Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis

Abstract

Background: The freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model.

Results: In this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes.

Conclusion: This study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at http://www.Lymnaea.org/.

Figure 1

The dissected central ring ganglia of Lymnaea stagnalis. LBuG and RBuG: left and right buccal ganglia; LCeG and RCeG: left and right cerebral ganglia; LPeG and RPeG: left and right pedal ganglia; LPIG and RPIG: left and right pleural ganglia; LPaG and RPaG: left and right parietal ganglia; VG: visceral ganglion. Scale bar: 1 mm.

Figure 2

Overall statistics of the Lymnaea cDNA library.(A) Distribution of the sequence length of the cDNA sequences before and after assembly by the CAP3 program. (B) Distribution of the maximum length of translated peptide sequences.

Figure 3

Distribution of BLAST hits of Lymnaea cDNA sequences to NCBI non-redundant protein (nr) and nucleotide sequence databases (nt). Analyses were limited to cDNA sequences with a length of at least 100 nucleotides (A) and 500 nucleotides (B).

Figure 4

Distribution of BLAST hits of Lymnaea cDNA sequences in other model organisms. The Venn diagram shows the occurrence of BLAST hits in the protein, mRNA and noncoding RNA sequences in worm (C. elegans), fruit fly (D. melanogaster) and human, at two different BLAST e-value cutoffs. (A) e-value < 1e-6, (B) e-value < 0.01.

Figure 5

Protein sequence alignment and phylogenetic tree of clathrin adapter-protein-2 (AP-2) mu-1 subunit. The alignment (A) and the phylogenetic tree (B) were generated by Clustalw. FPS013.CR.J08 is the Lymnaea orthologue identified in this study.

Figure 6

Protein sequence alignment and phylogenetic tree of clathrin adapter-protein-1 (AP-1) theta-1 subunit. The alignment (A) and the phylogenetic tree (B) were generated by Clustalw. FPS0112.CR.F02 is the Lymnaea orthologue identified in this study.

Figure 7

Protein sequence alignment and phylogenetic tree of syntaxin 7. The alignment (A) and the phylogenetic tree (B) were generated by Clustalw. FPS0110.CR.A09 is the Lymnaea orthologue identified in this study.