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Background Cytochrome P450s (CYPs) in animals fall into two categories: those that synthesize or metabolize endogenous molecules and those that interact with exogenous chemicals from the diet or the environment. The latter form a critical component of detoxification systems. Results Data mining and manual curation of the Daphnia pulex genome identified 75 functional CYP genes, and three CYP pseudogenes. These CYPs belong to 4 clans, 13 families, and 19 subfamilies. The CYP 2, 3, 4, and mitochondrial clans are the same four clans found in other sequenced protostome genomes. Comparison of the CYPs from D. pulex to the CYPs from insects, vertebrates and sea anemone ( Nematostella vectensis ) show that the CYP2 clan, and to a lesser degree, the CYP4 clan has expanded in Daphnia pulex , whereas the CYP3 clan has expanded in insects. However, the expansion of the Daphnia CYP2 clan is not as great as the expansion observed in deuterostomes and the nematode C. elegans . Mapping of CYP tandem repeat regions demonstrated the unusual expansion of the CYP370 family of the CYP2 clan. The CYP370s are similar to the CYP15s and CYP303s that occur as solo genes in insects, but the CYP370s constitute ~20% of all the CYP genes in Daphnia pulex . Lastly, our phylogenetic comparisons provide new insights into the potential origins of otherwise mysterious CYPs such as CYP46 and CYP19 (aromatase). Conclusion Overall, the cladoceran, D. pulex has a wide range of CYPs with the same clans as insects and nematodes, but with distinct changes in the size and composition of each clan.

Background The Wnt genes encode secreted glycoprotein ligands that regulate a wide range of developmental processes, including axis elongation and segmentation. There are thirteen subfamilies of Wnt genes in metazoans and this gene diversity appeared early in animal evolution. The loss of Wnt subfamilies appears to be common in insects, but little is known about the Wnt repertoire in other arthropods, and moreover the expression and function of these genes have only been investigated in a few protostomes outside the relatively Wnt -poor model species Drosophila melanogaster and Caenorhabditis elegans . To investigate the evolution of this important gene family more broadly in protostomes, we surveyed the Wnt gene diversity in the crustacean Daphnia pulex , the chelicerates Ixodes scapularis and Achaearanea tepidariorum , the myriapod Glomeris marginata and the annelid Platynereis dumerilii . We also characterised Wnt gene expression in the latter three species, and further investigated expression of these genes in the beetle Tribolium castaneum . Results We found that Daphnia and Platynereis both contain twelve Wnt subfamilies demonstrating that the common ancestors of arthropods, ecdysozoans and protostomes possessed all members of all Wnt subfamilies except Wnt3 . Furthermore, although there is striking loss of Wnt genes in insects, other arthropods have maintained greater Wnt gene diversity. The expression of many Wnt genes overlap in segmentally reiterated patterns and in the segment addition zone, and while these patterns can be relatively conserved among arthropods and the annelid, there have also been changes in the expression of some Wnt genes in the course of protostome evolution. Nevertheless, our results strongly support the parasegment as the primary segmental unit in arthropods, and suggest further similarities between segmental and parasegmental regulation by Wnt genes in annelids and arthropods respectively. Conclusions Despite frequent losses of Wnt gene subfamilies in lineages such as insects, nematodes and leeches, most protostomes have probably maintained much of their ancestral repertoire of twelve Wnt genes. The maintenance of a large set of these ligands could be in part due to their combinatorial activity in various tissues rather than functional redundancy. The activity of such Wnt 'landscapes' as opposed to the function of individual ligands could explain the patterns of conservation and redeployment of these genes in important developmental processes across metazoans. This requires further analysis of the expression and function of these genes in a wider range of taxa.

Background Numerous cases of predator-induced polyphenisms, in which alternate phenotypes are produced in response to extrinsic stimuli, have been reported in aquatic taxa to date. The genus Daphnia (Branchiopoda, Cladocera) provides a model experimental system for the study of the developmental mechanisms and evolutionary processes associated with predator-induced polyphenisms. In D. pulex , juveniles form neckteeth in response to predatory kairomones released by Chaoborus larvae (Insecta, Diptera). Results Previous studies suggest that the timing of the sensitivity to kairomones in D. pulex can generally be divided into the embryonic and postembryonic developmental periods. We therefore examined which of the genes in the embryonic and first-instar juvenile stages exhibit different expression levels in the presence or absence of predator kairomones. Employing a candidate gene approach and identifying differentially-expressed genes revealed that the morphogenetic factors, Hox3 , extradenticle and escargot , were up-regulated by kairomones in the postembryonic stage and may potentially be responsible for defense morph formation. In addition, the juvenile hormone pathway genes, JHAMT and Met , and the insulin signaling pathway genes, InR and IRS-1 , were up-regulated in the first-instar stage. It is well known that these hormonal pathways are involved in physiological regulation following morphogenesis in many insect species. During the embryonic stage when morphotypes were determined, one of the novel genes identified by differential display was up-regulated, suggesting that this gene may be related to morphotype determination. Biological functions of the up-regulated genes are discussed in the context of defense morph formation. Conclusions It is suggested that, following the reception of kairomone signals, the identified genes are involved in a series of defensive phenotypic alterations and the production of a defensive phenotype.

