Unusual duplication of the insulin-like receptor in the crustacean Daphnia pulex

Abstract

Background: The insulin signaling pathway (ISP) has a key role in major physiological events like carbohydrate metabolism and growth regulation. The ISP has been well described in vertebrates and in a few invertebrate model organisms but remains largely unexplored in non-model invertebrates. This study is the first detailed genomic study of this pathway in a crustacean species, Daphnia pulex.

Results: The Daphnia pulex draft genome sequence assembly was scanned for major components of the ISP with a special attention to the insulin-like receptor. Twenty three putative genes are reported. The pathway appears to be generally well conserved as genes found in other invertebrates are present. Major findings include a lower number of insulin-like peptides in Daphnia as compared to other invertebrates and the presence of multiple insulin-like receptors (InR), with four genes as opposed to a single one in other invertebrates. Genes encoding for the Dappu_InR are likely the result of three duplication events and bear some unusual features. Dappu_InR-4 has undergone extensive evolutionary divergence and lacks the conserved site of the catalytic domain of the receptor tyrosine kinase. Dappu_InR-1 has a large insert and lacks the transmembranal domain in the β-subunit. This domain is also absent in Dappu_InR-3. Dappu_InR-2 is characterized by the absence of the cystein-rich region. Real-time q-PCR confirmed the expression of all four receptors. EST analyses of cDNA libraries revealed that the four receptors were differently expressed under various conditions.

Conclusions: Duplications of the insulin receptor genes might represent an important evolutionary innovation in Daphnia as they are known to exhibit extensive phenotypic plasticity in body size and in the size of defensive structures in response to predation.

Figure 1

Daphnia pulex insulin and TOR signalling pathways, adapted from Drosophila melanogaster [39]. The D. pulex insulin/insulin-like growth factors signalling (IIS) comprises a group of insulin-like peptides (ILPs), four insulin receptors (InR1, InR2, InR3 and InR4) genes, an insulin receptor substrate (IRS), the type phosphatidylinositol-3-kinase (PI3K p85/p60 and PI3K p110), the lipid phosphatase PTEN, the protein kinase PKB/AKT, the phosphoinositide-dependent protein kinase 1 (PDK-1) and the transcriptional factor FOXO. The TOR- pathway includes a TSC complex (TSC1 and TSC2), a small GTPase RHEB, the target of rapamycin (TOR), two S6 kinase (S6K1 and S6K2), the 4E-binding protein (4EBP) and the Pol I transcription factor TIF-1A. The AMPK-pathway involves the activation of AMP-dependent kinase (AMPK) by the LKB1 protein kinase.

Figure 2

Phylogenetic tree for insulin receptors tyrosine kinase constructed with maximum likelihood and Bayesian inference methods under the WAG+G model of evolution. Numbers represent percent bootstrap values (maximum likelihood/Bayesian inference); unlabeled branches indicate a value less than 75. GenBank or Ensembl accession numbers are in brackets.

Figure 3

Amino acid alignment of the four Dappu_InR with Drosophila and Homo sapiens sequences.

Figure 4

Amino acid alignment of the four Dappu_InR with Drosophila and Homo sapiens sequences (continued).