Wild ciliates differ in susceptibility to JR32.

We investigated how (Lp) JR32 interacts with CS11A and E6, two ciliates that we isolated from sewage and sink trap sludge, respectively, using a handmade maze device containing a 96-well crafting plate. Our 18S rDNA-based phylogenetic analysis showed that CS11A and E6 formed distinct clades. Scanning electron microscopy showed that CS11A had a bigger-sized body than E6 and, unlike IB (the reference strain), neither ciliate produced pellets, which are extracellular vacuoles. Fluorescence microscopic observations revealed that although the intake amounts differed, all three ciliates rapidly ingested LpJR32 regardless of the presence or absence of the virulence genes, indicating that they all interacted with LpJR32. In co-cultures with CS11A, the LpJR32 levels were maintained but fell dramatically when the co-culture contained the LpJR32 deletion mutant instead. CS11A died within 2 days of co-culture with LpJR32, but survived co-culture with the deletion mutant. In co-cultures with E6, LpJR32 levels were maintained but temporarily decreased independently of the virulence gene. Concurrently, the E6 ciliates survived by forming cysts, which may enable them to resist harsh environments, and by diminishing the sensitivity of trophozoites to Lp. In the IB co-cultures with LpJR32 or Δ, the Lp levels were maintained, albeit with temporal decreases, and the IB levels were also maintained. We conclude that unlike IB with pellet production, CS11A can be killed by LpJR32 infection, and E6 can resist LpJR32 infection through cyst formation and the low sensitivity of trophozoites to Lp. Thus, the two ciliates that we isolated had different susceptibilities to LpJR32 infection.