Ion channel profiling of the Lymnaea stagnalis ganglia via transcriptome analysis

Abstract

Background The pond snail Lymnaea stagnalis (L. stagnalis) has been widely used as a model organism in neurobiology, ecotoxicology, and parasitology due to the relative simplicity of its central nervous system (CNS). However, its usefulness is restricted by a limited availability of transcriptome data. While sequence information for the L. stagnalis CNS transcripts has been obtained from EST libraries and a de novo RNA-seq assembly, the quality of these assemblies is limited by a combination of low coverage of EST libraries, the fragmented nature of de novo assemblies, and lack of reference genome. Results In this study, taking advantage of the recent availability of a preliminary L. stagnalis genome, we generated an RNA-seq library from the adult L. stagnalis CNS, using a combination of genome-guided and de novo assembly programs to identify 17,832 protein-coding L. stagnalis transcripts. We combined our library with existing resources to produce a transcript set with greater sequence length, completeness, and diversity than previously available ones. Using our assembly and functional domain analysis, we profiled L. stagnalis CNS transcripts encoding ion channels and ionotropic receptors, which are key proteins for CNS function, and compared their sequences to other vertebrate and invertebrate model organisms. Interestingly, L. stagnalis transcripts encoding numerous putative Ca2+ channels showed the most sequence similarity to those of Mus musculus, Danio rerio, Xenopus tropicalis, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that many calcium channel-related signaling pathways may be evolutionarily conserved. Conclusions Our study provides the most thorough characterization to date of the L. stagnalis transcriptome and provides insights into differences between vertebrates and invertebrates in CNS transcript diversity, according to function and protein class. Furthermore, this study provides a complete characterization of the ion channels of Lymnaea stagnalis, opening new avenues for future research on fundamental neurobiological processes in this model system.

Transcriptome analysis provides genome annotation and expression profiles in the central nervous system ofLymnaea stagnalisat different ages

“…The significant up-regulation of genes encoding these transmitter receptors as well as synaptotagmin, gap junctions, ion channels, FMRFamide and Mollusc insulin-related peptides (Supplemental Table 5 and results) clearly indicates the active engagement of intercellular communication and synaptic plasticity in these adult animals. Transcript regulation of synaptic genes may reflect animal behavioural changes; compared to young, adult and old animals normally exhibit more vigorous and diverse behaviours including reproduction, feeding, locomotion, respiration, and associative learning, for which the abovementioned synaptic machinery components play major roles (Dong et al, 2021;Ha, Kohn, Bobkova, & Moroz, 2006;Hoek et al, 2005;Ito et al, 2012;Kojima et al, 2015;Mersman et al, 2020;Murakami et al, 2013;Yeoman et al, 1994).…”

“…This result was mainly attributed to the lack of sufficient molluscan sequence coverage in the public databases for valid dataset comparison, urging the need for continued genetic studies of L. stagnalis and other gastropods. A very recent effort by Dong et al (Dong et al, 2021) has established an updated L. stagnalis transcriptome of adult CNS by RNA-Seq. However, the above studies have focused on only one developmental time point, predominantly in adults.…”

A combined bioinformatics and LC-MS based approach for the development and benchmarking of a comprehensive database for CNS proteins inLymnaea stagnalis

“…In this investigation, we set out to develop an extensive proteomics database based on the mRNA transcript assemblies from the NCBI Bioproject PRJDB98. A recently published paper by Dong et al 2021 presented a more comprehensive L. stagnalis transcriptomic database but included RNA transcripts from the ring without the buccal ganglia and the identified protein sequences were focused only on different ion channels (Dong et al, 2021). In our approach, we have developed a more general proteomics database that includes proteins involved in several CNS functions such as learning, fundamental decision making, motivational states, encoding hunger states and others.…”

“…To prepare a more comprehensive and representative database for CNS proteins of Lymnaea stagnalis, which will enable us to accurately identify proteins by LC-MS/MS analysis, we have selected the transcriptome dataset of Sadamoto et al, 2012 that is among the available transcriptome datasets (Bouetard et al, 2012;Davison and Blaxter, 2005;Dong et al, 2021;Feng et al, 2009;Sadamoto et al, 2012) published from the NCBI database and contains the transcriptome of the whole CNS including the buccal "learning" ganglia and the central cerebral ring.…”

Transcriptome analysis provides genome annotation and expression profiles in the central nervous system of Lymnaea stagnalis at different ages

“…Despite the importance of L. stagnalis to brain network, behaviour, and development studies, genetic information is mostly limited to the identification and cloning of individual genes. Only in the past decade several groups have tried to study the L. stagnalis transcriptome using expressed sequence tags (EST) [ 28 – 30 ] and RNA sequencing (RNA-Seq) [ 31 , 32 ] . However, all these studies have focused on only one developmental time point, predominantly in adults.…”