Transcriptome profiling of Lymnaea stagnalis (Gastropoda) for ecoimmunological research

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.

Examining adaptive evolution of immune activity: opportunities provided by gastropods in the age of ‘omics’

“…To enable such work at the gene expression level, L. stagnalis has recently been subjected to extensive transcriptome sequencing [109]. That work has provided a broad picture of the immune system of this species and identified multiple targets for future ecoimmunological work.…”

“…This approach allowed the identification of components of the immune system that respond to different immune challenges/environmental conditions. For instance, bacterial challenge activated the Toll-like receptor (TLR) signalling pathway, signalling through cytokines, antibacterial defences through cytolytic β pore-forming toxins and melanisation-type reaction [109]. Similarly, exposure to protein extracts from trematode parasites increased the gene expression of some components of the TLR signalling pathway and melanisation-type reaction.…”

“…Similarly, exposure to protein extracts from trematode parasites increased the gene expression of some components of the TLR signalling pathway and melanisation-type reaction. Additionally, apart from immune challenges, altered temperature and resource availability modified the expression levels of cytokines and effectors contributing to antibacterial defence [109]. These findings indicate a potentially important role of these components in the snail immune system against parasites and pathogens, as well as in determining context-dependence of immune activity.…”

“…Detecting variation in transcription that arises through causes such as genetic background and/or physiological condition of individuals is, however, easily overlooked in typical RNA-seq studies that aim to expose study organisms that are as genetically homogeneous as possible to highly controlled experimental treatments. To be able to detect such among-individual variation in immune activity, L. stagnalis transcriptomes [109] were specifically sequenced using a genetically diverse laboratory population of snails (see [106]). Interestingly, the results indicated high among-individual variation in the transcription of many components of the snail immune system, including non-self recognition, signalling through TLR pathway and cytokines, components of the production of reactive oxygen species (ROS), factors regulating apoptosis and effectors representing antibacterial defence and melanisation-type reaction [109].…”

“…To be able to detect such among-individual variation in immune activity, L. stagnalis transcriptomes [109] were specifically sequenced using a genetically diverse laboratory population of snails (see [106]). Interestingly, the results indicated high among-individual variation in the transcription of many components of the snail immune system, including non-self recognition, signalling through TLR pathway and cytokines, components of the production of reactive oxygen species (ROS), factors regulating apoptosis and effectors representing antibacterial defence and melanisation-type reaction [109]. In addition to immunological mechanisms that showed clear responses to immune challenges (see the previous paragraph), immune factors with high among-individual variation in transcription should be included in future ecoimmunological studies on this species.…”