Equivalent non-normalized cDNA libraries were constructed from a genetically clonal Daphnia isolate sampled from a natural pond along the Oregon coast. The clone was cultured under growing conditions favoring parthenogenetic reproduction. Consequently, the animals were predominantly juvenile females, adult females and brood-carrying females with a small proportion of males.

The strength of conclusions derived from the comparative analysis of expressed gene sequences rests, in large part, on the quality of cDNA libraries. Therefore, we performed quality control tests on 768 randomly chosen cDNA isolates. The cDNA size distribution was determined by agarose gel electrophoresis of PCR amplified inserts. The average molecular weight of inserts sampled from the libraries was 825 bp. To assess the cDNA diversity within the libraries, we sequenced single pass 5' reads from the cDNA inserts. Of the 768 sequence reads, 619 were informative. Only four plasmids were void of inserts and the failed reads (19%) were the result of capillary failures of the sequencer. Following an assembly of the ESTs, unique sequences comprised 68% of the total, reflecting the relative abundances of specific cDNA within the non-normalized libraries. This number diminished to 43% with over twice as much sequencing effort (see below).

To assess potential contamination of cDNA clones derived from prokaryotes and mitochondria, and to measure the distribution of full length ORFs, we aligned the translated sequences to proteins in Genbank. Of the unique sequences, 50% matched Genbank entries with e-values < 1 × 10^-10. A separate query of the NCBI non-redundant protein database identified 204 sequences with e-value scores < 1 × 10^-27. A total of 34 ESTs (6%) were identified as mitochondrial gene transcripts. No ESTs were identified as non-Daphnia. Thus, the cDNA libraries are high quality with a high level of diversity and low levels of contaminant sequences.

To investigate whether the libraries contained full-length or nearly full-length inserts, sequences from 170 clones with high similarity to known proteins (Blastx < 1 × 10^-27) were investigated for the presence of a translational start site. Of these sequences, 109 ESTs (64%) contained unambiguous open reading frames with an annotated ATG translational start site at their 5' end, and 44 ESTs (26%) did not contain an ATG that aligned with the start sites of corresponding database sequences. Of the remaining sequences, 7 ESTs (4%) were likely full-length because gapped alignments of the amino acids suggested poor evolutionary conservation at the N-terminus of the proteins, and 10 ESTs (6%) were unresolved because alignments failed altogether. We therefore estimate that 64–68% of the cDNAs are full-length, or close to full-length. This result may be an overestimate since many conserved genes within our non-normalized libraries encode for ribosomal proteins (34% of 170) which seldom have long transcripts. Indeed, the maximum length of investigated cDNA for open reading frames was < 2 kb, whereas the maximum length of PCR amplified inserts was nearly 3.5 kb. However, when the number of cDNA with and without annotated start sites were compared and sorted by their molecular weights, no association was found between the proportion of full-length transcripts and the size of cDNA, neither by including ribosomal genes (t = 0.39, df = 86; p = 0.70) nor by excluding these genes in the comparison (t = 0.19; df = 60; p = 0.85). A separate investigation of the consistency in our production of full-length or near full-length cDNA was conducted by calculating the proportions of sequences that shared nucleotides within the first 50 bases of the longest EST within contigs (see below for assembly of contigs). Of 233 ESTs forming 81 separate contigs, 202 (87%) shared the first 50 bp of the longest EST from each contig. These data suggest that the majority of the cDNA clones are near full-length.

In total, we produced 5' sequence reads from 1,648 cDNA isolates. In addition to the 768 randomly selected clones, 880 were selected on the basis of their transcription profiles in microarray experiments. After the removal of vector, poly-A tails and poor quality reads (Table 1), 1,546 high quality ESTs with an average size of 540 bp (SD = 188, min = 107, max = 852 bp) remained to be clustered (Genbank accession numbers EE681877-EE683416). The ESTs were assembled into 804 clusters (including 568 singletons) with an average of 1.93 sequences/cluster (SD = 3.95, min = 1, max = 95). After excluding clusters identified as mitochondrial DNA sequences, 787 nuclear genes remained. These non-redundant sequences are hereafter referred to as assembled sequences. We expect that some pairs of assembled sequences will be found to derive from the same locus, either due to excessive polymorphisms between alleles, or because of the alternative use of 5'-exons, or due to sequences from truncated cDNA clones that failed to overlap. However, given the high proportion of estimated full-length clones in the libraries, we anticipate the latter class to be small.

We investigated the proportion of assembled sequences that may be composed of alternative transcripts of the same genes by further clustering these sequences using more relaxed parameters (see methods). Forty-seven additional clusters were discovered. Eleven were composed of 2 assembled sequences that are likely allelic variants of the same genes, based on their matches to a single location in a preliminary draft assembly of the Daphnia genome sequence at the mid-point of the genome sequencing project (4 × coverage; deposited at wFleaBase). These allelic sequences were 84% to 97% similar to each other over a range from 77 to 665 overlapping nucleotides. By contrast, 21 of the additional clusters were of transcripts derived from duplicated genes or from conserved gene families, based on their matches to different locations in the draft genome sequence. In 14 cases, the clusters were composed of 2 sequences. Six clusters consisted of 3 sequences and a single cluster contained 4 similar sequences that shared 86–93% of their nucleotides in pair-wise comparisons. Overall similarities between sequences from closely related genes ranged from 62% to 93% among 220 to 807 overlapping bases. As expected for sequences originating from separate loci, their average similarity (85.5%) was significantly lower than that of allelic sequences (91%)(t = 2.02; df = 44; p = 0.01). Finally, 4 of the additional clusters were composed of paired splice variants from unique loci, while 8 clusters contained from 2 to 4 alternatively spliced transcripts from multiple loci. Therefore, the ESTs from this survey provided sequence tags for up to 787 new Daphnia genes, where some genes represent alternative transcripts or are closely related transcripts from duplicated genes.