Background The large gene superfamily of ABC (ATP-binding cassette) transporters encodes membrane proteins involved in trafficking processes across biological membranes and further essential cell biological functions. ABC transporters are evolutionary ancient and involved in the biochemical defence against toxicants. We report here a genome-wide survey of ABC proteins of Daphnia pulex , providing for the first time information on ABC proteins in crustacea, a primarily aquatic arthropod subphylum of high ecological and economical importance. Results We identified 64 ABC proteins in the Daphnia genome, which possesses members of all current ABC subfamilies A to H. To unravel phylogenetic relationships, ABC proteins of Daphnia were compared to those from yeast, worm, fruit fly and human. A high conservation of Daphnia of ABC transporters was observed for proteins involved in fundamental cellular processes, including the mitochondrial half transporters of the ABCB subfamily, which function in iron metabolism and transport of Fe/S protein precursors, and the members of subfamilies ABCD, ABCE and ABCF, which have roles in very long chain fatty acid transport, initiation of gene transcription and protein translation, respectively. A number of Daphnia proteins showed one-to-one orthologous relationships to Drosophila ABC proteins including the sulfonyl urea receptor ( SUR ), the ecdysone transporter ET23 , and the eye pigment precursor transporter scarlet . As the fruit fly, Daphnia lacked homologues to the TAP protein, which plays a role in antigene processing, and the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a chloride channel. Daphnia showed two proteins homologous to MDR (multidrug resistance) P-glycoproteins (ABCB subfamily) and six proteins homologous to MRPs (multidrug resistance-associated proteins) (ABCC subfamily). However, lineage specific gene duplications in the ABCB and ABCC subfamilies complicated the inference of function. A particularly high number of gene duplications were observed in the ABCG and ABCH subfamilies, which have 23 and 15 members, respectively. Conclusion The in silico characterisation of ABC transporters in the Daphnia pulex genome revealed that the complement of ABC transporters is as complex in crustaceans as that other metazoans. Not surprisingly, among currently available genomes, Daphnia ABC transporters most closely resemble those of the fruit fly, another arthropod.

Background DNA tandem repeats (TRs) are not just popular molecular markers, but are also important genomic elements from an evolutionary and functional perspective. For various genomes, the densities of short TR types were shown to differ strongly among different taxa and genomic regions. In this study we analysed the TR characteristics in the genomes of Daphnia pulex and 11 other eukaryotic species. Characteristics of TRs in different genomic regions and among different strands are compared in details for D. pulex and the two model insects Apis mellifera and Drosophila melanogaster . Results Profound differences in TR characteristics were found among all 12 genomes compared in this study. In D. pulex , the genomic density of TRs was low compared to the arthropod species D. melanogaster and A. mellifera . For these three species, very few common features in repeat type usage, density distribution, and length characteristics were observed in the genomes and in different genomic regions. In introns and coding regions an unexpectedly high strandedness was observed for several repeat motifs. In D. pulex , the density of TRs was highest in introns, a rare feature in animals. In coding regions, the density of TRs with unit sizes 7-50 bp were more than three times as high as for 1-6 bp repeats. Conclusions TRs in the genome of D. pulex show several notable features, which distinguish it from the other genomes. Altogether, the highly non-random distribution of TRs among genomes, genomic regions and even among different DNA-stands raises many questions concerning their functional and evolutionary importance. The high density of TRs with a unit size longer than 6 bp found in non-coding and coding regions underpins the importance to include longer TR units in comparative analyses.

Background Branchiopod crustaceans in the genus Daphnia are key model organisms for investigating interactions between genes and the environment. One major theme of research on Daphnia species has been the evolution of resistance to pathogens and parasites, but lack of knowledge of the Daphnia immune system has limited the study of immune responses. Here we provide a survey of the immune-related genome of D. pulex , derived from the newly completed genome sequence. Genes likely to be involved in innate immune responses were identified by comparison to homologues from other arthropods. For each candidate, the gene model was refined, and we conducted an analysis of sequence divergence from homologues from other taxa. Results and conclusion We found that some immune pathways, in particular the TOLL pathway, are fairly well conserved between insects and Daphnia , while other elements, in particular antimicrobial peptides, could not be recovered from the genome sequence. We also found considerable variation in gene family copy number when comparing Daphnia to insects and present phylogenetic analyses to shed light on the evolution of a range of conserved immune gene families.

