Comparative Biochemistry and Physiology Part D: Genomics and Proteomics
The transcriptome of the early life history stages of the California Sea Hare Aplysia californica
Introduction
Opisthobranch molluscs of the genus Aplysia, and most especially the California Sea Hare, Aplysia californica, are important model systems in the study of the molecular and cellular basis of neural memory and learning (Kandel, 2001). The utility of this model system derives from a number of factors, most notably the relatively small number and large size of cells in its nervous system, allowing repeatable identification of neurons and direct linkage of these with specific behaviors. Despite the importance of A. californica as a neurological model, genomic and other DNA sequence information has been relatively slow to accumulate for this species. This pace is also surprising given the interesting phylogenetic position of the Aplysia. Sequencing efforts on over 30 bilaterian species in the deuterostomes and ecydosozoa have been undertaken but Aplysia californica will be the first member of the Lophotrochozoa to have its entire genome sequenced (Aplysia Genome Sequencing Project, 2009, Genbank Accession AASC00000000.2 GI:225542573). In advance of the completed annotation of this full genome project, there is other important and interesting DNA sequence information available. The complete mitochondrial genome of A. californica is known (Knudsen et al., 2006), and molecular phylogenies at the order and genus level have been constructed based on mtDNA and other sequences (Medina and Walsh, 2000, Medina et al., 2001, Medina et al., 2005). More recently, two rather comprehensive expressed sequence tag (EST) projects have focused exclusively on the adult neural transcriptome in A. californica (Moroz et al., 2006) and A. kurodai (Lee et al., 2008).
A. californica is a simultaneous hermaphrodite and reproduces by copulation, and the life cycle consists of a fertilized egg, which develops in a capsule in a benthic egg mass, and then hatches into a free-swimming veliger larvae. Veliger larvae metamorphose to a juvenile stage, resembling a miniature adult, which grow and mature to the adult form. This life cycle has been reproduced in captivity (Kriegstein et al., 1974) and thus the model can be bred and manipulated in a number of ways (see Capo et al., 2009, for a recent example). One benefit of this closed life cycle is the ability to obtain cultured individuals from all life stages, and thus there is potential to further employ A. californica as a developmental model. Since there is no published information specifically on the transcriptome of the early developmental stages of this species, we undertook an EST project for these developmental stages in order to possibly identify genes not detected in earlier EST projects and which might be expressed only in the early development stages of these animals. Specifically, we analyzed ESTs from eggs, veliger larvae, metamorphic individuals, juveniles and adults and obtained 5507 quality-filtered ESTs that clustered into 1988 unigenes, which are annotated and deposited into GenBank. In general, these new sequences indicate that the expression of a large number of known genes has likely not yet been archived or reported for A. californica, and this new information likely stems from the fact that different developmental phases and tissues (i.e., non-neural) have been used.
Section snippets
Animals
All developmental stages of A. californica were obtained from the National Resource for Aplysia at the University of Miami, supported by the National Center for Research Resources of the U.S. National Institutes of Health. The developmental phases and the stages within each phase used in this study adhere to the nomenclature of Kriegstein (1977) and were: (1) EGGS consisting of fertilized egg strands collected each day from the date laid through pre-hatching, encompassing all embryonic stages.
Results and discussion
A total of five directional cDNA libraries were constructed, containing expressed sequences from all major phases/stages of the A. californica life cycle. Each library consists of between 300,000 and 500,000 clones with an average insert size between approximately 800 and 1000 base pairs. From these libraries a total of 7971 sequence reads were generated from randomly selected clones, and after culling reads of vector only, or of < 100 bp, or below a phred score of 20, the number of quality ESTs
Acknowledgements
The authors wish to acknowledge NCRR grant P40 RR10294 to MCS, NSF grant (OCE-0215667) to PJW, and a University of Miami Board of Trustees Innovation Fund award to PJW and MCS. AH also thanks the Korein Foundation for their generous donations to the project and Julia Radic for assistance in library construction. PJW is supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada, the Canada Research Chairs Program, and the Canada Foundation for Innovation.
References (28)
- et al.
A conserved Six-Eya cassette acts downstream of Wnt signaling to direct non-myogenic versus myogenic fates in the C. elegans postembryonic development
Dev. Biol.
(2009) - et al.
Larval growth, development, and survival of laboratory-reared Aplysia californica: effects of diet and veliger density
Comp. Biochem. Physiol. C
(2009) - et al.
Complete DNA sequence of the mitochondrial genome of the sea-slug, Aplysia californica, Conservation of the gene order in Euthyneura
Mol. Phylogenet. Evol.
(2006) - et al.
Wnt signaling and the evolution of embryonic posterior development
Curr. Biol.
(2009) - et al.
Molecular systematics of the order Anaspidea based on mitochondrial DNA sequence (12S, 16S and COI)
Mol. Phylogenet. Evol.
(2000) - et al.
Neuronal transcriptome of Aplysia: neuronal compartments and circuitry
Cell
(2006) - et al.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs
Nucleic Acids Res.
(1997) - et al.
dbEST–database for “expressed sequence tags”
Nat. Genet.
(1993) - et al.
