De novo transcriptome sequencing of triton shell Charonia lampas sauliae: Identification of genes related to neurotoxins and discovery of genetic markers
Introduction
Charonia lampas is a marine gastropod snail belonging to the family Ranellidae. The species is widely distributed in tropical and semi-tropical areas of the Atlantic, Indian and Pacific Oceans. Charonia lampas sauliae (Reeve, 1844), commonly called trumpet shellfish, is known mainly from Japan, the East China Sea, Korea (south coast and Jeju-do), Philippines, and Taiwan (Seon et al., 2005). Its shell has been exploited to make souvenirs and jewelry. Due to its low growth rate under natural environmental conditions, sustainable production of this species is difficult. Some studies have addressed artificial larval development methods to support the utilization of C. lampas sauliae in aquaculture (Montresor et al., 2012;Horiguchi et al., 2014). In Korea, C. lampas sauliae is the largest triton shell species; it is found in rocky areas with sand and gravel beds of 20–30 m depth outside the intertidal zone. A recent survey by the National Institute of Biological Resources (NIBR) in Incheon, Korea, reported a decline in the number of C. lampas sauliae populations due to illegal collection for ornamental and edible use, as well as changes in the marine environment (NIBR National Institute of Biological Resources, 2014). This species is listed as vulnerable due to high risk of extinction in the wild and is thus protected as an endangered species under regional laws.
In recent years, conservation of biological resources has focused on securing bioinformation, such as genetic sequences of useful marine species, preserving such information, and establishing databases for its access (Kang et al., 2008; Morton, 2012). One type of highly valued and exploited biological resource is the neurotoxins, such as tetrodotoxin (TTX). Due to its function as a sodium channel blocker, TTX has been explored for biomedical applications (Nguyen et al., 2015; Lago et al., 2015). Although TTX-bearing animals are phylogenetically diverse (including annelids, gastropods, crabs, frogs, and newts), the origin and biosynthesis mechanisms of TTX remain unresolved. Biosynthesis of TTX by symbiotic microorganisms associated with TTX-bearing animals is possible (Chau et al., 2011). No evidence to date suggests that TTX synthesis genes are encoded in the genomes of marine gastropods such as C. lampas; rather, it is secreted through an exogenous pathway by symbiotic bacteria (Hanifin, 2010; Jal and Khora, 2015). The exogenous hypothesis was successfully proved in C. sauliae, which accumulates TTX via the food chain through ingestion of toxic starfish (Matsui et al., 1985) and puffers (Noguchi et al., 2006). Moreover, many TTX-containing marine species, such as puffer fish and xanthid crabs, have been found to harbor TTX-producing bacteria (Vibrio sp. and Pseudomonas sp., are the major taxa) within their microbiomes, as determined through chemical analysis and toxicity assays of media inoculated with isolated bacteria (Lago et al., 2015). Analytical studies using high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), and gas chromatography–mass spectrometry (GC–MS) have revealed the origin of TTX from microorganisms inhabiting TTX-producing animals (Kudo et al., 2012; Wang et al., 2010). Therefore, we speculate that most transcripts identified through mRNA sequencing of higher eukaryotes are channel proteins that bind to TTX and that understanding the origin and production of TTX would be best accomplished by focusing on the bacterial genome.
