*3.4. Detection of Native F. hepatica SOD*

Western blot analysis of adult parasite E/S and somatic extract using polyclonal antibodies raised in rabbits to the recombinant FhSODs revealed detectable FhSOD1 in both extracts at the expected size (Figure 4). In agreement with transcriptome and proteome data from adult parasites, native FhSOD3 was not detected in either fraction by polyclonal antibodies against rFhSOD3. These results are consistent with the immuno-detection of the proteins in adult parasite sections, where FhSOD1 was distributed throughout the muscle, tegument, and parenchyma, while limited FhSOD3 was visible in the tegument (Figure 5). The anti-pFhSOD1 and pFhSOD3 antibodies demonstrated protein-specific binding against rFhSOD1 and rFhSOD3 by Western blot analysis but did not reveal detectable fluorescence when applied to fixed parasite specimens, suggesting recognition of a linear epitope in NEJ (Figure S5).

**Figure 4.** Immune detection of native *F. hepatica* SODs in adult parasite E/S and somatic extracts. (**A**) Native (10 ug/well) *F. hepatica* adult worm E/S and somatic extracts were resolved in a 4–20% SDS-PAGE gel and stained with Biosafe Coomassie. Lane 1: adult E/S; lane 2: adult somatic extract. (**B**–**D**) Western blot analysis of native FhSOD1 and FhSOD3. Lane 1: adult E/S; lane 2: adult somatic extract. Immuno blots were probed with (**B**) rabbit pre-immune sera (negative control), (**C**) anti-rFhSOD1 polyclonal antibodies raised in rabbit, and (**D**) anti-rFhSOD3 polyclonal antibodies raised in rabbit. M molecular weight in kDa (Precision Plus Protein Dual, Bio-Rad).

**Figure 5.** Immuno-localisation of native FhSOD1 and FhSOD3 in *F. hepatica* adult sections. Sections were probed with (**A**) anti-rFhCL1 polyclonal antibodies (non-related positive control), (**B**) rabbit pre-immune sera (negative control), (**C**) anti-rFhSOD1 polyclonal antibodies, and (**D**) anti-rFhSOD3 polyclonal antibodies raised in rabbit. Immuno-localisation of native *F. hepatica* proteins is represented by green fluorescence (FITC staining) and indicated with white arrows. G; gut, P; parenchyma, T; tegument, VS; ventral sucker, scale bars 50 μM.

Whole mount immuno-localisation of NEJ 3 h post-excystment using anti-rFhSOD1 and anti-rFhSOD3 antibodies revealed the presence of FhSOD1 and FhSOD3 on the outer surface, gut and tegument as indicated by diffuse fluorescence (Figure 6). This is in

contrast to NEJ stained with anti-rFhCL3 polyclonal antibodies, which locates the FhCL3 cysteine peptidase solely within the bifurcated gut (Figure 6). Despite a potential crossreaction between the two FhSOD polyclonal antibodies, these results are consistent with the transcriptome and proteome analyses of the juvenile parasites, which show abundant expression by the metacercariae and 1, 3, and 24 h post-excystment NEJ (Figure 2A,C).

**Figure 6.** Immuno-localisation of native FhSOD1 and FhSOD3 in newly excysted juveniles (NEJ). Whole-mount *F. hepatica* NEJ 3 h post-excystment were probed with (**A**) rabbit pre-immune sera (negative control), (**B**) anti-rFhCL3 polyclonal antibodies (non-related positive control), (**C**) antirFhSOD1 polyclonal antibodies, and (**D**) anti-rFhSOD3 polyclonal antibodies. Immuno-localisation of native *F. hepatica* proteins is represented by green fluorescence (FITC staining). All samples were counter-stained with phalloidin-TRITC to stain muscle tissue (red fluorescence). OS; oral sucker, VS; ventral sucker, scale bars; 25 μM.