Confident over the quality of the D. pulex cDNA libraries and EST sequences, we explored the range of likely biological or biochemical functions of the genes represented by the ESTs sequences by querying the NCBI non-redundant protein databank (NR) using Blastx 21. Of the 787 assembled sequences, 452 (58%) matched at least one known protein with an e-value threshold of 1 × 10^-3 and a minimal value of 33 aligned amino acids (Additional file 1). The distribution of their e-value scores showed that 26% of matched sequences have scores < 1 × 10^-50, while 79% have scores < 1 × 10^-10 (Figure 1a). Therefore, searches for putative homologues in the protein database gave strong to suggestive information regarding possible biological and biochemical functions. As expected, a survey of the distribution of best Blastx matches against the NCBI taxonomic domains showed that the majority (72%) of assembled Daphnia sequences matched best with those derived from other invertebrates (Figure 1b), whereas 25% of the highest scoring hits matched best with vertebrate sequences (including those from rodents, primates and other mammals). The cDNA libraries are free of contaminants, as only 6 assembled sequences (1%) matched bacterial proteins. A closer examination of the distribution of the best Blastx hits within the classes of invertebrates showed that 79% of 323 assembled sequences matched annotated proteins from insects (Figure 1c): 23% from Drosophila, 16% from Anopheles, 15% from Apis, 4% from Bombyx and 21% from other insects. This large insect constituency within the best Blastx matches is clearly a consequence of the limited representation of sequences from Crustacea in the databanks. A survey of the NCBI protein database revealed that out of 6,897,314 archived sequences, only 10,485 (or 0.1%) were from Crustacea. Only 19 assembled sequences (6%) best matched proteins from Branchiopoda, the class that includes Daphnia, while an additional 10 assembled sequences best matched proteins from other classes of Crustacea (Malacostraca, Ostracoda).

The assembled Daphnia sequences that matched annotated proteins from genetic model species were assigned Gene Ontology (GO) terms using Blast2GO 22. Their putative functions spanned a spectrum of biological and biochemical processes (Figure 2a). A total of 227 assembled sequences were assigned 799 biological process terms from the fourth level of the GO. The predominant terms were for metabolic processes ascribed to 190 assembled sequences. These terms included cellular metabolism (22%), primary metabolism (21%), macromolecule metabolism (18%), biosynthesis (10%), biopolymer metabolism (7%), catabolism (5%) and regulation of metabolism (< 1%). From among these processes, 72 assembled sequences were annotated to involve protein biosynthesis, while 30 sequences involved the catabolism of proteins. Sixteen sequences were attributed roles in chitin metabolism, including chitinases and peritrophins. The next most predominant biological process terms were related to the localization of cellular components (establishment of localization, transport, protein localization), which were ascribed to 33 assembled sequences. Four of these sequences coded for genes involved in oxygen transport (hemoglobin). Thirteen of these sequences encoded genes with putative homologues in insects that specifically transport charged atoms like metals, of which seven were also ascribed the functions of cell and ion homeostasis (Figure 2a). Finally, the remaining assembled sequences with GO biological process terms were likely involved in cell communication (such as signal transduction, cell signaling and adhesion), development, and physiological processes that specify a response to external stimuli, stress and cell death.

A total of 288 assembled sequences were additionally assigned 371 GO molecular function terms (Figure 2b). One hundred and thirteen sequences were suggested to have catalytic activities that included hydrolase (17%), transferase (6%), oxidorectuctase (4%), ligase (1%) and isomerase activities, among others. Another 105 assembled sequences were likely involved in structural activities. Their GO terms included structural constituent of the ribosome (18%), structural constituent of the cuticle (9%) and structural constituent of the cytoskeleton (1%). Indeed, Daphnia genes matched to 67 of the total number of 194 listed Drosophila ribosomal components. The next major functional class was represented by 86 sequences putatively involved in binding, which included 27 sequences coding for nucleic acid binding proteins, 18 carbohydrate (also listed as pattern and chitin) binding proteins, 17 nucleotide binding proteins, and 15 proteins that bind ions (calcium, zinc, iron). The remaining 11 sequences within this class were protein or lipid binding. The final major functional class contained 26 assembled sequences assigned to have transporter or carrier activities; twelve sequences were annotated as ion transporters.

