Parasitic and Adult Lamprey
Adult sea lampreys were collected from tributaries to lakes Huron and Michigan by the staff of the U. S. Fish and Wildlife Service (Marquette Biological Station, Marquette, Michigan, USA). Parasitic sea lampreys were captured in Lake Huron attached to fish caught by commercial and recreational anglers and were collected by the staff of U.S. Geological Survey Hammond Bay Biological Station (Millersburg, Michigan). The animals were transported to Michigan State University and held in flow-through tanks supplied with chilled, aerated well water (5°C).
Olfactory epithelium, brain, liver, kidney, gills and gonads were collected for study. Tissues were immediately placed in liquid nitrogen for DNA and RNA preparation. Olfactory organs were dissected to include the accessory olfactory organ that is immediately adjacent and posterior to main olfactory epithelia. Tissues for cryosectioning were embedded in Tissue Tek O.C.T compound (Sakura Finetek, Torrance, CA) after fixation by 4% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.5.
Whole Genome Shotgun assembly of the sea lamprey (Petromyzon marinus) genome was undertaken with sequence data generated by the Washington University Genome Sequencing Center (WUGSC). A total of 18,787,613 sequence reads deposited in the NCBI Trace Archive were downloaded; 382,756 were excluded from the assembly for various reasons. The average read length was 660 nucleotides which yields a total input of 12,147,205,620 nucleotides. Genome coverage is between 5.9 and 9.3X depending on the estimated genome size. 93% of the input reads were paired. The assembly was performed with the Arachne WGS assembly program (v3) written at the Broad Institute and was carried out on the Michigan State University High Performance Computing Center (MSU HPCC) Silicon Graphics Altix 3700 BX2. Total running time for the assembly was 494 hours, 38 minutes (20.6 days). Including computer downtime and repeating select steps of the assembly the total time required was approximately 30 days. To estimate the degree of genome coverage a total of 90,955 ESTs were mapped to the draft 2 assembly with the program BLAT  using stringent alignment parameters (minimum score = 400, minimum identity = 98%). 61,904 ESTs (70%) mapped to this assembly. These ESTs had previously been clustered using stackPACK  which generated 9,649 multisequence consensi from 65,029 ESTs. The other 22,833 remained as singletons. These 32,482 unique sequences were also mapped to the new draft, 19,056 aligned to the assembly; these 19,056 represent 66,738 ESTs (76% of the total).
Prediction of Chemosensory Receptor Genes
Candidate odorant receptor gene sequences were identified in the WGS Petromyzon marinus Draft Assembly (Draft v.2, 2007-02) and the NCBI Trace database http://www.ncbi.nlm.nih.gov/Traces/trace.cgi. A flowchart of the gene identification process is provided [see Additional File 6]. TBLASTN http://www.ncbi.nlm.nih.gov/BLAST searches were used to identify intact olfactory genes in sea lamprey using known olfactory receptor, trace amine-associated receptor, and pheromone receptor amino acid sequences as queries [Additional File 7]. Contiguous sequences producing alignment hits (E-values < 1 × 10-10) were added to a non-redundant list of queries and searching continued in this manner until no new contig hits were found in the assembly. Genes were predicted using GENSCAN http://genes.mit.edu/GENSCAN.html and predicted amino acid sequences (>200 aa) were tentatively identified by batch BLASTP searches against the non-redundant (nr) NCBI Protein database. 7-transmembrane domains were confirmed using Phobius http://phobius.cbr.su.se/ and TMHMM Server v. 2.0 http://www.cbs.dtu.dk/services/TMHMM/. Gene names are composed of supercontig numbers from the genome draft assembly, followed by gene family designation and initial predicted amino acid length.
Predicted lamprey CR genes were included for analysis if they possessed start and stop codons, a complete 7-transmembrane domain, and an open reading frame ≥690 nucleotides. CR-like sequences not meeting these criteria were considered pseudogenes. Sequences identified by BLASTP as G-coupled protein receptors that were not potential CR genes, i.e., GABA-receptors, muscarinic acid receptors, and 5-hydroxytryptamine receptors, were excluded from the analysis.
