods. Some Cardinium strains spread by increasing female fitness through the induction of cytoplasmic incompatibility (CI), which kills offspring of crosses between infected males and uninfected female hosts. CI is a two-step manipulation: first, sperm from male hosts are modified by the symbiont in a manner that causes offspring death. Second, when females are also infected, the symbiont reverses sperm modification in the egg cytoplasm, allowing offspring of infected females to survive and spread the symbiont. While Cardinium causes CI in many arthropod hosts, most of what is known about Cardinium CI stems from the symbiosis between the Cardinium strain cEper1 and its minute parasitoid host, Encarsia suzannae. Here, we study a second Cardinium CI system in the wasp Encarsia partenopea. Encarsia partenopea harbors two Cardinium strains, cEina2 and cEina3, with the cEina3 present at a much lower density than cEina2. Using antibiotic treatments and crossing assays, we find that the low-density cEina3 strain is responsible for CI, and that cEina3 appears to modify sperm during the pupal stage, like the better known cEper1. However, cEina3 shows a markedly different localization pattern in male reproductive tissues. Instead of infecting sperm cells, cEina3 is found in somatic cells at the base of the testis and around the seminal vesicle. This localization pattern suggests that cEina3 may use a different sperm modification strategy from cEper1, and highlights variation between these closely related symbioses