The transcriptome of the early life history stages of the California Sea Hare Aplysia californica

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Abstract

Aplysia californica is a marine opisthobranch mollusc used as a model organism in neurobiology for cellular analyses of learning and behavior because it possesses a comparatively small number of neurons of large size. The mollusca comprise the second largest animal phylum, yet detailed genetic and genomic information is only recently beginning to accrue. Thus developmental and comparative evolutionary biology as well as biomedical research would benefit from additional information on DNA sequences of Aplysia. Therefore, we have constructed a series of unidirectional cDNA libraries from different life stages of Aplysia. These include whole organisms from the egg, veliger, metamorphic, and juvenile stages as well as adult neural tissue for reference. Individual clones were randomly picked, and high-throughput, single pass sequence analysis was performed to generate 7971 sequences. Of these, there were 5507 quality-filtered ESTs that clustered into 1988 unigenes, which are annotated and deposited into GenBank. A significant number (497) of ESTs did not match existing Aplysia ESTs and are thus potentially novel sequences for Aplysia. GO and KEGG analyses of these novel sequences indicated that a large number were involved in protein binding and translation, consistent with the predominant biosynthetic role in development and the presence of stage-specific protein isoforms.

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)

  • R.D. Finn et al.

    Pfam: clans, web tools and services

    Nucleic Acids Res.

    (2006)
  • B.J. Haas et al.

    Full-length messenger RNA sequences greatly improve genome annotation

    Genome Biol.

    (2002)
  • A. Heyland et al.

    Signaling mechanisms underlying metamorphic transitions in animals

    Integr. Comp. Biol.

    (2006)
  • X. Huang et al.

    CAP3: A DNA sequence assembly program

    Genome Res.

    (1999)
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