Conotoxins or conopeptides (CTX) are a group of neurotoxic peptides isolated from the venom of marine cone snails. These peptides have been the focus of pharmacological research for decades due to their efficacy as anti-cancer and pain relief agents for various ailments (Dave and Lahiry, 2012; Leonardi et al., 2012; Akondi et al., 2014). In fact, these peptides display selectivity for molecular targets such as ion channels, G protein-coupled receptors (GPCRs), or neurotransmitter receptors (Lewis et al., 2012). Conotoxin diversity, including conotoxin precursors and hormones, can be illustrated based on CTX gene superfamilies, cysteine frameworks or pharmacological families. The pharmacological family classification is based on the receptor target and its interactions, and 26 cysteine framework patterns have been described (Robinson and Norton, 2014). Furthermore, Conoserver (the CTX database) reports 34 CTX gene superfamilies (namely A, B, B2, B3, C, D, E, F, G, H, I1, I2, I3, I4, J, K, L, M, N, O1, O2, O3, P, Q, R, S, T, U, V, Y, con-ikot-ikot, conoCAP, conopressin, conkunitzin, and conodipine) based on the conserved signal sequence. Moreover, the structural and functional diversity within a single CTX gene superfamily can be surprisingly high (Azam and McIntosh, 2009; Jacob and McDougal, 2010). CTX precursors and hormones obtained from the venom gland of the magician's cone, Pionoconus magus, from Japan through transcriptome sequencing were assigned to 53 CTX precursor and hormone superfamilies, wherein a majority of them were classified under M, O1, T, A, O2, and F superfamily (Pardos-Blas et al., 2019). Earlier, venom diversity and differential expression of venom components including CTX were reported for the snail genus Profundiconus (Profundiconus cf. vaubani and Profundiconus neocaledonicus), and these components were classified into 27 gene superfamilies including the H, I1, I2, L, M, O1, P and T superfamilies (Fassio et al., 2019). CTX transcripts across 22 superfamilies have been obtained from three vermivorous cone snails, including Conus caracteristicus (118 CTX), C. generalis (61 CTX), and C. quercinus (48 CTX) (Yao et al., 2019).
In this study, we performed high-throughput transcriptome sequencing, assembly, and annotation of the endangered triton shell C. lampas sauliae to support genetic information cataloguing and informed conservation planning for this species. This study is critical, as no genetic information has been reported for C. lampas sauliae to date, except for a mitochondrial genome assembly (Cho et al., 2017). The present study aimed to identify various ion channel-associated genes that binds to TTX and conotoxin hormones and their precursors in conserved superfamilies for biomedical and evolutionary applications. The findings may accelerate research on the utilization of pharmacologically active substances from marine snails. This is the first report of a comprehensive transcriptome obtained from C. lampas sauliae using the Illumina HiSeq 2500 platform.
Section snippets
Sample collection
Experiments were conducted with C. lampas sauliae, an endangered species in Korea. The sea snails (n=3, 5–8 g body weight) were collected during August 2014 near the Jeju Island coast of South Korea using scuba diving and gill-net fishery techniques. The snails were maintained in laboratory tanks at the Division of Marine Technology, Chonnam National University, Gwangju, South Korea. After transferring the specimens to the laboratory, the shells of C. lampas sauliae were broken and the visceral
Illumina sequencing and de novo assembly
An mRNA-Seq library was constructed after isolation of total RNA from the visceral mass tissue of C. lampas sauliae. The sequencing was conducted on the Illumina HiSeq 2500 platform for transcriptome characterization of this species in order to explore the regulatory mechanisms underlying conotoxin synthesis. Sequencing of the C. lampas sauliae visceral mass transcriptome resulted in a total of 257,693,556 raw read sequences (32,469,388,056 bases). The raw read sequences were pre-processed,
Discussion
Transcriptome analysis of non-model species using the Illumina HiSeq/MiSeq short-read platform has attracted considerable attention over the last decade (Mazumdar and Chattopadhyay, 2015; Patnaik et al., 2015; Al-Qurainy et al., 2019). Most of these non-model organisms have been either species of commercial interest (González-Castellano et al., 2019; Huang et al., 2019; Lin et al., 2019) or those that are protected by law due to their endangered status (Vidotto et al., 2013; Li et al., 2018;
Conclusion
This study describes the transcriptomic characterization of the marine gastropod species C. lampas sauliae, which is protected under South Korean law as an endangered species. The datasets generated here will contribute to transcriptomic research in marine invertebrates at risk of extinction. The screening of toxins allows elucidating of the mechanisms related to invertebrate feeding. The presence of high value-added toxins in these species indicates that they may have future applications and
Acknowledgements (or funding information)
This work was supported by the Soonchunhyang University Research Fund and the Basic Science Research Program of the National Research Foundation of South Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (NRF-2017-R1D1A3B06034971).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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