A number of assembled sequences likely encode conserved proteins involved in gene regulatory functions. Daphnia genes with such potential functions based on sequence homologies included 19 sequences involved in transcription regulation and 14 sequences with translational regulator activity (Additional file 2). Examples of regulatory genes involved in arthropod development included a putative homologue to maf-S, which is a basic-leucine zipper (bZIP) transcription factor in Drosophila that is required for the development of pharyngeal structures 23. A Daphnia sequence also matched closely to the Dorsal switch protein 1 (Dsp1) gene that regulates a number of homeotic genes in Drosophila 24 and is therefore involved in many developmental pathways. The putative homologue to the fly gene shaggy (sgg) was identified, which is part of the Notch, Wnt and Smoothened signaling pathways. Interestingly, two other regulators of the Notch signaling pathway called Cdc42 25 and neurotic (nti or O-fut1) were also identified (Additional file 3). The fly gene nti is specifically required for the proper localization of Notch at the cell surface 26, is essential for the physical interaction of Notch with its ligand Delta, and is an essential component for neurogenesis 27. A second gene involved in neurogenesis was identified as homologous to similar to Deadpan (Side), which is a basic helix-loop-helix (bHLH) transcription factor. A Daphnia gene was also matched with the fly zinc-finger-C4 transcription factor (ZnFC4) called ftz transcription factor 1 (ftz-f1), which coordinates stage-specific responses to the steroid hormone ecdysone during metamorphosis 28 and directs key developmental events at the transition between prepupal and pupal stages of Drosophila development 29. The putative functions for the other identified transcription regulators (Additional file 2) included the regulation of mitotic progression (the TFIIH transcription factor Cdk7) and important roles during gametogenesis (Rab11, bic, and the C2H2-zinc finger transcription factor Meics). Other putative transcription factors included genes matching CG1876I that contains a helix-turn-helix (HTH) DNA-binding motif, CG18619 that contains a bZIP DNA-binding motif and CG3224 that contains a putative zinc-finger DNA-binding motif. Finally, among the regulators of translation (Additional file 2), Daphnia genes matched to 7 of the total number of 24 listed Drosophila translational elongation genes, yet matched only to 2 of the 58 listed translational initiation genes in flies. Their putative functions include DNA repair (RpLP0), autophagic cell death (eIF-5A, Ef1gamma), immune response (RpS6, Thor), regulation of cell growth (Thor) and germ-line stem cell division (piwi).

To discover Daphnia genes that may jointly participate in conserved biological processes or within gene interaction networks, we investigated GO classes that were highly represented within our list of putative homologues to fly proteins. Significant functional groupings of Daphnia genes were expected, because more than half of the sequenced ESTs were chosen based on their differential expression patterns in separate microarray experiments examining developmental differences among males and females, juveniles and adults, and toxicological responses to metals (Eads et al. submitted; Shaw et al. submitted). Seventeen genes were identified as candidates for gametogenesis (Additional file 4); the majority of these loci are involved in oocyte development in flies. Daphnia loci matching the fly genes mago, Rab11, chic, Tm1, tsu and bic may play conserved evolutionary roles in specifying the anterior-posterior axis of the oocyte. All 6 genes save bicaudal (bic) coordinate to assemble the pole plasm at the posterior end of the Drosophila oocyte by localizing maternally derived transcripts for oskar. Moreover, mago, Tm1 and Rab11 are known to interact in genetic screens 30, while a two-hybrid-based fly protein interaction study 31 implicates tsu and possibly a translational elongation RpLP1 homologue (Additional file 3) within this network. One other Daphnia gene with weak sequence similarity to Sop2 may have transport functions during oogenesis 32 and two genes similar to the signaling gene Cdc42 and to tsr were respectively identified, which are involved in follicle cell development 3334. Other genes similar to snf, RpS3A and sgg in flies are also candidate for oogenesis. Only two genes from our survey have known homologues in flies that function in spermatogenesis. The gene Act5C has a role in sperm individualization 35 and Meics is associated with central spindle and mid-body microtubules during meiosis 36. The chic gene in flies is important in gametogenesis for both sexes 37. These Daphnia genes sharing sequence similarities with Drosophila loci, which are known to coordinate conserved developmental processes, are prime candidates for functional investigations of early crustacean development.

In contrast to genes that participate in biological processes that are shared between Crustacea and Insecta, gene families that have expanded in Daphnia compared to insects may be indicative of new gene functions linked to their specific biology and ecological setting. Therefore, we identified GO classes that were overrepresented within our dataset compared to the Gene Ontologies for the D. melanogaster proteome using Fisher's exact test, and counted multiple assembled sequences that matched to unique Drosophila proteins (Additional file 1). Some lineage expansions seemed to occur primarily by tandem duplication, while other radiations implied interesting functional specializations or innovations. Among the 53 Daphnia sequences that were provisionally annotated as cuticle proteins and genes involved in chitin metabolism and molting, only 13 singularly matched to a fly gene (Additional file 5). In most cases, 2–4 assembled sequences matched to the same protein in the fly genome. Yet in another case, 15 sequences matched with the D. melanogaster gene CG6305, which contains an insect cuticle protein domain. Because we could not produce a reliable sequence alignment for all 15 assembled sequences, we conclude that the observed gene expansion was not an artificial result from inadequate clustering of redundant ESTs. Yet, from the pairwise comparisons of these 15 sequences, alternative splice variants for three genes were identified: Contigs 20 and 180 showed >90% sequence similarity, Contigs 23, 241 and 257 were >94% similar, and Contigs 19 and 24 were >85% identical over shared exons. Additional cDNA sequence data aligned to a completed genome sequence assembly for Daphnia is needed to confirm that cuticle proteins are expanded gene families compared to insects. However, our study also uncovered clearer examples of gene expansions.

