Primers used in this study.

<p>Primers used in this study.</p

BgTLR immunolocalization in haemocytes and Bge cells.

<p>Haemocytes (A-C) and Bge cells (D-E) were labelled with DAPI and anti-BgTLR primary antibody. Control haemocyte (C) and Bge (E) samples were stained with DAPI but the primary antibody step omitted. Scale bars represent 20 μM.</p

siRNA oligonucleotide sequences.

<p>siRNA oligonucleotide sequences.</p

Knockdown of BgTLR in BS-90 snails decreases haemocyte phagocytic response.

<p>(A) Mean number of phagocytosed beads per haemocyte. Five BS-90 snails each were injected with siRNA targeting BgTLR or GFP (control) and 96 hours later, haemolymph was extracted from the snails and immediately mixed with ~1 x 10⁶ 1μM FITC-labelled streptavidin-coated beads pre-incubated with biotinylated <i>S</i>. <i>mansoni</i> excretory/secretory products and sporocyst. After 3 hours, haemocytes from each snail were counted from a random field of view on the slide, and 30 haemocytes for each snail were assessed for the number of beads within each cell from which mean number of beads per haemocyte was calculated. Asterisk (*) indicates significant reduction (P < 0.05) in the mean number of beads per haemocyte in BgTLR knockdown snails. (B) Frequency of number of beads observed in 30 haemocytes from one field of view (FOV). Asterisk (*) indicates significant difference (P < 0.05) in the average number of beads phagocytosed between BgTLR and GFP knockdown.</p

siRNA-mediated knockdown of BgTLR transcripts.

<p>(A) Graphic view of BgTLR (not drawn to scale) showing the leucine-repeat motifs and Toll/IL-1 domain. Red bars indicate the approximate positions targeted by siRNA oligonucleotides used to inject the snails, while the blue bar indicates the targeted position of the antibody used. (B) Confirmation of BgTLR (~135 kDa) knockdown in BS-90 haemocytes and Bge cells respectively. Protein was detected via Western blot with primary antibody developed against the extracellular region of BgTLR. BgActin (~42 kDa) served as the loading control.</p

BgTLR displayed increased transcript expression in BS-90 snails following challenge with <i>S</i>. <i>mansoni</i>.

<p>(A) BgTLR transcript expression with and without <i>S</i>. <i>mansoni</i> challenge. Snails (BS-90 and M-line strains) were individually exposed to ~5 miracidia or left unexposed (control). Five snails were collected at indicated time points over the incubation period of the parasite. RNA was extracted from whole snails, converted to cDNA and BgTLR expression was measured by quantitative PCR. Expression was quantified in fold changes normalized to time 0-hour controls. Bars represent standard error (n = 5). Asterisk (*) indicates significant difference (P < 0.05) between experimental and control samples, while hash (#) indicates significant difference between BS-90 and M-line snails at the respective time points. (B) Knockdown of BgTLR or GFP (control) in BS-90 snails responding to <i>S</i>. <i>mansoni</i> parasite challenge. (C) Knockdown of BgTLR in unexposed M-line and BS-90 snails. Five snails were collected for each time point in B and C for RNA extraction and cDNA synthesis. Bars represent standard error (n = 5). Asterisks indicate significant difference (P < 0.05) from 0-hour time points.</p