An evolutionary genomics view on neuropeptide genes in Hydrozoa and Endocnidozoa (Myxozoa)

Abstract

Background: The animal phylum Cnidaria consists of six classes or subphyla: Hydrozoa, Scyphozoa, Cubozoa, Staurozoa, Anthozoa, and Endocnidozoa. Cnidarians have an early evolutionary origin, diverging before the emergence of the Bilateria. Extant members from this phylum, therefore, are important resources for understanding the evolution of the nervous system. Cnidarian nervous systems are strongly peptidergic. Using genomics, we have recently shown that three neuropeptide families (the X₁PRX₂amides, GRFamides, and GLWamides) are wide-spread in four (Scyphozoa, Cubozoa, Staurozoa, Anthozoa) out of six cnidarian classes or subphyla, suggesting that these three neuropeptide families emerged in the common cnidarian ancestor. In the current paper, we analyze the remaining cnidarian class, Hydrozoa, and the subphylum Endocnidozoa, to make firm conclusions about the evolution of neuropeptide genes in Cnidaria.

Results: We analyzed sixteen hydrozoan species with a sequenced genome or transcriptome, using a recently developed software program for discovering neuropeptide genes. These species belonged to various hydrozoan subclasses and orders, among them the laboratory models Hydra, Hydractinia, and Clytia. We found that each species contained three to five neuropeptide families. A common feature for all hydrozoans was that they contained genes coding for (i) X₁PRX₂amide peptides, (ii) GRFamide peptides, and (iii) GLWamide peptides. These results support our previous conclusions that these three neuropeptide families evolved early in evolution. In addition to these three neuropeptide families, hydrozoans expressed up to two other neuropeptide gene families, which, however, were only occurring in certain animal groups. Endocnidozoa (Myxozoa) are microscopically small endoparasites, which are strongly reduced. For long, it was unknown to which phylum these parasites belonged, but recently they have been associated with cnidarians. We analyzed nine endocnidozoan species and found that two of them (Polypodium hydriforme and Buddenbrockia plumatellae) expressed neuropeptide genes. These genes coded for neuropeptides belonging to the GRFamide and GLWamide families with structures closely resembling them from hydrozoans.

Conclusions: We found X₁PRX₂amide, GRFamide, and GLWamide peptides in all species belonging to the Hydrozoa, confirming that these peptides originated in the common cnidarian ancestor. In addition, we discovered GRFamide and GLWamide peptide genes in some members of the Endocnidozoa, thereby linking these parasites to Hydrozoa.

Keywords: Buddenbrockia; Cnidaria; Evolution; Fish parasite; Genomics; Hydrozoa; Myxozoa; Nervous system; Neuropeptide; Polypodium.

Fig. 1

Schematic drawing showing the phylogenetic positions of the subclasses Ceriantharia, Hexacorallia and Octocorallia (class Anthozoa), the classes Hydrozoa, Cubozoa, Scyphozoa, Staurozoa, and the subphylum Endocnidozoa. Cnidarians are a sister group to Bilateria. The figure also shows that X₁PRX₂amide (highlighted in purple), GRFamide (highlighted in yellow), and GLWamide peptides (highlighted in blue) are present in all tested Ceriantharia, Hexacorallia, Cubozoa, Scyphozoa, and Staurozoa species. However, the Octocorallia have apparently lost their GLWamide genes [52]. In the current paper, we are investigating the presence of these three neuropeptide families in Hydrozoa and Endocnidozoa. We found that hydrozoans contain all three neuropeptide families (X₁PRX₂amides, GRFamides, GLWamides). The endocnidozoans contain GRFamides and GLWamides, but have apparently lost their X₁PRX₂amide genes. In this figure we have presented the Endcnidozoa as two sister taxa, the Polypodiozoa and Myxozoa, but their mutual phylogenetic relationship is unclear

Fig. 2

The proposed intron/exon organization of the gene coding for the GRFamide preprohormone-C from H. magnipapillata and H. vulgaris. A. Protein sequences encoded by exons one to ten. The immature neuropeptide sequences are highlighted in red, while the C- and N-terminal cleavage sites are highlighted in blue. Note that the N-terminal peptide cleavage sites are always at the C-terminus of a Glu residue. The gene has ten exons that each code for a fragment of the preprohormone. Exon one codes for the N-terminus of the protein, which also includes the signal sequence. Exons two codes for one copy of Hydra-RFamide-1. Exons three codes for a protein sequence that includes one copy of the non-amidated neuropeptide pQWFSGRFGLX sequence that combines with the XX sequence at the N-terminus of exon four to yield pQWFSGRFGLXXX. Exon four codes for a pQWLSGRFGLX sequence that combines with the XX sequence at the N-terminus of exon five to yield pQWLSGRFGLXXX. This sequence of events continues until exon ten. Exon ten codes for the C-terminus of the protein, which also includes an XX sequence and one copy of Hydra-RFamide-1. B. Intron/exon organization. This genomic organization of the preprohormone-C gene is based on the assumption that H. magnipapillata and H. vulgaris are one species. The genomic organization is supported by our identification of a contig sc4wPfr_569 in the genomic database from H. magnipapillata, containing exons one to eight plus exon ten. Exon nine was found on a different contig sc4wPfr_90. We assume that this was due to a technical problem in the genome sequencing or assembly. All exons are flanked by consensus donor and acceptor splice sites. The possibility of alternative splicing is supported by our findings that different transcripts exist of the preprohormone-C gene: H. magnipapillata transcript-5 contains exons one to ten. H. vulgaris transcript-3 contains exons one, three to seven and exon ten. H. vulgaris transcript-8 contains exon one and exon two. Exon two contains a stop codon, which explains the absence of other exon combinations involving this exon