- Ecoimmunological studies, however, typically use one/few phenotypic immune assays and thus do not consider the complexity of the immune system. - Results: Immune defence factors identified in the de novo assembly covered elements broadly described in other gastropods. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - Full list of author information is available at the end of the article. - Most of the attention on molecular immunology in gastropods has focused on Biomphalaria glabrata (Planorbidae, Hygrophila, Panpulmonata), a tropical freshwater snail that transmits the human blood fluke, Schistosoma mansoni [36–38]. - Attention to other species has been comparatively limited although several gastropods, including pond snails of the family Lymnaei- dae (Hygrophila, Panpulmonata), transmit medically and veterinary-relevant parasites (e.g., Fasciola hepatica, Diplostomum spp., Trichobilharzia spp in temperate regions. - After that, we evaluated variation in the transcription of those candi- dates to detect transcripts that responded to experimen- tal treatments and/or that showed high among- individual variation. - were deposited in the NCBI Sequence Read Archive (ac- cession number PRJNA664475). - The de novo assembly (trinity v2.0.6) of the reads from nine libraries (one per each experimental treatment. - BUSCO analysis indicated high completeness of the reference transcriptome based on the detection of 98.8% (complete plus partial) of the set of metazoan uni- versal single-copy orthologs [complete: 96.3% (dupli- cated: 28.2. - The reference transcriptome included sequences that represent parts of the snail rDNA cassette, including the complete ITS1 and ITS2 regions (GenBank accession numbers MT864603 and MT864602, respectively). - Lym- naea stagnalis was confirmed as the species identity of the experimental snails by the highest sequence similar- ities (BLASTN) of these sequences with GenBank entries from this species. - The mining of the L. - of the detected lectin families included two FREPs of the VIgLs (variable immunoglobulin and lectin- domain-containing molecules), galectins comprising of either one, two or four carbohydrate recognition do- mains, multiple Chi-lectins, as well as L- and M-type lectins. - 1 Summary of the identified immune defence factors in Lymnaea stagnalis reference transcriptome. - Factors are organised across different immunological mechanisms/pathways and steps of the immune response (i.e., non-self recognition, signalling/. - The search of the complete reference transcriptome for antimicrobial defences yielded a single family of macin antimicrobial peptides (AMPs, 6 variants), as well as several families of antimicrobial proteins, with an abundant representation of lipopolysaccharide-binding /bactericidal permeability-increasing proteins (LBP/BPI, 3 variants), L-amino acid oxidases (LAAOs, 3 variants), lysozymes (4 variants), and transcripts encoding for cytolytic β pore-forming toxins. - Representatives of the intrinsic signalling. - Heatmaps for the transcription of the annotated immune factors (see Additional file 1) across experimental treatments indicated some immune- elicitor specific responses (Fig. - coli cells increased the transcription of TLR, as well as of some components of the TLR sig- nalling pathway (IκB, NF-κB), when compared to the levels in snails injected with physiological saline (com- parison 1 in Fig. - 3, Additional file 3). - 3, Additional file 5). - the transcription of the lectin FREP (3 out of 6 individ- uals. - 3, Additional file 7), one component of the TLR signalling pathway (IκB;. - Additionally, wounding led to upregu- lation in the transcription of DUOXA (Additional file 8) that contributes to ROS production. - This was the case for PRRs GNBP (Additional file 6), galectin (Additional file 6) and TLR (Additional file 3), as well as for some components of the TLR signalling pathway (Myd88, TRAF, NF-κB p65. - Additional file 9),. - Interestingly, within certain components of the immune system, individual snails expressed distinct gene transcripts, which suggests dif- ferent defence strategies against pathogens. - coli showed high transcription in two to four of the examined six antibacterial defence factors and the factors with high levels were different among individuals (Additional file 4). - Similarly, snails ex- posed to trematode-infected snail tissue extracts showed high transcription in only one to two of the examined non-self recognition components (GNBP, FREP, galec- tins, TLR), and the expressed factors were different among individuals (areas 10 in Fig. - The examined environmental changes apart from im- mune challenge affected several components of the snail immune system. - The elevated temperature increased the transcription of the cytokine MIF (comparison 11 in Fig.. - 2 Principal component analysis (PCA) plot showing variation in transcriptome-wide expression profiles of the experimental snails. - PCA plots for the first five PCs, as well as the proportion of total variance each of them explained in the data, are presented in Additional file 2. - Additionally, some of the factors that contribute to stress-responses, antioxidation and metab- olism were found to be affected by the experimental treatments (Fig. - BUSCO analysis, how- ever, indicated representation of 98.8% of the universally shared metazoan genes in the assembly. - This is remark- able because of the great diversity and numbers of AMP genes and gene families that are usually recorded from other organisms, including bivalve molluscs (e.g., [64]).. - 3 Expression levels of selected transcripts that represent different components of the immune system. - Heatmap shows transcripts that deviated in their transcription between certain experimental treatments and their specific controls and components of the immune system in which