Unlike insects, which have three ferritin genes that play important roles in iron homeostasis of cells (Fer1HCH, Fer2LCH) and of organelles (Fer3HCH), the annotation of Daphnia sequences revealed seven assembled sequences with strong matches to Drosophila ferritin proteins (Table 2). Singlet 73 showed a strong match to the Drosophila Fer1HCH protein via a Blastx alignment (bit score = 137), but was poorly matched to crustacean sequences, even to a D. pulex ferritin sequence within Genbank (AJ245734; bit score = 71). The remaining six Daphnia sequences, plus the Genbank entry, all aligned best to other crustacean sequences and to the single Fer3HCH locus of Drosophila. Therefore, Singlet 73 represents the first sequence of an orthologous crustacean Fer1HCH gene. This result was verified by constructing a phylogeny using representative insect and crustacean protein sequences and by including additional Daphnia ferritin-like sequences that were extracted from an ongoing D. pulex cDNA sequencing project (Colbourne et al. in preparation).

The Neighbor-Joining tree of 35 aligned amino acid sequences clustered the pancrustacean ferritins into three main groups (Figure 3). Ferritin 1 contained insect genes plus two cDNA from Daphnia libraries; the Singlet 73 amino acid sequence was identical to a sequence extracted from other cDNA libraries (branch G, Figure 3), while branch F was a unique gene that stemmed at the base of the group. The ferritin 2 group was solely composed of insect genes. However, both crustacean and insect sequences clustered into the third group containing the insect ferritin 3 genes. Although this group contained single copies of the insect genes, the Daphnia genes were further subdivided among five branches representing distinct ferritin 3 loci within the D. pulex genome. Branches D and E were gene sequences derived from the other libraries and showed 45% sequence divergence from each other, while branches A, B, C each contained at least one sequence from this present study. Further investigations indicated that the multiple sequences clustering within branches A, B and C are different alleles of the same locus. Given that insects and more distant outgroups have only three ferritin genes, Daphnia ferritins clearly expanded to include possibly one additional ferritin 1 locus and minimally four additional ferritin 3 genes.

To examine the conservation of genes represented by the EST sequences across Pancrustacea, we used tBlastx to match the Daphnia assembled sequences to the NCBI UniGene sets for Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Apis mellifera and included Caenorhabditis elegans for an outgroup. The assembled sequences were clustered into 27 groups, based on the strength of these sequence matches across these taxonomic data sets. This arrangement identified a variety of gene classes that share patterns of sequence conservation (Figure 4). The first class of interest was composed of 124 genes (16%) that are conserved equally among all species included in this study. This class was mostly enriched by genes that participate in protein metabolism including protein modifications (67 genes to GO:0044267; p = 4.8 × 10^-7) plus 32 genes that were likely involved in cellular metabolism (total of 99 genes to GO:0044237; p = 3.2 × 10^-8). Other genes enriched within this class included transcriptional regulators (12 genes to GO:0045449; p = 2.4 × 10^-3). The second class of interest was composed of Daphnia sequences that matched a nematode protein plus at least one insect locus (183 genes) and others that had no matches to nematode proteins yet matched to at least one insect proteome (167 genes). Therefore, 21% of the sequences were derived within the Pancrustacea – thus shared by Daphnia plus at least one insect in our set – or lost within the nematode. Especially noticeable were 43 assembled sequences that had no detectable homologues in worms and were uniformly conserved across the four insects (Figure 4). Genes that were absent in nematodes were enriched with structural constituents of the cuticle (26 genes to GO:0042302; p = 1.1 × 10^-12) and loci having serine-type endopeptidase activity (10 genes to GO:0004252; p = 3.6 × 10^-4), of which 8 genes were annotated as also having chymotrypsin activity (GO:0004263; p = 0.003). The third class of interest consisted of 309 genes (39%) that had no matches to insect proteomes. At face value, this result suggests that these orphaned genes are unique to Daphnia or Crustacea; either they have been lost in the insects – as suggested by 17 Daphnia genes (2%) showing sequence similarity to proteins in the nematode database – or acquired by Daphnia or Crustacea since diverging from their last common ancestor.

There are many potential sources of errors that can inflate our estimate of the fraction of unique Daphnia genes compared to the selected pancrustaceans. For instance, sequences may fail to align for technical reasons. This may occur if the Daphnia sequences included untranslated regions (UTR) of the cDNA and not the coding regions. Indeed, the mean size of predicted open reading frames (ORFs) within this class differed significantly from that of genes having sequence matches to insect proteomes (t = 12; p < 0.0001; df = 785). For example, over half of the assembled sequences with no matches had ORFs smaller than 225 bases compared to 16% of matched sequences (Figure 5). Therefore, the trivial explanation that these sequences were mostly UTR cannot be dismissed for a large fraction of these genes. Other technical explanations for the absence of matches include genes that had not been annotated or included in the insect UniGene sets. Further analysis by aligning the non-matching sequences to all predicted Drosophila gene translations uncovered 9 additional matches with e-values ranging from 4 × 10^-3 to 2 × 10^-10. Another 4 Daphnia sequences were found to have matches to Drosophila proteins, based on tBlastx searches against the full genome sequence (e-values ranging from 4 × 10^-3 to 9 × 10^-29). Finally, given the tremendous evolutionary divergence between Daphnia and insects, matches may not have been detected from loci that are not under similar evolutionary constraints. We are unable to investigate this last point with the current data.

