Selection of candidate genes

In D. pulex defense morph formation, it is thought that morphogenetic factors such as those that have been identified in many arthropods are expressed downstream of physiological regulation [30, 31]. The following 31 candidate genes were identified in the D. pulex genome: Hox genes [labial (lb), proboscipedia (pb), Hox3, Deformed (Dfd), Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B)], morphogenetic genes [Distal-less (Dll), aristaless (al), homothorax (hth), dachshund (dac), extradenticle (exd), escargot (esg), teashirt (tsh), epidermal growth factor receptor (EGFR1, 2), spitz (spi), decapentaplegic (dpp), wingless (wg) and hedgehog (hh)], endocrine genes [juvenile hormone acid methyltransferase (JHAMT), Methoprene-tolerant (Met), ultraspiracle (USP), ecdysone receptor (EcR), insulin-like receptor (InR), insulin receptor substrate-1 (IRS-1) and forkhead box O (FOXO)] and neuronal genes [tyramine beta-monooxygenase (TBM) and dopamine beta-monooxygenase (DBM)]. BLASTX searches http://blast.ncbi.nlm.nih.gov/Blast.cgi confirmed that the predicted D. pulex sequences are homologues of the candidate genes (Table 1, Additional file 1).

Table 1 Expression profiles of investigated candidate genes Full size table

Expression profiles of candidate genes

The relative expression levels of the candidate genes were quantified using real-time quantitative RT-PCR to examine whether these genes were differentially expressed after exposure to the predator kairomone in the embryonic stage (stage 4) and the postembryonic instar (first-instar). 18S ribosomal RNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were tested as reference genes for real-time qPCR. Since GAPDH was considered to be the most stable gene and its expression levels were closest to those of the candidate genes (data not shown), GAPDH was used as the reference gene. This result was consistent with a previous report of expression stability in D. magna following exposure to the drug, ibuprofen [32].

Real-time qPCR revealed that 21 of the 31 candidate genes were up-regulated in the presence of kairomones in first-instar juveniles. Conversely, none of the candidate genes were up-regulated by more than 1.5-fold at the embryonic stage 4 (Table 1, Additional file 1). Two genes (abd-A and FOXO) were not amplified by PCR, probably because the designed primer sites were inappropriate. Since a number of traits in first-instar juveniles change in response to defense morph formation (e.g. crest epithelial hyperplasia), the candidate genes were thought to be up-regulated in the preceding developmental stage (i.e. embryonic stage 4), although the expected results were not obtained. It is therefore suggested that the up-regulated genes are involved in neckteeth development, which is most pronounced at the second- and third-instar [6]. Furthermore, these results show that the candidate gene approach using a Daphnia genome database can be used for the analysis of the molecular mechanisms responsible for the defense morph formation.

To further clarify differences in the expression of the six candidate genes showing the most marked up-regulation (TBM, JHAMT, exd, InR, esg, Hox3) and the two gene candidates thought to be associated with JHAMT (Met) and InR (IRS-1), we reperformed the real-time qPCR using biological replicates to analyze the detailed expression profiles of kairomone-responsive genes whose functions were suspected of being involved in defense morph formation (Figure 2). Unfortunately, since the level of expression of TBM varied between trials (possibly due to low expression level), we excluded TBM from further analyses.

Figure 2 Relative expression levels of JHAMT , Met , InR , IRS-1 , Hox3 , exd , esg , DD1 , DD2 and DD3 in stage 4 embryos and the first-instar juveniles treated with kairomone and control media, analyzed by real-time quantitative PCR. DD1 showed higher levels of expression after exposure to kairomone medium (black) than in control medium (white) during embryonic development. Other genes showed higher levels of expression after exposure to the kairomone medium than in the control during juvenile development. Y-axes indicate relative expression levels normalized by comparison with GAPDH expression (internal control gene). Technical triplicates were performed for all reactions. Bars indicate standard errors. Asterisks indicate significant differences (P < 0.05, based on [57]). Full size image

Endocrine genes (JHAMT, Met, InR and IRS-1)

Compared to when no kairomones were present, the expression levels of JHAMT, Met, InR and IRS-1 in first-instar juveniles exposed to kairomones increased by approximately 2.5-, 1.5-, 1.8- and 1.6-fold, respectively (Figure 2). JHAMT encodes the methyltransferase that mediates the final step of juvenile hormone synthesis [33], Met encodes a candidate receptor for juvenile hormone [34, 35], InR encodes an insulin/insulin-like growth factor receptor, and IRS-1 encodes a downstream element that interacts directly with InR [36].

In addition to polyphenism [37–39], juvenile hormones (juvenoids) constitute a group of acyclic sesquiterpenoids that are key hormones in the regulation of a variety of physiological regulations in insect development and morphogenesis [28, 40]. In crustaceans, methyl farnesoate (MF) is known to act as a juvenile hormone and plays important roles in the regulation of development [41]. For example, male production can be induced in female daphnids treated with MF [42]; however, little is known about other functions of MF in D. pulex. In addition to JH, the insulin-signaling pathway in many animals is also important for the regulation of a variety of developmental processes, including body-size and allometry controls [30, 36, 43, 44]. It has been suggested that the crosstalk between the JH and insulin-signaling pathways is responsible for the expression of morphogenetic factors in the development of beetle horns [30]. Thus, it appears that physiological regulation by these endocrine factors may induce the expression of morphogenetic genes resulting in neckteeth formation in D. pulex.