Background Two major biological stressors of freshwater zooplankton of the genus Daphnia are predation and fluctuations in food quality. Here we use kairomones released from a planktivorous fish ( Leucaspius delineatus ) and from an invertebrate predator (larvae of Chaoborus flavicans ) to simulate predation pressure; a microcystin-producing culture of the cyanobacterium Microcystis aeruginosa and a microcystin-deficient mutant are used to investigate effects of low food quality. Real-time quantitative polymerase chain reaction (QPCR) allows quantification of the impact of biotic stressors on differential gene activity. The draft genome sequence for Daphnia pulex facilitates the use of candidate genes by precisely identifying orthologs to functionally characterized genes in other model species. This information is obtained by constructing phylogenetic trees of candidate genes with the knowledge that the Daphnia genome is composed of many expanded gene families. Results We evaluated seven candidate reference genes for QPCR in Daphnia magna after exposure to kairomones. As a robust approach, a combination normalisation factor (NF) was calculated based on the geometric mean of three of these seven reference genes: glyceraldehyde-3-phosphate dehydrogenase, TATA-box binding protein and succinate dehydrogenase . Using this NF, expression of the target genes actin and alpha-tubulin were revealed to be unchanged in the presence of the tested kairomones. The presence of fish kairomone up-regulated one gene ( cyclophilin ) involved in the folding of proteins, whereas Chaoborus kairomone down-regulated the same gene. We evaluated the same set of candidate reference genes for QPCR in Daphnia magna after exposure to a microcystin-producing and a microcystin-free strain of the cyanobacterium Microcystis aeruginosa . The NF was calculated based on the reference genes 18S ribosomal RNA , alpha-tubulin and TATA-box binding protein . We found glyceraldehyde-3-phosphate dehydrogenase and ubiquitin conjugating enzyme to be up-regulated in the presence of microcystins in the food of D. magna . These findings demonstrate that certain enzymes of glycolysis and protein catabolism are significantly upgregulated when daphnids ingest microcystins. Each differentially regulated gene is a member of an expanded gene family in the D. pulex genome. The cyclophilin , GapDH and UBC genes show moderately large sequence divergence from their closest paralogs. Yet actin and alpha-tubulin genes targeteted by our study have nearly identical paralogs at the amino acid level. Conclusion Gene expression analysis using a normalisation factor based on three reference genes showed that transcription levels of actin and alpha-tubulin were not substantially changed by predator-borne chemical cues from fishes or invertebrates, although changes in expression on the protein level were shown elsewhere. These changes in protein level could be caused by others than the investigated paralogs, showing the importance of the construction of phylogenetic trees for candidate gene approaches. However, fish kairomones caused an up-regulation, and Chaoborus kairomone caused a down-regulation of cyclophylin , which proved to be a potential target gene for further analysis of kairomone effects on the life history of daphnids. Changes in food quality required a different set of reference genes compared to the kairomone experiment. The presence of dietary microcystins led to an up-regulation of two genes involved in the basic metabolism of D. magna , i.e. glyceraldehyde-3-phosphate dehydrogenase and ubiquitin conjugating enzyme , which suggests that microcystins in cyanobacteria have more general effects on the metabolism of D. magna than previously thought. Phylogenetic trees resolving relationships among paralogs that share the same gene name are shown to be important for determining the identity of the candidate genes under investigation.