Multiple amino acid and nucleotide data sets were assembled to evaluate the overall topologies of the predicted gene trees in the context of other lineages with relevant estimated divergence times. BLASTN searches of the Ciona intestinalis v2.0 and Branchiostoma floridaev1.0 draft genomes were performed locally using a representative list of predicted odorant receptors from lamprey in addition to published V1R and V2R from Danio and Rattus. Putative Ciona and Branchiostoma pheromone receptor, TAAR and OR sequences were obtained by TBLASTN and TBLASTX searches of the most recent draft genomes available from the Joint Genome Institute http://genome.jgi-psf.org/euk_cur1.html using lamprey CR queries. Searches were also performed of both databases using teleost, sea urchin, and mammalian pheromone receptor sequences as queries. Searches for potential echinoderm CR gene orthologs were carried out using lamprey CRs as BLAST queries against the Strongylocentrotus purpuratus GLEAN3 gene predictions available at the Baylor College of Medicine Human Genome Sequencing Center http://www.hgsc.bcm.tmc.edu/blast.hgsc.
Predicted protein sequences of Ca++-sensing receptors (CASR) and metabotropic glutamate receptors (MGR) from lamprey were analyzed alongside Danio, Takifugu and higher vertebrate V2R, CASR and MGR amino acid sequences (n = 86) by neighbor-joining to detect possible clustering of V2R orthologs [Additional File 5] [35, 36]. Similar phylogenetic analyses were conducted with lamprey OR and TAAR genes (n = 55, Figure 2), lamprey and vertebrate V1R genes (n = 25, see Additional File 3) and vertebrate OR and TAAR genes with best-hit predicted genes from Stronglyocentrotus (n = 204, see Additional File 2). To provide evolutionary context for the predicted CR repertoire in sea lamprey, functional representatives from nine vertebrate olfactory receptor (OR) families representing both Class I and II OR as described by Niimura and Nei  were also included in this phylogenetic analyses.
Neighbor-joining trees were constructed using MEGA4 [35, 36]. Amino acid sequences were aligned using CLUSTAL V , alignment gaps and missing data were eliminated only in pairwise sequence comparisons, and distances were computed using the JTT protein matrix,.
Preparation of cDNAs for 454 sequencing
cDNA was prepared from pooled olfactory organ RNA from sexually mature female sea lamprey (n = 5) and both male and female parasitic-stage sea lamprey (n = 5, 3 females). Olfactory organ epithelial tissue was flash frozen in liquid nitrogen and stored at -80°C until extraction. Total RNA was extracted using Perfect Pure RNA Tissue Kit (5 Prime, Gaithersburg, MD) according to the manufacturer's protocol. The quality of the extracted RNA was verified by gel electrophoresis and RNA concentration was quantified using a NanoDrop ND-1000 spectrophotometer. 1 ug of total RNA was used as a template for first strand cDNA synthesis using the SMART™ cDNA Synthesis Kit (Clontech Laboratories, Inc.). 13 cycles of LD-PCR was performed to amplify single-strand cDNA using the Advantage® 2 PCR Kit (Clontech Laboratories, Inc.) according to manufacturer's instructions. PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Inc) and concentrated on Millipore YM-30 (MWCO 30,000) columns. 5200 ng of cDNA were submitted to the Research Technology Support Facility at Michigan State University for Roche 454 GS 20 sequencing.
Bioinformatics processing of cDNA reads generated by Roche 454 GS 20
The TIGR SeqClean1 sequence trimming pipeline was used to remove low quality, low complexity, polyA and adapter sequences from the cDNA sequences prior to any analyses. Sequences were aligned to the draft genome assembly (Draft v.2, 2007-02) using BLAT2 and resulting alignments were further filtered using the associated pslSort and pslReps tools. The stringent filtering threshold was set for 95% sequence identity and 90% coverage of the cDNA sequence.
Clustering and consensus assembly of the cDNA sequences was performed using the TIGR Gene Indices clustering tools (TGICL) . Clusters may be generated ab-initio or by using known full or partial cDNA sequences to "seed" the clustering. Two complete 454 runs were performed. After screening for vector, low complexity, low quality sequences, these runs resulted in a total of 373,391 high quality reads with an average read length of 93 nucleotides. This resulted in a total read length of 35,035,388 nucleotides and a total unique length of 10,420,310 nucleotides that were mapped to the assembly. The NJ tree (Figure 2) includes 454 expression data for lamprey OR and TAAR. Table 1 summarizes the available CR expression data.