Morphogenetic genes (Hox3, extradenticle, escargot)

Compared to conditions without kairomones, the expression levels of Hox3, extradenticle and escargot in first-instar juveniles increased by approximately 1.7-, 1.9- and 1.8-fold, respectively, in the presence of kairomones (Figure 2). Hox3 is a member of the Hox cluster and appears to have a typical Hox-like role in the centipede, whereas the insect Hox3 ortholog, zerknüllt (zen), has lost the function of specifying segmental identity during embryogenesis [31, 45–47]. Although little is known about the functions of crustacean Hox3, expression in D. pulex has been reported in the nuchal area (where the neckteeth subsequently form) and in the mandibular mesoderm during the early- and mid-embryonic stages, respectively [48], suggesting a possible role in establishing the position of neckteeth development. Furthermore, although Daphnia neckteeth cannot be considered to be homologous to appendages, it is possible that the molecular mechanisms for appendage development are co-opted for neckteeth development.

Exd and esg respectively encode a homeobox transcription factor and a zinc finger transcription factor, and both are known to determine the proximal segmental identity of appendages (coxa and trochanter) in Drosophila melanogaster [49]. Furthermore, up-regulation of dac, a known selector gene for the femur and tibia in D. melanogaster, and Dll, which defines tarsus and pretarsus, did not produce as conspicuous a response as exd and esg in first-instar juveniles (1.6- and 1.2-fold, respectively) (Table 1, Additional file 1). This evidence showed that the genes responsible for the determination of proximal appendages were up-regulated in juveniles with neckteeth, implies that these genes might be co-opted for neckteeth formation. However, our results also showed that the expression level of al, a known selector gene for the most distal region of appendage [49], was also higher (Table 1, Additional file 1). This is probably because, in Drosophila, al is also expressed in the proximal regions of appendages [49]. It has recently been reported that Dll and al are both involved in the development of beetle horns, which are not homologous to appendages [29]. This is similar to the situation in the Daphnia neckteeth formation, except that the co-opted regions of appendages are different (proximal or distal). Indeed, further analyses of this hypothesis will provide us with insights, not only into defense morph formation in Daphnia, but also into the evolution of appendage morphology in arthropods.

Exploring novel genes by differential display

Next, differential display was performed to identify any novel genes that were related to neckteeth formation, but which were not included in the candidate gene approach. As a result, we obtained 22 fragments exhibiting differential expressions in response to kairomone exposure. To refine these results further, BLASTN searches were used to compare these fragments against wFleaBase and their coding sequences were predicted using a gene prediction software joined to wFleabase (Gnomon, Dappu v1.1 gene models, SNAP gene predictor). For the single fragment for which the functional sequence could not be predicted by the gene predictor, the full sequence was determined by rapid amplification of cDNA ends (RACE)-PCR. Of these 22 sequences, we performed real-time quantitative RT-PCR to confirm their responsiveness as described for the candidate genes above. As a result, three genes (DD1, DD2 and DD3) showed marked up-regulation in response to kairomone exposure (Figure 2, Table 2, Additional file 2).

Table 2 Expression profiles of the genes obtained by differential display (DD) Full size table

DD1

In stage 4 embryos, DD1 expression was up-regulated approximately 1.9-fold after exposure to kairomones (Figure 2); among all the genes examined in this study, this was the only gene that responded to kairomones in the embryonic stage. BLAST searches suggested that there were no genes homologous to DD1 in other crustaceans and insects. Motif searches using the InterPro database http://www.ebi.ac.uk/Tools/InterProScan/ revealed that DD1 has a signal peptide and a dopamine beta-monooxygenase N-terminal (DOMON) domain. DOMON domains are ubiquitous among plants and animals, and exist in a variety of proteins, including dopamine beta-monooxygenase, in which this domain was originally found [50]. In D. pulex, DD1 is thus considered to be a novel gene containing a DOMON domain. In addition to the aforementioned dopamine beta-monooxygenase (DBM), representative proteins containing a DOMON domain also include tyramine beta-monooxygenase (TBM), which is involved in the biosynthesis of biogenic amines [51]. However, the sequence and the domain structure of DD1 were completely different to those of DBM and TBM (data not shown), and the expression profiles of DD1 also did not correspond to those of DBM and TBM (Table 1, 2, Additional file 1, 2). Consequently, DD1 is considered to play a different role than either DBM or TBM. It is possible that DD1 is involved in kairmone reception and/or fate determination in the defense morph, because DD1 expression was initiated by the presence of kairomones at embryonic stage 4, which is considered to be a critical period for the reception of kairomones [4–6], before declining over the course of postembryonic development (Figure 2).

DD2, DD3

In postembryonic first-instars, expression levels of DD2 and DD3 in the presence of kairomones increased by approximately 3- and 2-fold, respectively (Figure 2). While DD2 showed extremely high homology to bacterial ribosomal RNA (Table 2, Additional file 2), the DD2 sequence in the D. pulex genome database was found to contain introns. Furthermore, the full DD2 sequence obtained by RACE-PCR had a 3' poly(A) tail, which is not typically present in bacterial transcripts and suggests that the identification of DD2 was not the result of contamination. Interestingly, in addition to kairomone responsiveness, the expression levels of DD2 were more than 100-fold higher in the first-instar juveniles than in the embryos (Figure 2). Based on these findings, it is possible that DD2 may have been acquired by horizontal transfer from bacteria.

DD3 exhibited similarity to growth and transformation-dependent protein (GTD-P) (Table 2, Additional file 2). Although GTD-P homologues have been identified in some arthropod species, little is known about its functions. However, it was reported that GTD-P was strongly expressed when a rat pheochromocytoma cell line (PC12) was exposed to nerve growth factor (NGF) [52]. As it was reported that PC12 cells exposed to NGF undergo proliferation, it is possible that GTD-P is involved in the cellular proliferation observed during defense morph formation in D. pulex.