Background The nuclear receptor superfamily currently consists of seven gene subfamilies that encompass over 80 distinct receptor proteins. These transcription factors typically share a common five-domain structure with a highly conserved DNA-binding domain. Some nuclear receptors are ubiquitous among the metazoans, while others are unique to specific phylogenetic groups. Crustaceans represent the second largest group of arthropods with insects being the largest. However, relative to insects, little is known about the nuclear receptors of crustaceans. The aim of this study was to identify putative nuclear receptors from the first assembled genome of a crustacean Daphnia pulex http://wFleaBase.org . Nuclear receptor expression was evaluated and receptors were subjected to phylogenetic analyses to gain insight into evolution and function. Results Twenty-five putative nuclear receptors were identified in D. pulex based on the presence of a conserved DNA-binding domain. All of the nuclear receptor protein sequences contain a highly homologous DNA-binding domain and a less conserved ligand-binding domain with the exception of the NR0A group. These receptors lack a ligand-binding domain. Phylogenetic analysis revealed the presence of all seven receptor subfamilies. The D. pulex genome contains several nuclear receptors that have vertebrate orthologs. However, several nuclear receptor members that are represented in vertebrates are absent from D. pulex . Notable absences include receptors of the 1C group (peroxisome proliferators-activated receptors), the 3A group (estrogen receptor), and the 3C group (androgen, progestogen, mineralcorticoid, and glucocorticoid receptors). The D. pulex genome also contains nuclear receptor orthologs that are present in insects and nematodes but not vertebrates, including putative nuclear receptors within the NR0A group. A novel group of receptors, designated HR97, was identified in D. pulex that groups with the HR96/CeNHR8/48/DAF12 clade, but forms its own sub-clade. Gene products were detected in adult female D. pulex for 21 of the 25 receptors. Conclusion Nuclear receptors are ancient proteins with highly conserved DNA-binding domains. The DNA-binding domains of the nuclear receptors of D. pulex contain the same degree of conservation that is typically found within nuclear receptors of other species. Most of the receptors identified in D. pulex have orthologs within the vertebrate and invertebrate lineages examined with the exception of the novel HR97 group and the Dappu-HR10 and potentially the Dappu-HR11 receptors found in D. pulex . These groups of receptors may harbour functions that are intrinsic to crustacean physiology.

Background Shotgun sequences of DNA extracts from whole organisms allow a comprehensive assessment of possible symbionts. The current project makes use of four shotgun datasets from three species of the planktonic freshwater crustaceans Daphnia : one dataset from clones of D. pulex and D. pulicaria and two datasets from one clone of D. magna . We analyzed these datasets with three aims: First, we search for bacterial symbionts, which are present in all three species. Second, we search for evidence for Cyanobacteria and plastids, which had been suggested to occur as symbionts in a related Daphnia species. Third, we compare the metacommunities revealed by two different 454 pyrosequencing methods (GS 20 and GS FLX). Results In all datasets we found evidence for a large number of bacteria belonging to diverse taxa. The vast majority of these were Proteobacteria. Of those, most sequences were assigned to different genera of the Betaproteobacteria family Comamonadaceae. Other taxa represented in all datasets included the genera Flavobacterium, Rhodobacter, Chromobacterium, Methylibium, Bordetella, Burkholderia and Cupriavidus . A few taxa matched sequences only from the D. pulex and the D. pulicaria datasets: Aeromonas, Pseudomonas and Delftia . Taxa with many hits specific to a single dataset were rare. For most of the identified taxa earlier studies reported the finding of related taxa in aquatic environmental samples. We found no clear evidence for the presence of symbiotic Cyanobacteria or plastids. The apparent similarity of the symbiont communities of the three Daphnia species breaks down on a species and strain level. Communities have a similar composition at a higher taxonomic level, but the actual sequences found are divergent. The two Daphnia magna datasets obtained from two different pyrosequencing platforms revealed rather similar results. Conclusion Three clones from three species of the genus Daphnia were found to harbor a rich community of symbionts. These communities are similar at the genus and higher taxonomic level, but are composed of different species. The similarity of these three symbiont communities hints that some of these associations may be stable in the long-term.

Background Chemoreception is vitally important for all animals, yet little is known about the genetics of chemoreception in aquatic organisms. The keystone species Daphnia pulex , a well known crustacean, is the first aquatic invertebrate to have its genome sequenced. This has allowed us the initial investigation of chemoreceptor genes in an aquatic invertebrate, and to begin the study of chemoreceptor evolution across the arthropod phylum. Results We describe 58 Grs (gustatory receptors), belonging to the insect chemoreceptor superfamily, which were identified bioinformatically in the draft genome of the crustacean waterflea Daphnia pulex . No genes encoding proteins similar to the insect odorant receptors (Ors) were identified. These 58 Grs form 3 distinctive subfamilies of 37, 12, and 5 genes, as well as a highly divergent singleton (Gr58). In addition, Grs55–57 share distinctive amino acid motifs and cluster with the sugar receptors of insects, and may illuminate the origin of this distinctive subfamily. ESTs, tiling array, and PCR amplification results support 34 predicted gene models, and preliminary expression data comparing the sexes indicates potential female-biased expression for some genes. Conclusion This repertoire of 58 chemoreceptors presumably mediates the many chemoperception abilities of waterfleas. While it is always possible that the entire Or gene lineage was lost at some point in the history of Daphnia pulex , we think it more likely that the insect Or lineage is indeed a relatively recently expanded gene lineage concomitant with the evolution of terrestriality in the insects or their hexapod ancestors.