It was previously shown that gene preservation is correlated with gene function. In particular, correlations have been found between the level of gene conservation and sex-biased gene expression among insects 3839. There is reason to believe that such correlations are extended to other biological functions. In comparing 787 Daphnia assembled sequences to those of insects, 39% of the genes were characterized as orphans because no sequence matches were detected. Interestingly, the orphan genes were not randomly distributed among the gene expression classes. Three specific observations were made by incorporating the gene expression datasets (Table 3). First, 47% of male-biased genes did not match insect proteins compared to only 22% of female-biased genes. The two fold difference in sequence similarity among the sex-biased genes in Daphnia is consistent with differences seen among the insects, reflecting the overall accelerated evolution of male reproductive genes 40. Second, 46% of metal responsive genes did not match insect proteins compared to only 34% of non metal responsive genes. Third, genes that were responsive to metals and not sex-biased included the greatest proportion of orphans (50%), whereas genes that were female biased and not responsive to metals included the fewest (12%). These results suggest that lineage specific genes are correlated with certain biological functions associated with an organism's ecological challenges.

In this study, we characterize two non-normalized cDNA libraries from a clonal population reared under standard laboratory conditions. As a result, the diversity of biological processes and molecular functions represented among the sequenced Daphnia genes is relatively modest. Almost one quarter of the genes are likely involved in metabolic processes. More than one quarter of the genes are predicted to have catalytic or structural activities. Although a large fraction of arthropod genomes is composed of genes having these basic cellular functions (35–40% of Drosophila genes for instance), the diversity of transcribed genes discovered in Daphnia would be augmented by creating libraries from animals under a variety of environmental conditions. Yet, our libraries do contain cDNA from daphniids of mixed life stages, including gravid females, embryos, juveniles and a small number of males. Therefore, some Daphnia genes have sequence similarity to insect proteins associated with reproduction, development and growth. These include regulatory genes like Dsp1, which operates in patterning the developing fly embryo by acting as a corepressor of the transcriptional regulator Dorsal protein 47. Under different circumstances, Dsp1 can also act as an activator or repressor of thorax-group and polycomb-group homeotic genes in Drosophila 24. Because all known homeotic targets of Dsp1 are conserved in sequence and function in metazoans, Daphnia's putative orthologue is likely to share this regulatory function. As expected, this gene transcript is enriched in pregnant females compared to males in Daphnia microarray experiments (Additional file 2). Putative homologues to three regulatory genes (sgg, Cdc42, O-fut1) within the Notch signaling pathway are identified, which is one of a small number of signal transduction pathways that are highly conserved in insects and throughout animal evolution. The gene sgg is a point of convergence between the Notch and Wnt/wingless signaling pathways 48. We predict that further sequencing of Daphnia cDNA will uncover more genes operating within these and other conserved signaling mechanisms: nuclear receptors, Sonic Hedgehog, receptor tyrosine kinases, JAK/STAT, and BMP/TGF-beta. An example is a homologue to the ftz-f1 nuclear hormone receptor that is also found on the microarray. Like all arthropods, Daphnia growth is synchronized with molting and the regeneration of the cuticle, which is governed by pulses of ecdysteroid hormones. Although one isoform of ftz-f1 is a transcriptional regulator of the embryonic segmentation gene fushi tarazu, a second isoform is necessary for larval molting in Drosophila and its premature expression results in the disruption of the epicuticle, suggesting that targets for this transcription factor in flies include genes involved in cuticle formation 49. The function of this gene is conserved in Caenorhabditis, where it is also required for epidermal development and regulates molting 50. No differential expression is observed on the microarray for the signaling genes discussed above (Additional file 3). Overall, our survey of Daphnia ESTs uncovers genes expected to be present in crustacean genomes based on their important regulatory roles in conserved cellular and developmental processes.

Roughly half of the cDNA isolates that are sequenced for this study are chosen based on their differential expression patterns between males and females and on their responses to toxic metals. These experimental conditions reflect two research interests of our labs involving Daphnia: the genetic basis of environmental sex determination and cyclical parthogenesis, and understanding how populations adapt to environmental change in aquatic habitats including industrial pollutants. Therefore, we expect that the provisional annotations of 787 assembled sequences include a fraction of genes sharing functional attributes that would in part be shared with other arthropods and others that are more reflective of Daphnia's unique biology. Certainly, 17 genes are candidates for gametogenesis. The majority of these genes play significant roles in oogenesis. In particular, six sequences have strong matches to conserved genes that specify the oocyte polarity and four loci are known to genetically interact in flies. By contrast, only three of these 17 Daphnia sequences match genes that are known to function during spermatogenesis in Drosophila. This discrepancy between the numbers of sex specific transcripts for genes involved in reproduction is likely caused by the small representation of males within the Daphnia cultures used to create the cDNA libraries. Equally impressive are the large number of cuticle proteins identified.

