Anti fluorescein ap

To amplify the genomic fragments corresponding to the loci targeted in ISH, we designed three primer sets for Locus_D, four for Locus_H and one for Locus_N. These primers produced 5.5–10.9 kb of PCR fragments covering the target locus as a whole (Figure 1). The PCR fragments were labelled with fluorescein or digoxigenin using nick translation kits from Enzo Life Sciences or Roche, respectively (see Supplementary Methods for details). For whole-mount in situ hybridization (WISH) for genomic loci, the fixed gill filaments of two host individuals from Myojin Knoll (Supplementary Table S1) were used. The hybridization reaction with the fluorescein labelled probe was carried out overnight at 37 °C. After incubation in anti-fluorescein-AP (Roche) in PBST with blocking, hybridization was detected using NBT/BCIP solution (Roche) in TMNT buffer (see Supplementary Methods for details). For fluorescent in situ hybridization (FISH), the fixed gills of three host mussels from Myojin Knoll, different individuals from those used for WISH (Supplementary Table S1), were embedded in paraffin and sliced with a microtome into 4-μm-thick sections. Two-colour FISH of the gill sections was performed using probes labelled with fluorescein or digoxigenin and TSA Plus Cyanine 3/Fluorescein System (Perkin Elmer, Waltham, MA, USA) (see Supplementary Methods for details).