Base-calling of automated sequencer traces using phred. II. Error probabilities
Genome Res.
(1998) - et al.
Base-calling of automated sequencer traces using phred. I. Accuracy assessment
Genome Res.
(1998)
Pfam: clans, web tools and services
Nucleic Acids Res.
Full-length messenger RNA sequences greatly improve genome annotation
Genome Biol.
Signaling mechanisms underlying metamorphic transitions in animals
Integr. Comp. Biol.
CAP3: A DNA sequence assembly program
Genome Res.
Cited by (31)
The transcriptome analysis of the whole-body of the gastropod mollusk Limax flavus and screening of putative antimicrobial peptide and protein genes
2020, GenomicsCitation Excerpt :Matches against Lottia gigantean and Crassostrea gigas were limited to about 3.1% and 3.0%, respectively. It is not surprising that most of the top hits match Aplysia sequences, a related gastropod mollusk subject to considerable genomic/transcriptomic annotation, as compared to Lottia and Crassostrea, which have also been deeply annotated but are more phylogenetically distant [34,35,36,37]. In total, 75.8% of the unigenes showed similarity with the sequences from gastropod mollusk.
Description and initial characterization of metatranscriptomic nidovirus-like genomes from the proposed new family Abyssoviridae, and from a sister group to the Coronavirinae, the proposed genus Alphaletovirus
2018, VirologyCitation Excerpt :In this report we describe the discovery and characterization of one of the nidoviruses prototyping a new family along with another putative nidovirus. We used BLAST searches to scan the publicly available transcriptomes and expressed sequence tag libraries available at the US National Center for Biotechnology Information, and revealed two novel nido-like virus sequences from the frog Microhyla fissipes developmental transcriptome (Zhao et al., 2016) and from several transcriptome studies dealing with the marine gastropod Aplysia californica (Fiedler et al., 2010; Heyland et al., 2011; Moroz et al., 2006). We describe the bioinformatics of the new virus-like sequences, and demonstrate that the Aplysia virus-like sequence encodes a functional proteinase, and a translational termination-suppression signal.
Sequencing and de novo assembly of visceral mass transcriptome of the critically endangered land snail Satsuma myomphala: Annotation and SSR discovery
2017, Comparative Biochemistry and Physiology - Part D: Genomics and ProteomicsCitation Excerpt :A. californica is an aquatic gastropod with the most extensive genomic information. In fact, the species was the first mollusc to be sequenced at the genome level and the information has led to rich insights into invertebrate evolution, developmental biology and neurobiology of molluscs (Choi et al., 2010; Fiedler et al., 2010; Heyland et al., 2011). Other top hit species with sufficient genomic resources included the freshwater snail, Lottia gigantea (Simakov et al., 2013) and the oyster, Crassostrea gigas (Zhang et al., 2012).
Transcriptome-wide analysis of the response of the thecosome pteropod Clio pyramidata to short-term CO<inf>2</inf> exposure
2015, Comparative Biochemistry and Physiology - Part D: Genomics and ProteomicsCitation Excerpt :In the context of ocean acidification, these targeted analyses may include the identification of pteropod homologs of genes associated with calcification in other mollusk species. A number of recent papers have focused on the characterization of biomineralization proteins and genes in mollusks (Gardner et al., 2011; Jackson et al., 2010; Joubert et al., 2010; Shi et al., 2012; Zhang and Zhang, 2006; Zhao et al., 2012a), while other studies have increased the number of available molluscan transcriptomes to include pearl oysters (Huang et al., 2012; Zhao et al., 2012a, 2012b), sea hares (Fiedler et al., 2010; Heyland et al., 2011), freshwater snails (Sadamoto et al., 2012), abalone (Franchini et al., 2011; Picone et al., 2015), scallops (Artigaud et al., 2014), mussels (Freer et al., 2014), and clams (Clark et al., 2010; Sleight et al., 2015). These studies confirm that molluscan biomineralization is a complex process with some conserved pathways shared between distantly related animal groups, and other proteins and compounds that are taxonomically restricted (Jackson et al., 2010), as has been shown in more distantly related animal groups (Jackson et al., 2007; Moya et al., 2008, 2012).
Characterization of GdFFD, a D-amino acid-containing neuropeptide that functions as an extrinsic modulator of the Aplysia feeding circuit
2013, Journal of Biological ChemistryCitation Excerpt :Here, we combined MS/MS with off-line multidimensional chromatographic separations to create a screening assay for potential endogenous DAACPs in small volume tissue extracts. To identify candidate DAACPs, we applied bioinformatics as a data mining technique to identify peptides from Aplysia that are similar to known DAACPs; Aplysia is well suited as the model because of the large amount of genomic information available for this animal from the RNAseq transcriptome assembly, the Broad Institute Genomic Database, and others (35, 39, 40). Based on the results of our data mining, we targeted two putative neuropeptides in Aplysia (35), GFFD and YAEFL-amide (YAEFLa), which are structurally similar to two known DAACPs in the land snail Achatina fulica: achatin-I (GdFAD versus GFAD) (9) and fulyal (YdAEFLa versus YAEFLa), respectively (24).