In the comparative study of the Anopheles and Drosophila proteomes 51, cuticular proteins were noted to be particularly active in their lineage specific expansions and deletions. This present study identifies 53 sequences that are either structural components of the cuticle or involved in chitin metabolism. Their abundance within our dataset is a consequence of their transcriptional responses to the microarray experiments; all but four of the assembled sequences were differentially regulated on the arrays. Yet as noted in the comparisons between the two dipteran insects, many Daphnia sequences share similarities to single loci within insect genomes. Among these cuticle loci are 15 assembled sequences that are best aligned to a single Drosophila gene when compared to the rest of the fly proteome. Alternative transcripts account for only 4 sequences. Thus, no less than 11 loci remain as possible representatives of a large lineage specific gene expansion of Daphnia cuticle genes. Further investigations are obviously required to verify this finding, including more thorough sampling of the Daphnia transcriptome and functional data such as in situ hybridizations to support the notion that these genes may have contributed to biological innovations. Regarding the 15 transcripts on the current array, all but one is enriched in males compared to females, and with the addition of differential expression patterns under metal stress, these transcripts can be grouped into five separate expression profiles.

A first compelling case for lineage specific gene expansion is made by investigating the diversity of ferritin genes within the Daphnia ESTs. Except for Aedes aegypti, their number of ferritin genes are evolutionarily conserved 52. Ferritins are the principle iron storage proteins for nearly all animals, and their abundance within cells is controlled in part by iron-regulatory proteins that interact with iron-regulatory elements (IREs) within alternatively spliced 5' UTRs of certain mRNAs 5354. In insects, ferritins consist of a heavy-chain homolog (HCH) and a light-chain homolog (LCH) forming heterodimers that function in the secretory pathways of cells, and which also appear to act as iron transporters 52. The genes encoding the subunits (Fer1HCH, Fer2LCH) are positioned in the Drosophila genome in a back-to-back orientation, enabling coordinated regulation of their transcription 55. This feature is conserved in all insects studied thus far 52. Except in Bombyx, which has IREs within the UTRs of both subunits, insect IREs are predominantly localized to the Fer1HCH locus. Recently, a third Drosophila ferritin (Fer3HCH) has been described that controls iron homeostasis of the mitochondria, yet its transcription is not responsive to iron treatment 56. As in humans and mice, the gene is predominantly expressed in adult testis. Our phylogeny of Daphnia ferritin gene transcripts uncovers six or seven distinct Daphnia loci (Figure 3). The branch F locus cannot be unequivocally included as part of the ferritin expansion until a description of the gene is available based on its alignment to the genome sequence. However, all of the other loci are defined based on their sequence alignments to distinct genome scaffolds assembled at this point in the Daphnia genome sequencing project (not shown).

Branch G of the ferritin phylogeny represents the first characterized crustacean orthologue to the insect Fer1HCH genes. Following naming conventions, we designate this gene as Dpu_Fer1HCH. The gene has four introns and is the only Daphnia ferritin on the array showing differential expression for all three experimental conditions; its transcripts are enriched in males, depleted when exposed to cadmium and enriched when challenged by arsenic (Eads et al. submitted; Shaw et al submitted and in prep). Therefore like the insect subunit, this locus responds to metal ion treatments. The other five Daphnia ferritin genes are homologous to the insect Fer3HCH loci and are arranged within a monophyletic cluster, suggesting that they originate from a series of gene duplications. Indeed, each has retained two introns, despite showing amino acid sequence divergences from 14 to 56%. These genes are designated Dpu_Fer3HCH-1 to Dpu_Fer3HCH-5. Regrettably, expression data is not available for genes representing branches D and E. However, the microarray results show that the remaining three loci differ in their transcriptional responses to the experimental treatments. Like the Drosophila Fer3HCH gene, elements on the array whose sequences cluster within branch C do not respond to metals, yet unlike the fly gene, Contig 26 transcripts are enriched in females. By contrast, a single element on the array representing branch B is enriched in males, and all 4 cDNA elements from this gene respond to arsenic treatment. These differences are likely the result of undetected splice variants. Lastly, all elements representing branch A show elevated expression patterns when treated with arsenic, yet no sex specific expression is detected. These additional observations strongly support the existence of a Fer3HCH gene expansion that diversified within a crustacean lineage leading to D. pulex. A homologue to the insect Fer2LCH genes has yet to be discovered in Crustacea.

