Flow cytometry analyse of the hemocyte response in sympatric and allopatric interactions.

A) Flow cytometry was used to count in vivo EdU-labeled hemocytes at 24 h and 96 h after infection in sympatric and allopatric interactions. A total number of hemocytes of n = 10,000were recoveredfor6 biological replicates of 3 snails. Control naïve snails (BgBRE, yellow) were compared to those subjected to the sympatric interaction (BB, BgBRE/SmBRE, blue) or an allopatric interaction (BV, BgBRE/SmVEN, green). B) The experiment described in A was repeated using the BgVEN snail strain. Control naïve snails (BgVEN, yellow) were compared to those subjected to the sympatric interaction (VV, BgVEN/SmVEN, green) or an allopatric interaction (VB, BgVEN/SmBRE, blue). C) FSC (forward-scattered light, representing cell size) and SSC (side-scattered light, representing cell granularity) circulating hemocyte patterns in BgBRE snails under the naïve condition (yellow) or 24 h and 96 h after infections in sympatry (BB24/96, BgBRE/SmBRE, blue) or allopatry (BV24/96, BgBRE/SmVEN, green). D) FSC and SSC circulating hemocyte patterns in BgVEN snails under the naïve condition (yellow) or 24 h and 96 h after infections in sympatry (VV24/96, BgVEN/SmVEN, blue) or allopatry (VB24/96, BgVEN/SmBRE, green). The red dots correspond to EdU-positive hemocytes. Between-group differences in the percentage of proliferation were tested using the Mann-Whitney U-test, with statistical significance accepted at p<0.05. The “a” indicates a significant difference between the naïve and infective condition, “b” indicates a significant difference between the infective conditions at 24h, and “c” indicates a significant difference between the infective conditions at 96h.