individuals expressed distinct transcripts. - Red (injections with bacteria), blue (injections with snail/trematode tissue extracts), purple (injections with bacteria and snail/trematode tissue extracts) and black (exposures to environmental change) rectangles (dashed line), arrows and numbers refer to the specific results mentioned in the text. - stagnalis is similar to that of the physid Physella acuta (Schultz et al., 2017) but differs from the planorbid B. - For example, lectins and GNBP receptors are available to detect pathogens and signal through the TLR pathway and cytokines to activate defence re- sponses that encompass cellular (e.g., ROS production) and humoral (antimicrobial proteins) branches of the immune system.. - Purple (immune challenge) and black (exposures to environmental change) rectangles (dashed line), arrows and numbers refer to the specific results mentioned in the text. - Visual examination of the transcription patterns of the annotated immune defence factors suggested different responses both against Gram-negative (E. - Two of the annotated factors (laccase, IκB), how- ever, showed broader responses to various immune elici- tors. - The transcription patterns did not indicate activation of the heat shock proteins at elevated temperature (25 °C vs. - Inter- estingly, transcription of the heat shock proteins was re- duced under food deprivation, which indicates their general role in stress responses and that the resource level of snails could affect their ability to tolerate expos- ure to high temperature. - Such variation may arise, for example, from differences in the genetic background (expressed within and/or among populations) and physiological condition of snails. - The heat map analysis suggested high variation in transcription among snail individuals in several compo- nents of the snail immune system from pathogen recog- nition (GNBP, lectins, TLR), to signalling/regulation. - Second, these components of the snail immune system are expressed at a constant level within each individual, but the actual level varies among individuals [84], perhaps due to differences among alleles that they carry. - Despite high among-individual variation in the tran- scription of many components of the snail immune sys- tem, our data did not reveal any individuals with generally very low or very high immune activity across the annotated factors. - However, individuals within some of the immune activation treatments showed investment in different components of defence. - coli showed high transcription in two to four of the examined six antibacterial defence factors.. - snails exposed to trematode-infected snail tissue ex- tracts, each individual typically showed high transcrip- tion in only one, but not the same of the examined non- self recognition components (GNBP, FREP, galectins, TLR). - Table 1 Summary of the experimental design. - Interestingly, many examined im- mune factors (PRRs, lectins, TLR pathway, cytokines, ROS production, apoptosis, PO enzymes, antibacterial proteins) showed considerable among-individual vari- ation in the genetically diverse experimental snails used in this study. - Snails were fed with fresh lettuce ad libitum, and half of the water in each water bath was changed every second day. - tissues for RNA extractions (i.e., duration of the treat- ments was not the same).. - In the experiment, the snails were randomly assigned to nine experimental treatments (Table 1). - RNA extraction, library preparation and sequencing At the end of the experimental treatments, each snail was removed from its shell, and whole-body soft tissues were submerged in self-made RNAlater solution (see protocol at https://www.protocols.io/view/RNAlater- Recipe-c56y9d) in which they were cut into small pieces.. - Completeness of the produced reference transcriptome was assessed by examining the detection of core BUSCO genes [95, 96]. - Components of the immune system queried included non-self recogni- tion through pathogen-recognition receptors (PRRs. - all abbreviations are explained at the end of the article), Toll-like receptor (TLR) pathway, cytokines, antibacter- ial peptides and proteins, production of reactive oxygen species (ROS), phenoloxidase (PO)/melanisation-type defence, and apoptosis. - From the statistically best Blast hits (E-value ≤0.05), transcripts that were at least 60% of the length of the shortest reference sequence and showed minimum 40%. - ORFs that were at least 60% of the length of the shortest reference. - To examine variation in the expression levels of different transcripts among experimental snails, sequencing reads from each library were first indexed to transcripts in the reference transcriptome using Kallisto v.0.44.0 with 100 bootstraps [98]. - This measure calculates the proportion of counts per base for each transcript in the whole data set (multiplied by million).. - Additional file 1. - Summary of the annotated transcripts linked to proteins with a role in immune defence, stress-responses, or metabolism.. - Additional file 2. - Additionally, the proportion of total variance each PC ex- plained in the data is presented.. - Additional file 3. - Additional file 4. - Additional file 5. - Additional file 6. - Additional file 7. - Additional file 8. - Additional file 9. - Additional file 13 GenBank accession numbers of the sequences of proteins/genes relevant for immune function, stress responses, antioxidation and metabolism that were used as references in BLAST similarity searches to identify orthologs in the L. - OS and CMA examined the variation in transcription and wrote the first draft of the manuscript. - The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - The raw sequencing reads are deposited in the NCBI Sequence Read Archive (accession number PRJNA664475). - 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