PMC

The Brazilian strain of albino B. glabrata (BgBRE) is 100% susceptible (for 10 miracidia and upwards) to its corresponding strain of S. mansoni (SmBRE). When a snail is infected with 10 miracidia of S. mansoni within the same individual compatible and incompatible interactions occur, 3 to 4 miracidia develop normally in the snail’s tissues while the others are recognized and encapsulated by the snail’s cellular immune response. A. Six-day-old sporocyst in a compatible interaction. B. Encapsulated sporocyst 48 h after primary infection in an incompatible interaction. C. Six-day-old sporocyst in a primed snail. Primed BgBRE are 100% protected against a secondary challenge with SmBRE. Sporocysts from secondary challenge were neutralized by immune humoral factors.

Heatmap showing differentially represented transcripts compared to naïve snails, as identified by DESeq2 analysis (p < 0.1). Color scale indicates the Log2FC ratio from under-represented (blue) to over-represented (red) transcripts. Transcripts were grouped into six clusters based on their expression patterns during the process of innate immune memory. Samples were recovered at 1DPPI, 4DPPI, 15DPPI and 25DPPI following primo-infection. Following secondary challenge samples were recovered at 1 day, 4 days and 15 days and pooled into DPC sample. Six clusters are identified: Cluster 1: transcripts over represented more than once all along infection and challenge. Cluster 2: transcripts exclusively over represented in single one condition. Cluster 3: transcripts exclusively over represented after immune challenge (DPC). Cluster 4: transcripts exclusively under represented after immune challenge (DPC). Cluster 5: transcripts exclusively under-represented in single one condition. Cluster 6: transcripts under represented more than once all along infection and challenge. FREP: Fibrinogen-related protein, HSP: Heat-shock protein, PGRP 1-like: Pathogenesis-related protein 1-like, LBP/BPI: lipopolysaccharide-binding protein/bactericidal/permeability-increasing protein, BgLBP/BPI: Biomphalaria glabrata LBP/BPI, TEP: thioester-containing protein.

A. Cumulative expression [Log2FC (fold change) from the DESeq2 analysis] of FREP transcripts showed that FREPs were over-represented after the secondary challenge (DPC; 5.096 log2 fold change enrichment of FREPs transcripts). Green points corresponded to the differentially represented FREPs transcripts in each samples. Purple bars represent the cumulated Log2FC of FREP transcripts. At 4DPPI no FREP transcripts were differentially expressed, thus no value appeared in the graph. B. siRNA injection against FREP2, FREP3 & FREP4 was carried out and mRNA abundance was monitored during 4 days by Q-RT-PCR. Snails were injected with siRNAs against FREP 2, 3, and 4 or GFP (control), the relevant mRNA levels were assessed following normalization with respect to the S19 gene in siGFP injected snails versus siFREPs injected snails. Knock-down of the three FREPs tested was confirmed at 96h. C. Naïve B. glabrata and siFREP-injected snails were subjected to a typical priming experiment: Snails were infected with 10 miracidia of S. mansoni as a primo-infection, 21 days later they were injected with siGFP, or SiFREP or not treated and 4 days later they were infected with another 10 miracidia as a secondary challenge. FREP siRNA-injected snails show a significant proportion of non-primed snails (15%; *, binomial test, P < 0.05).

A. 2D gel electrophoresis of plasma proteins. One gel of each plasma sample analysed was shown. Spot numbers of qualitative and quantitative differences were indicated. Four plasma samples were analysed from naïve (uninfected snails), 15DPPI and 25DPPI (recovered at 15 and 25 days after primo-infection) and 15DPC (recovered at 15 days after secondary challenge). B. Heat-Map of the qualitative and quantitative ratio versus naïve sample. Ratios were calculated using PDQuest software between all differentially regulated spots. Blue to red scale indicate ratio values from lower to higher represented spots. Four clusters are identified: Cluster 1: higher-represented proteins exclusively following secondary challenge (15 DPC). Cluster 2: sustained response: higher-represented proteins after the primo-infection and secondary infection. Cluster 3: higher-represented proteins at 15DPPI and thereafter down regulated at 25DPPI and 15DPC. Cluster 4: lower-represented proteins. C. Plasma transfer and effect on prevalence of S. mansoni infection. Four conditions were tested: untreated snails (Control group, n = 48); saline injected snails (control of injection, n = 25); naïve-plasma injected snails (n = 22); and primed-plasma injected snails (n = 25). For all the experimental groups, 15 days following injection, snails were infected with 10 miracidia of SmBRE. * indicated significant differences (P< 0.05).

Innate immune memory experiments were carry out. For primo-infection, Brazilian Biomphalaria glabrata (BgBRE) snails were individually exposed to 10 miracidia of their sympatric Brazilian Schistosoma mansoni trematode parasite (SmBRE). Following infection depending on the compatibility status of the snail/parasite couples, some of the miracidia were encapsulated by the hemocytes (snail immune cells) or developed into primary sporocysts (intra-molluscan stage of the parasite). Intramolluscan parasite stages include two generations of sporocysts (primary sporocyst (SPI) and secondary sporocyst (SPII)) and the production of cercariae. SPII developed inside SPI and migrated to reach the snail hepato-pancrea. Cercariae developed inside SPI and migrated back into the snail to reach the aquatic environment. Twenty-five days after primo-infection, the snails were challenged for a second time with again 10 SmBRE miracidia. In this case all miracidia degenerated in snail tissues, demonstrating the activation of a humoral immune response. Immune phenotypes observed during innate immune memory process were analyzed using a histological approach (see ). In order to explore the molecular mechanisms of innate immune memory several experimental procedures were designed. A RNAseq experiment was realized with samples recovered from uninfected snails (Naive 1, Naive 2), samples recovered at 1, 4, 15 and 25 days post primo-infection (DPPI) and at 1, 4 and 15 days post-secondary challenge (DPC) (see ). Based on RNaseq results, functional validation of the FREP immune recognition receptor was undergone. First, individual quantification were made for all FREPs annotated on transcriptomic analysis (see ). FREP knockdown was then carried out by siRNA injection, normalized by siGFP and monitored by Q-RT-PCR (see ). Finally, to confirm the involvement of plasmatic factors in innate immune memory, snail hemolymph was recovered (Naive, 15, 25 DPPI and 15 DPC) and plasmatic fraction was characterized by 2D-gel electrophoresis (see ). Plasma samples were also injected to naïve snails to demonstrate that immune protection could be acquired following primed snail plasma transfer (see ).