Integrative and Comparative Biology Advance Access originally published online on May 17, 2006
Integrative and Comparative Biology 2006 46(6):912-918; doi:10.1093/icb/icl002
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Gene discovery in Carcinus maenas and Homarus americanus via expressed sequence tags
Center for Marine Functional Genomics, Mount Desert Island Biological Laboratory Salsbury Cove, Maine 04672, USA
Correspondence: 1E-mail: dtowle{at}mdibl.org
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Synopsis |
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Expressed sequence tags (ESTs) were produced for normalized cDNA libraries prepared from several tissues of 2 marine crustaceans, the green shore crab Carcinus maenas and the American lobster Homarus americanus. Tissues represented in the Carcinus library were anterior and posterior gills, hypodermis, heart, hepatopancreas, antennal gland, brain, testis, and skeletal muscle, obtained from animals acclimated to 35 and 10
salinity. Tissues represented in the Homarus library were gill, epipodite, branchiostegite, heart, ovary, testis, antennal gland, skeletal muscle, hepatopancreas, and brain, obtained from intermolt and post-molt animals. Directional libraries from oligo-dT-primed cDNA were constructed in the pCMVsport6.1 vector and normalized by self-subtraction at 2 different Cot values. Randomly picked clones were single-pass sequenced from the 5' end. Raw sequence data were trimmed and prepared for submission to dbEST using phred, cross-match, and blastx protocols embedded within trace2dbest software. A total of 4604 Homarus and 12 401 Carcinus ESTs produced 540 and 2651 clusters, respectively, as determined by TIGR Gene Index Clustering software. Gene Ontology analysis with reference to a Drosophila melanogaster database using GOblet revealed 187 positive hits (35%) with the Homarus clusters and 1037 positive hits (39%) with the Carcinus clusters. Combining the number of assembled sequences with the number of singlets obtained after cluster analysis suggested that transcripts representing as much as 25% of the total number of genes in Carcinus have been isolated. |
Introduction |
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The green shore crab Carcinus maenas is a highly invasive portunid crab, originating in coastal regions of northern Europe and now occupying marine and estuarine habitats around the globe (Carlton and Cohen 2003
). Nominated as 1 of the "World's 100 Worst Invaders" by the IUCN Species Survival Commission Invasive Species Specialist Group, the green shore crab is capable of disrupting coastal ecosystems as a result of its aggressive behavior as a predator (Grosholz and Ruiz 1996). For example, recent studies have suggested that C. maenas may compete with juvenile and sub-adult lobsters for limited food supplies, negatively impacting lobster growth and survival (Rossong and others 2006; Williams and others 2006). A sister species, Carcinus aestuarii, with its origin in the Mediterranean and Adriatic Seas, has also invaded distant ecosystems (Geller and others 1997).
Clues regarding the adaptability of Carcinus species have come from physiological studies that detail their capacity to tolerate wide ranges of salinity (Siebers and others 1982; Lucu 1990; Henry and others 2003), oxygen concentration (Truchot 1975; Johnson and Rees 1988; Zinebi and others 1990; Lucu and Pavicic 1995), temperature (Aagaard 1996; Queiroga and others 1997), and ammonia levels (Weihrauch and others 1998; Weihrauch and others 2002).
The American lobster Homarus americanus, in addition to providing an important commercial fishery, is the subject of many studies in neurobiology, developmental biology, behavioral biology, and physiology (Factor 1995). The lobster fishery in the United States was valued dockside at 
$375 million in 2004, with 76% of the catch obtained by Maine lobstermen (Diaby 2004). In southern New England, the lobster fishery has declined over the last decade, possibly in part due to increasing problems with disease (Floreto and others 2000) and pesticide toxicity (De Guise and others 2004).
Prior to the expressed sequence tag (EST) study described here, <70 nucleotide sequences from C. maenas and 100 sequences from H. americanus were known, making gene-based studies of their physiology and development difficult and time-consuming. The goal of the EST project described here was to alleviate, at least in part, this paucity of nucleotide sequence data, facilitating gene discovery and gene expression analysis in these 2 genera.
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cDNA library construction and normalization |
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Source tissues were selected to provide a diverse collection of transcripts representing a broad range of tissue function and physiological state. To prepare material for the Carcinus library, crabs were either maintained in 35
seawater or transferred to 10 for 1 week. Anterior and posterior gills were sampled from both groups, in addition to hypodermis, heart, hepatopancreas, antennal gland, brain, testis, and skeletal muscle. Tissues were stored overnight in RNAlater (Ambion) at 4°C then kept at –20°C for a maximum of 2 weeks prior to RNA extraction.