RT-PCR Amplification of Predicted Odorant Receptor Genes
To analyze tissue-specific expression of CR genes, primers designed from predicted CR gene sequences were used to perform RT-PCR on RNA samples extracted from different sea lamprey tissues (liver, brain, testis, kidney and gill) of adult and parasitic phases. One μg total RNA from each tissue was reverse-transcribed using oligo (dT) primers. The resulting cDNA was subjected to PCR using the gene-specific primers. A partial β-actin sequence was amplified from the same cDNA template as a positive control. The PCR cycling parameters are as follows: 45 sec. at 94°C, 45 sec. at 52–58°C, 45 sec. at 72°C; 35 cycles; and final step: 10 min. at 72°C. PCR products were visualized on a 1% agarose gel by ethidium bromide staining. Results for 6425.OR330 and 16230.TAAR353 are shown in Figure 3. RT-PCR results, primer sequences and annealing temperatures are shown in Table 1.
Synthesis of Digoxigenin- labelled cRNA Probes
Digoxigenin labelled antisense RNA probes were generated from sea lamprey OR clones using the Riboprobe In vitro Transcription System (Promega). Golf mRNAs are expressed specifically in odorant receptors neurons , and because immunoreactive Golf are widely distributed in odorant receptor neurons of both larval and adult sea lampreys , a positive control probe was generated from a Golf cDNA clone kindly provided by Dr. R. Reed (Johns Hopkins University, Baltimore, MD). In brief, 2 μg linearized vector were transcribed in the presence of 700 nmol digoxigenin-11-UTP. The cRNA was collected by ethanol precipitation and resuspended in DEPC water. The sense RNA was prepared by a similar procedure and used as the negative control.
Olfactory rosettes dissected from adult, parasite and larval lamprey and testis dissected from adult sea lamprey were fixed in 4% paraformaldehyde PBS solution for 3 h. Following cryoprotection in 25% sucrose buffer overnight at room temperature, the tissues were embedded in Tissue Tek O.C.T compound and rapidly frozen in -80°C. Cross sections of 12 μm were cut using Leica1850 cryostat at -25°C, adhered to Superfrost plus microslides (Fisher Scientific; Orangeburg, NY) and stored at -80°C.
Tissue sections were brought to room temperature, treated with proteinase K (20 μg/ml in PBS) for 5 min and post fixed for 15 min in 4% paraformaldehyde/PBS solution. Sections were rinsed three times for 10 min. each in PBS before a 2 h incubation in prehybridization solution [50% deionized formamide, 1× Denhart's solution, 750 mM sodium chloride, 25 mM ethylenediaminetetraacetic acid (EDTA), 25 mM piperazine-N,N'-bis (2-ethanesulfonic acid; PIPES), 0.25 mg/ml calf thymus DNA, 0.25 mg/ml Poly A acid and 0.2% sodium dodecyl sulfate (SDS)]. Sections were then hybridized with antisense or sense RNA probe in hybridization solution (prehybridization solution containing 5% dextran sulfate) at 60°C for 16–20 h. After hybridization, sections were washed three times for 10 min each in 2×SSC with 0.3% polyoxyethylenesorbitan monolaurate (Tween-20) followed by three washes in 0.2×SSC with 0.3% Tween-20 at 65°C.
Immunovisualization of Digoxigenin
For detection of digoxigenin-labeled probes, the sections were blocked for 1 h in 4% dry milk, 2% bovine albumin and 0.3% triton. The sections were incubated for 3 h with alkaline phosphatatase-conjugated sheep anti-digoxigenin Fab fragments, 1:1000 in blocking solution (Boehringer Mannheim; Indianapolis, IN). The color was developed with incubation in nitroblue tetrazolium chloride and 5-bromo-4-chloro-3 indolyl phosphate substrate (NBT/BCIP, Boehringer Mannheim) for 20–30 min. Sections were mounted in DPX Mountant.