The present study is a preliminary annotation of the emerging D. pulex genome using comparative and functional data to predict the fraction of genes unique to Daphnia. Aside from gene expansions that are suggestive of adaptations specific to aquatic environments, accounts of orphan genes (defined here has having no matches to insects) can offer equally important insights into crustacean biology from the perspective of a class of genes that usually has the shortest average lengths and are most rapidly evolving 5157. Our searches using Blastx of 787 assembled sequences against the proteome of four insects suggest that ~68% of the Daphnia genes are shared with at least one insect. Less than half of these show matches to the individual protein databases of Bombyx, Apis, Anopheles and Drosophila. This discrepancy is likely a combined effect of incomplete datasets and of lineage specific gene losses among the insects. Taking into account associated matches to proteins from our chosen outgroup (Caenorhabditis), we discover that 21% of the Daphnia genes are either derived within Pancrustacea or lost within the nematodes. Those genes that are uniformly conserved across all four insect species are primarily cuticle proteins and serine proteases having trypsin activity. These gene families are also listed as two of the top 20 most significant expansions or reductions between the Anopheles and Drosophila proteomes 51, which diverged some 250 million years ago. It is tempting to speculate that, in both crustaceans and insects, a fraction of gene families are equally active in their evolutionary diversification. Such gene families would be candidates for detailed investigations leading to a better understanding of how Pancrustacea succeeded in exploiting its range of ecological settings.

A careful evaluation of assembled sequences showing no matches to the insect proteomes suggests that ~1/3 of the genes are either derived in crustaceans or lost within insects. This estimation is admittedly from a very limited sampling of the total number of Daphnia genes and is derived from sequencing non-normalized cDNA libraries that were created under standard laboratory conditions and interrogated by microarrays. Although this fraction cannot be extrapolated to the genome, it is comparable to findings from other taxa. In D. melanogaster, 10% of the genes have homologous best hits in non-insect species plus 19% have no homologous hits to other species, while the combined estimate for A. gambiae is 21% 51. Within the nematodes, which diverged ca. 600 mya, 23% of the genes are estimated to be unique to species 14. Of course, the fraction of species specific genes declines dramatically when evaluating close allies; comparing two Caenorhabditis species reveals that 4% of their genes are unique 58, and the mouse gene set differs from the human set by only 1% 59. A future investigation of a larger D. pulex gene collection against an equivalent dataset for the congener D. magna 60 will help define the true estimate of species specific genes in Daphnia. Additional contributions of EST data for non-branchiopod crustaceans will further define the crustacean proteome and shed light on the biological factors that led to the group's divergence from insects. However, the sequences presented by our present study are accompanied by expression data from three microarray experiments, which authenticate the orphan sequences as genes and support the notion that ecological factors are more likely to contribute to sequence and functional divergences among genomes.

Combining the gene expression data obtained by Eads et al. (submitted) and Shaw et al. (submitted, and in prep) for the 787 assembled sequences reveals that the majority of genes with sex biased expression, including developmental and regulatory loci, do not respond to the cadmium and arsenic metal toxicity. A clear example is provided by genes predicted to regulate translation. All 15 genes save two are differentially expressed in males versus females and only three genes also show transcriptional responses to metal toxicity (Additional file 2). We find that this class of sex biased genes proportionally contains the fewest orphans. The relatively larger number of sequences with matches to the insect proteome suggests that genes functioning during development and reproduction are generally well conserved between crustaceans and insects. Further work is required to elucidate crustacean and Daphnia specific components of these central processes. By contrast, nearly half of the Daphnia genes that respond to metals, but show no differences between the sexes, are likely absent in insects. This is explicable in light of the fact that metal exposure is an ecological stressor that varies between aquatic and terrestrial environments 61, which has catalyzed the evolution of certain protein types (cuticles, iron metabolism, defense) to increasingly specialized functions. The extent to which ecology has shaped the genome organization of pancrustaceans is an important future direction for research. For example, the mosquito A. gambiae spends part of its larval stage in water; by comparing genes differentially expressed during this stage to expression patterns in D. pulex, it may be possible to examine the effects of an aquatic lifestyle on the expression of particular protein families.

For the purpose of creating a collection of cDNA for printing onto microarrays, a clonal isolate of a D. pulex /D. pulicaria hybrid (called log52) was cultured under standard laboratory conditions by Jim Haney (University of New Hampshire) within a large, aerated, 200 liter container of filtered lake water by feeding a concentrated monoculture of green algae (Scenedesmus acutus). Animals at all life stages were harvested and immediately processed. Total RNA was isolated using Trizol reagent (Invitrogen Life Sciences) and was subsequently purified using the RNeasy protocol (Qiagen). The cDNA libraries were constructed by Darren Bauer and Kelley Thomas (University of New Hampshire) using the Creator SMART (Clontech) system by following the manufacture's instructions. The cDNA was ligated into the pDNR-LIB vector supplied by Clontech.

To control for bias towards smaller fragments inserting during the ligation of cDNA into plasmids, reaction were performed on four cDNA size fractions. Size fractionation was performed as per the SMART cDNA protocol using the CHROMA SPIN-400 column. The column was prepared for drip procedure by inverting several times to completely resuspend the gel matrix and storage buffer was drained by gravity flow. Seven hundred microliters of column buffer were added to the column and allowed to drain out, then 100 μl of a mixture of Sfi I-digested cDNA and xylene cyanol dye were applied to the matrix and allowed to fully absorb. One hundred microliters of column buffer were added to the matrix and allowed to fully absorb, then 600 μl of column buffer were added and single-drop fractions were collected in 16 tubes. The profile of the fractions was verified by running 3 μl of each fraction on a 1.1% agarose/EtBr gel at 150v for 10 minutes. The samples were then pooled into two size classes; fractions 7 and 8 were pooled into the "large size" and fractions 9 & 10 were pooled into the "small size".