To prepare material for the Homarus library, 1 male and 1 female intermolt lobster were transferred from 35 seawater to 20 18 h prior to tissue removal to induce salinity-sensitive transcripts. To include molt-related and calcifying transcripts, tissues from 1 male and 1 female post-molt (soft-shell) lobster were sampled as well. From each animal, 0.12 g of individual tissues were obtained, including gill, epipodite, branchiostegite, heart, ovary or testis, antennal gland, skeletal muscle, hepatopancreas, and brain.
Total RNA extracts were prepared according to Chomczynski and Sacchi (1987), using materials supplied in the RNAgents Total RNA Isolation System (Promega). Total RNA samples were analyzed by microfluidic electrophoresis in an Agilent 2100 Bioanalyzer. Electropherograms of total RNA from Carcinus tissues revealed 3 sharp peaks of ribosomal RNA, the 28S rRNA appearing as 2 discrete fragments typical of crustacean rRNA (Skinner 1968) (Fig. 1A). In electropherograms of total RNA from Homarus tissues, 1 of the 28S fragments apparently overlaps the 18S peak, giving 2 sharp peaks (Fig. 1B). All tissue RNA preparations used for library construction showed no indication of degradation. For the Carcinus library, 100 µg of each tissue RNA preparation were combined into one pool, and for the Homarus library, 175 µg.
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The Carcinus and Homarus multiple-tissue RNA preparations were submitted to Invitrogen where reverse transcription of mRNA transcripts was carried out by oligo-dT priming. Normalization of the cDNA libraries was accomplished by subtractive hybridization at 2 different Cot values (15 and 7.5) using Invitrogen's proprietary technology. Normalization reduced the abundance of a highly expressed gene by 400-fold for the Carcinus library and 53-fold for the Homarus library (Table 1), thus enriching the libraries in rare transcripts. The cDNAs remaining after normalization were directionally ligated into the pCMV Sport 6.1 vector and electroporated into competent DH10B tonA Escherichia coli, yielding 6.6 x 107 colony forming units (cfu) for the Carcinus library and 2.5 x 106 cfu for the Homarus library (Table 1).
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Sequencing and EST submission |
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An aliquot of each library was diluted with 2x LB broth, spread on LB agar plus carbenicillin (100 mg l–1), and incubated at 37°C overnight. Using a QPix2 robot (Genetix), colonies were picked at random into 96-well plates containing 100 µl 2x LB broth, 8% glycerol, and carbenicillin (100 mg l–1). After overnight incubation at 35°C, deep-well plates containing 1 ml 2x LB broth and carbenicillin (100 mg l–1) were inoculated from the original plates that were then stored at –80°C. Following a 16-h incubation at 37°C, plasmids were isolated from the 1-ml cultures by alkaline lysis using a BioMek 2000 robot (Beckman-Coulter) and Millipore miniprep reagents. Inserts were then single-pass sequenced from the 5' end using SP6 (5'-ATTTAGGTGACACTATAG-3') as primer on an ABI 3100 16-capillary sequencer (Applied Biosystems).
Sequence traces were processed for submission to dbEST using the trace2dbest component of partigene software from the University of Edinburgh (Parkinson and others 2004). This Linux-based software performs base-calling with the phred protocol, removes vector and low quality sequences with cross-match, performs local blastx analysis for putative identification by homology, and prepares records for direct submission to dbEST at NCBI. As of January 23, 2006, 14 880 clones from the Carcinus library have been processed, yielding 12 401 acceptable ESTs (83.3% of the clones sequenced). For the Homarus library, 5568 clones have been sequenced, producing 4604 acceptable ESTs (82.7% of the original total). All of the ESTs are searchable in dbEST, available at http://www.ncbi.nlm.nih.gov, to facilitate discovery of sequences of specific interest to individual investigators. Samples of the bacterial clones underlying these ESTs are available from the authors upon request.
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Clustering and preliminary blast analysis |
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Top
SynopsisIntroductioncDNA library construction and...Sequencing and EST submission Clustering and preliminary blast... Functional annotation of crab...Implications for future genome...REFERENCES |
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The Carcinus and Homarus ESTs were clustered using TIGR Gene Indices Clustering software (Pertea and others 2003
). The 12 401 Carcinus ESTs yielded 2651 contigs with greater than 1 member plus 1145 singlets. The 4604 Homarus ESTs yielded 540 contigs plus 3289 singlets (Table 2). Sequence clusters containing more than 40 ESTs were rare in both libraries while, as expected, clusters of 2 ESTs were the most frequent.
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The most populated cluster in both libraries (45 members in Homarus and 133 in Carcinus) produced no hit upon searching NCBI's non-redundant protein-sequence database with the blastx algorithm (Altschul and others 1997
). However, searching the nucleotide database with the blastn algorithm produced high-scoring matches between the most populated Homarus and Carcinus cluster and mitochondrial 16S rRNA genes (Schubart and others 2000; Giribet and others 2001). Blastx analysis showed that the second-most populated cluster in the Homarus ESTs (16 members) matched the sequence for Homarus cytochrome oxidase subunit 1 (Giribet and others 2001) and in Carcinus (67 members) matched the nuclear transcript encoding myosin 3 light chain of insects (Veiga and others 2005) (Table 3).
