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. 2021 Mar 1;22(1):144.
doi: 10.1186/s12864-021-07428-1.

Transcriptome profiling of Lymnaea stagnalis (Gastropoda) for ecoimmunological research

Affiliations

Transcriptome profiling of Lymnaea stagnalis (Gastropoda) for ecoimmunological research

Otto Seppälä et al. BMC Genomics. .

Abstract

Background: Host immune function can contribute to numerous ecological/evolutionary processes. Ecoimmunological studies, however, typically use one/few phenotypic immune assays and thus do not consider the complexity of the immune system. Therefore, "omics" resources that allow quantifying immune activity across multiple pathways are needed for ecoimmunological models. We applied short-read based RNAseq (Illumina NextSeq 500, PE-81) to characterise transcriptome profiles of Lymnaea stagnalis (Gastropoda), a multipurpose model snail species. We used a genetically diverse snail stock and exposed individuals to immune elicitors (injury, bacterial/trematode pathogens) and changes in environmental conditions that can alter immune activity (temperature, food availability).

Results: Immune defence factors identified in the de novo assembly covered elements broadly described in other gastropods. For instance, pathogen-recognition receptors (PRR) and lectins activate Toll-like receptor (TLR) pathway and cytokines that regulate cellular and humoral defences. Surprisingly, only modest diversity of antimicrobial peptides and fibrinogen related proteins were detected when compared with other taxa. Additionally, multiple defence factors that may contribute to the phenotypic immune assays used to quantify antibacterial activity and phenoloxidase (PO)/melanisation-type reaction in this species were found. Experimental treatments revealed factors from non-self recognition (lectins) and signalling (TLR pathway, cytokines) to effectors (e.g., antibacterial proteins, PO enzymes) whose transcription depended on immune stimuli and environmental conditions, as well as components of snail physiology/metabolism that may drive these effects. Interestingly, the transcription of many factors (e.g., PRR, lectins, cytokines, PO enzymes, antibacterial proteins) showed high among-individual variation.

Conclusions: Our results indicate several uniform aspects of gastropod immunity, but also apparent differences between L. stagnalis and some previously examined taxa. Interestingly, in addition to immune defence factors that responded to immune elicitors and changes in environmental conditions, many factors showed high among-individual variation across experimental snails. We propose that such factors are highly important to be included in future ecoimmunological studies because they may be the key determinants of differences in parasite resistance among individuals both within and between natural snail populations.

Keywords: Ecological immunology; Great pond snail; Mollusc.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 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/regulation, effectors). Numbers in brackets show how many transcripts with unique open reading frames (ORFs) were detected from those factors for which determining the completeness of ORFs was possible
Fig. 2
Fig. 2
Principal component analysis (PCA) plot showing variation in transcriptome-wide expression profiles of the experimental snails. The first two principal components (PCs) after internal normalisation in Sleuth are used. 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
Fig. 3
Fig. 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. Examined immunological pathways/mechanisms included non-self recognition, Toll-like receptor (TLR) signaling pathway, cytokines, antibacterial defence, production of reactive oxygen species (ROS), and phenoloxidase (PO)/melanisation-type reaction. Transcripts within each pathway/mechanism are clustered according to their similarity. Heatmap shows the variation for each factor among all experimental snails (each column represents one snail) using its dynamic range in units of transcripts per million (TPM). 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
Fig. 4
Fig. 4
Expression levels of selected transcripts that represent antioxidation (ANOX), stress responses and metabolism. Heatmap shows transcripts that deviated in their transcription between certain experimental treatments and their specific controls. Transcripts within each pathway/mechanism are clustered according to their similarity. Heatmap shows the variation for each factor among all experimental snails (each column represents one snail) using the dynamic range in units of transcripts per million (TPM). Purple (immune challenge) and black (exposures to environmental change) rectangles (dashed line), arrows and numbers refer to the specific results mentioned in the text

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