From the libraries, 768 colonies were chosen for quality assurance tests. The bacterial transformants were amplified in selective 2xYT media, plasmids were purified by an alkaline lysis protocol according to the manufacturer's instructions (PerfectPrep, Eppendorf) and quantified by spectrophotometry. The molecular weights of cDNA inserts were measured by PCR amplification of cDNA inserts using the M13 vector primers M13fw (GTG TAA AAC GAC GGC CAG TAG) and M13rev (AAA CAG CTA TGA CCA TGT TCA C) followed by agarose gel electrophoresis against standards and visualized using a Kodak 440cf imaging station. Sequencing reactions were performed by priming at the 5' end of cDNA using vector primer pDNRlib30-50 (TAT ACG AAG TTA TCA GTC GAC G), ABI BigDye chemistry and the 3730 sequencer. Vector and poor quality sequences were trimmed from the sequencing reads and ESTs were assembled into contigs using the SeqManII software (DNASTAR package). Homologies with Genbank entries were discovered using Blastx against the non-redundant (nr) protein database. Those sequences with expectation-values better than 1 × 10^-27 were further examined for the presence of an annotated ATG start codon at the 5' end of the open reading frame (ORF). This last step was accomplished using NCBI's ORF finder tool 62. Only those sequences whose Methionine aligned (including gaps) with the first amino acid of complete sequences were considered full-length transcripts.

On thousand twenty-eight additional cDNA samples were chosen for sequencing based on the microarray results obtained by Eads et al. (submitted) and Shaw et al. (submitted, and in prep). The sequencing reactions were carried out as outlined above. All 1,648 sequence reads from this study, with their quality scores, were obtained from ABI sequencer data files using phred 63 with default parameter values. The reads were then processed by discarding low quality and vector sequences using Lucy v1.19p 64 with default parameter values, by removing poly-A tails using EMBOSS trimest 65 and by discarding sequences with lengths under 100 bases. The remaining high quality EST set was reduced to a non-redundant set of unique gene transcripts by clustering with phrap 66 using the following parameters: mismatch penalty = -5; minimum match = 50; minimum score = 100. The resulting contigs and singlets that matched to mitochondrial gene transcripts (Genbank accession NC 000844) using Blastn were removed from subsequent analyses. To investigate whether the set of assembled sequences contain alternative transcripts of the same loci, the contigs and singlets were further clustered using the SeqManII software with the following relaxed parameters: match size = 12; maximum added gap length = 70; minimum percent match = 80; no gap penalty; gap length penalty = 0.70.

The putative open reading frames (ORFs) for the assembled sequences were determined in three steps using Prot4EST v2.2 67 with DECODER having been disabled, ESTScan – which is an integral component of Prot4EST – and getorf from EMBOSS. The ORFs were selected during the first step when the assembled sequence translations aligned to proteins within the NCBI NR database with a Blastx e-value better than 1 × 10^-8. Failing this first step, the ORFs where selected during the second step using ESTScan or simply by recording the longest uninterrupted ORFs when they were located on the positive stands of the sequences. Otherwise, the longest ORFs were selected during step three, based on the results obtained by using the EMBOSS program that restricted sequence translations from the negative strand. This restriction was justified by observing only 3 ORFs on the negative strand from among 376 predictions from step one.

Numerous sequence similarity searches were done for both the high quality EST set and the assembled sequences. First, queries were performed against the NCBI NR protein database (Genbank release 148) using a local installation of the WU-BLAST program 68. The taxonomic domains were added to the results by parsing the taxa ID from the top match for each query and by retrieving the associated information from the NCBI 69. Second, for a more confident assessment of whether the assembled sequences were shared with insects, they were compared to protein sequences archived in the NCBI UniGene sets for Bombyx mori (build #7), Apis mellifera (build #5), Anopheles gambiae (build #29) and Drosophila melanogaster (build #37) using tBlastx with an expectation threshold set at E < 0.005. The same search was performed against the Caenorhabditis elegans (build #23) UniGene database to judge whether the differences can be attributed to gains or losses within the representative insects or crustacean. The assembled sequences were clustered based on the distribution of bit scores across the databases using self organizing maps followed by k-means clustering within 28 nodes in Cluster v2.11 70. Third, to further ascertain whether the assembled sequences can be aligned to known proteins, queries were made against all Drosophila melanogaster gene translations that are predicted by the annotation v4.2.1 of the genome sequence assembly and against the genome nucleotide sequences of Drosophila melanogaster and Caenorhabditis elegans using tBlastx.

The assembled sequences were classified into gene ontology (GO)-defined functional classes using the program Blast2GO 22 and by extracting the GO annotations from FlyBase for sequences that strongly matched D. melanogaster gene transcripts. The putative gene annotations were examined for functional classes that are enriched within our lists of Daphnia genes compared to the total set of GO terms for all Drosophila genes using Gostat 71 and by testing for the enrichment of GO terms within subsets of the assembled sequences using Fisher's Exact Test executed within Blast2GO.