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Functional annotation of crab and lobster ESTs |
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All of the resulting contigs and singlets were submitted to blastx analysis using the Tera-BlastP algorithm (translated nucleotide sequence versus protein sequence) on a DeCypher server (TimeLogic), with reference to the non-redundant protein sequence database from NCBI. Positive hits with an E-value <0.0001 were obtained for 44.7% of the Carcinus contigs and 33.9% of the Homarus contigs, and for 47.4% of the Carcinus singlets and 39.3% of the Homarus singlets (Table 4). These results will be made available on a publicly available server at the Mount Desert Island Biological Laboratory (http://www.mdibl.org/~dtowle/DNA/DNAFacility.htm).
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The frequency of positive blastx hits in the range of 30–50% agrees with EST projects for other non-model arthropods. A study of 7210 ESTs from the water flea Daphnia magna produced 2958 clusters representing unique, non-redundant sequences of which 41% showed significant similarity to known nucleotide or protein sequences (Watanabe and others 2005
). Analysis of 14 642 high-quality ESTs from the brain of honeybee Apis mellifera revealed 8912 unique sequences, of which 39% were similar to known sequences in the non-redundant protein database (Whitfield and others 2002). A recent study of 11 761 high-quality ESTs from hypodermis and gill of blue crab Callinectes sapidus produced 2176 unique sequences, of which 55% could be identified by significant homology to known sequences (Coblentz and others 2006).
Gene Ontology (GO) annotation of the assembled sequences was accomplished using GOblet (http://goblet.molgen.mpg.de/) (Hennig and others 2003), employing the highly annotated genome of the arthropod Drosophila melanogaster as the reference (Adams and others 2000). Positive GO hits with an E-value <1e–10 were obtained for 39.1% of the Carcinus contigs and 34.6% of the Homarus contigs, roughly corresponding to the returns for blastx analysis. Assignment of GO terms within the "molecular function" category revealed representatives within each of 12 categories (Table 4), with the most frequent annotations occurring in 3 categories: binding activity (25%), catalytic activity (36–38%), and transporter activity (9–10%).
Because of the long-standing interest of our laboratory in osmoregulatory and transport physiology in marine crustaceans, we examined the distribution of GO terms within the transporter activity category more closely. Among the Carcinus assembled sequences, representatives of 15 transporter categories were identified, including 1 contig tentatively identified as encoding an aquaporin. The smaller number of contigs in the Homarus library resulted in a lower diversity of transporters, with 11 sub-categories represented. The 3 most abundant sub-categories of transporter activity identified in both libraries were the GO terms carrier activity (37–39%), electron transporter activity (9–10%), and ion transporter activity (24–26%) (Fig. 2). Among the latter were several cation transporters that we had identified in earlier experiments using degenerate primers with reverse transcription and the polymerase chain reaction (Towle and others 1997, 2001; Weihrauch and others 2001).
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Of particular interest for future studies of osmoregulatory and transport physiology are a number of newly identified sequences that were made possible by the EST projects at Mount Desert Island Biological Laboratory. These include ATPases, ion channels, and transporters that may play important roles in osmoregulatory adjustments to changing salinity (Table 5). The availability of species-specific sequence data for these transcripts will facilitate detailed studies of gene expression and its control.
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Implications for future genome-based studies |
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Top
SynopsisIntroductioncDNA library construction and...Sequencing and EST submissionClustering and preliminary blast...Functional annotation of crab...Implications for future genome...REFERENCES |
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The haploid genome size of C. maenas is estimated at 1.07 pg, the smallest known genome among the decapod crustaceans (Gregory 2001
). Thus C. maenas may serve as a suitable target for whole-genome shotgun sequencing if the necessary informatics resources become available. Among these resources should be an extensive, well-annotated EST library. The total of 3796 putative unique transcripts (assembled sequences plus singlets) presented in this study likely represents 25% or less of the total number of structural genes in the Carcinus genome. Thus, a more complete EST collection will require additional sequencing and analysis using cDNA libraries that may be more complex in their composition. |
Acknowledgements |
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The expressed sequence tag projects at Mount Desert Island Biological Laboratory are supported by the National Center for Research Resources through an IDeA Network of Biomedical Research Excellence grant (NIH P20 RR-016463, P. Hand, PI).
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Footnotes |
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From the symposium "Genomic and Proteomic Approaches in Crustacean Biology" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 4–8, 2006, at Orlando, Florida.
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