The porcelain crab transcriptome and PCAD, the porcelain crab microarray and sequence database.

BACKGROUND: With the emergence of a completed genome sequence of the freshwater crustacean Daphnia pulex, construction of genomic-scale sequence databases for additional crustacean sequences are important for comparative genomics and annotation. Porcelain crabs, genus Petrolisthes, have been powerful crustacean models for environmental and evolutionary physiology with respect to thermal adaptation and understanding responses of marine organisms to climate change. Here, we present a large-scale EST sequencing and cDNA microarray database project for the porcelain crab Petrolisthes cinctipes. METHODOLOGY/PRINCIPAL FINDINGS: A set of approximately 30K unique sequences (UniSeqs) representing approximately 19K clusters were generated from approximately 98K high quality ESTs from a set of tissue specific non-normalized and mixed-tissue normalized cDNA libraries from the porcelain crab Petrolisthes cinctipes. Homology for each UniSeq was assessed using BLAST, InterProScan, GO and KEGG database searches. Approximately 66% of the UniSeqs had homology in at least one of the databases. All EST and UniSeq sequences along with annotation results and coordinated cDNA microarray datasets have been made publicly accessible at the Porcelain Crab Array Database (PCAD), a feature-enriched version of the Stanford and Longhorn Array Databases. CONCLUSIONS/SIGNIFICANCE: The EST project presented here represents the third largest sequencing effort for any crustacean, and the largest effort for any crab species. Our assembly and clustering results suggest that our porcelain crab EST data set is equally diverse to the much larger EST set generated in the Daphnia pulex genome sequencing project, and thus will be an important resource to the Daphnia research community. Our homology results support the pancrustacea hypothesis and suggest that Malacostraca may be ancestral to Branchiopoda and Hexapoda. Our results also suggest that our cDNA microarrays cover as much of the transcriptome as can reasonably be captured in EST library sequencing approaches, and thus represent a rich resource for studies of environmental genomics.

A cDNA microarray analysis of the response to heat stress in hepatopancreas tissue of the porcelain crab Petrolisthes cinctipes.

Intertidal zone organisms experience thermal stress during periods of low tide, and much work has shown that induction of heat shock proteins and ubiquitination occurs in response to this stress. However, less is known of other cellular pathways that are regulated following thermal stress in these organisms. Here, we used a functional genomics approach to identify genes that were up- and downregulated following heat stress in the intertidal porcelain crab, Petrolisthes cinctipes using custom cDNA microarrays made from 13,824 cloned P. cinctipes ESTs representing 6717 unique consensus sequences. Statistically significant differences in gene expression between heat stressed and control groups were determined with R/maanova. Genes upregulated following heat stress were involved with protein folding, protein degradation, protein synthesis and gluconeogenesis, suggesting that heat stress accelerated protein turnover. Genes downregulated following heat stress were involved with detoxification, oxygen transport, oxidative phosphorylation, and lipid metabolism, suggesting that the animals were avoiding the generation of reactive oxygen species. ESTs matching hypothetical proteins and ESTs that had no GenBank match were also found to have been both upregulated and downregulated following heat stress, suggesting that novel genes may be involved in the heat stress response.

Construction and characterization of EST libraries from the porcelain crab, Petrolisthes cinctipes.

The thermal phenotype of an organism (heat and cold tolerance, thermal range, and thermal plasticity) is an essential feature of how the organism performs across thermal environments and in response to thermal stress. Porcelain crabs are of interest in addressing questions of thermal phenotype because of their high species diversity and the large variation in thermal phenotype among species, as well as the biogeographic patterning of these crabs along environmental stress gradients. We are studying the cellular bases of thermal phenotype and physiological responses to environmental stress using a functional genomics cDNA microarray approach. To do this, we have isolated total RNA from a range of tissues from 1 species of porcelain crab (Petrolisthes cinctipes) exposed to a suite of thermal conditions, and have used this RNA to construct a 13 824-clone EST library. Here, we describe construction, EST sequencing, assembly and clustering, and results of BLASTx homology search for our initial 13 824-clone library. From 12 060 usable ESTs, 6717 consensus sequences were identified, and roughly 50% of these have homology to known proteins. At present, an additional 50 000-75 000-clone library of P. cinctipes ESTs is being generated, with the aim of developing a library with near-complete coverage of the transcriptome. The libraries and sequence information that will be generated as a result of this project should be of value for crustacean biologists working across a broad range of scientific disciplines (for example, physiology, developmental biology, biological rhythms, ecology, fisheries biology), as well as in studies of molecular evolution and phylogeography.