Molecular evolution of the crustacean hyperglycemic hormone family in ecdysozoans

Abstract

Background: Crustacean Hyperglycemic Hormone (CHH) family peptides are neurohormones known to regulate several important functions in decapod crustaceans such as ionic and energetic metabolism, molting and reproduction. The structural conservation of these peptides, together with the variety of functions they display, led us to investigate their evolutionary history. CHH family peptides exist in insects (Ion Transport Peptides) and may be present in all ecdysozoans as well. In order to extend the evolutionary study to the entire family, CHH family peptides were thus searched in taxa outside decapods, where they have been, to date, poorly investigated.

Results: CHH family peptides were characterized by molecular cloning in a branchiopod crustacean, Daphnia magna, and in a collembolan, Folsomia candida. Genes encoding such peptides were also rebuilt in silico from genomic sequences of another branchiopod, a chelicerate and two nematodes. These sequences were included in updated datasets to build phylogenies of the CHH family in pancrustaceans. These phylogenies suggest that peptides found in Branchiopoda and Collembola are more closely related to insect ITPs than to crustacean CHHs. Datasets were also used to support a phylogenetic hypothesis about pancrustacean relationships, which, in addition to gene structures, allowed us to propose two evolutionary scenarios of this multigenic family in ecdysozoans.

Conclusions: Evolutionary scenarios suggest that CHH family genes of ecdysozoans originate from an ancestral two-exon gene, and genes of arthropods from a three-exon one. In malacostracans, the evolution of the CHH family has involved several duplication, insertion or deletion events, leading to neuropeptides with a wide variety of functions, as observed in decapods. This family could thus constitute a promising model to investigate the links between gene duplications and functional divergence.

Figure 1

Current hypotheses about the relationships of pancrustacean taxa. (A) Phylogeny built from 12 mitochondrial protein-encoding genes [19]. (B) Phylogeny built from the analysis of nuclear protein-encoding genes EF-1α, EF-2 and POLII [23].

Figure 2

Alignments of the putative ITP (ion transport peptide) and ITP-L sequences from branchiopods, collembolans, insects and chelicerates. (A) Multiple-sequence alignment of 17 ITP sequences with CHH (crustacean hyperglycemic hormone) sequence from the shore crab Carcinus maenas. (B) Multiple-sequence alignment of 16 ITP-L sequences with CHH-L sequence from Carcinus maenas. Aea: Aedes aegypti ; Ang: Anopheles gambiae ; Apm: Apis mellifera ; Bom: Bombyx mori ; Cam: Carcinus maenas ; Cup: Culex pipiens ; Dam: Daphnia magna ; Dap: Daphnia pulex ; Dev: Dermacentor variabilis ; Drm: Drosophila melanogaster ; Foc: Folsomia candida ; Ixs: Ixodes scapularis ; Lom: Locusta migratoria ; Mas: Manduca sexta ; Nav: Nasonia vitripennis ; Peh: Pediculus humanus ; Scg: Schistocerca gregaria ; Trc: Tribolium castaneum. Sequence accession numbers are given in Table 2.

Figure 3

Alignment of putative CHH family peptides of nematodes. ITP-like sequences deduced from nematode genomes were aligned with the ITP sequence of Schistocerca gregaria. Brm: Brugia malayi ; Cab: Caenorhabditis briggsae ; Cae: Caenorhabditis elegans ; Car: Caenorhabditis remanei ; Scg: Schistocerca gregaria ; Trs: Trichinella spiralis.

Figure 4

Structure of the genes encoding CHH family peptides. Exons are represented by open boxes: untranslated regions are in grey, regions encoding signal peptides are in yellow, those encoding precursor-related peptides in light blue, those encoding mature ITPs, CHHs, MIHs and MOIHs in purple, red, green and dark blue, respectively, and alternatively spliced exons encoding ITP-L and CHH-L C-terminus regions in violet and orange, respectively. Question marks indicate untranslated regions whose precise borders are not known.

Figure 5

Phylogeny of the CHH family in Pancrustacea, based on maximum likelihood analysis of an amino acid dataset (56 taxa, 73 characters). The analysis was carried out using a WAG+I+G model of protein evolution. Ixodes scapularis and Dermacentor variabilis putative ITPs were assigned as outgroup. Numbers at nodes are bootstrap values based on 100 replicates. Sequence accession numbers are given in Table 2. For taxa in which the genes have been sequenced, the number of exons (three, four or five) is indicated in a black circle after the name of the species. A four-exon pattern was assigned for taxa in which two peptides arising by alternative splicing have been described

Figure 6

Phylogeny based on Bayesian analysis of the amino acid dataset including CHH-L and ITP-L sequences (23 taxa, 109 characters). Trees obtained by maximum likelihood analysis of the amino acid dataset and by Bayesian analysis of the DNA dataset were fully congruent. Numbers above branches are posterior probabilities and bootstrap values (based on 100 replicates) obtained from the analysis of the amino acid dataset, and numbers below branches are posterior probabilities obtained from the analysis of the DNA dataset. Sequence accession numbers are given in Table 2.

Figure 7

Evolutionary scenarios of the CHH family genes in Ecdysozoa. (A) Hypothesis accounting for two independent exon duplications originating the four-exon genes, which may have occurred on one side in a common ancestor of decapods and on the other side in a common ancestor of insects and branchiopods. (B) Hypothesis accounting for a single exon duplication originating the four-exon genes of insects, branchiopods and decapods.