Hemocyanins are copper-containing respiratory proteins of the Arthropoda that have so far been thoroughly investigated only in the Chelicerata and the Crustacea but have remained unstudied until now in the Myriapoda. Here we report the first sequence of a myriapod hemocyanin. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two distinct subunits that are arranged in a 6 x 6 native molecule. The cloned hemocyanin subunit cDNA codes of for a polypeptide of 653 amino acids (75.5 kDa) that includes a signal peptide of 18 amino acids. The sequence closely resembles that of the chelicerate hemocyanins. Molecular phylogenetic analyses reject with high statistical confidence the integrity of the Tracheata (i.e., Myriapoda + Insecta) but give conflicting results on the position of the myriapod hemocyanin. While distance matrix and maximum-likelihood methods support a basal position of the Spirostreptus hemocyanin with respect to the other hemocyanins, parsimony analysis suggests a sister group relationship with the chelicerate hemocyanins. The latter topology is also supported by a unique shared deletion of an alpha-helix. A common ancestry of Myriapoda and Chelicerata should be seriously considered.

The velvet worms (Onychophora) are considered living fossils and are closely related to the Euarthropoda. Onychophora possess a tracheal system for respiratory function, but oxygen-transport proteins have been considered unnecessary. Here, we show that the hemolymph of the Epiperipatus sp. (Onychophora: Peripatidae) contains an arthropod-type hemocyanin, demonstrating that such protein exists outside the Euarthropoda. Thus, the evolution of oxygen carriers preceded the divergence of the Onychophora and Euarthropoda and was most likely linked to the evolution of an efficient circulatory system in a low-oxygen environment. The cDNA of the Epiperipatus hemocyanin subunit comprises 2,287 bp and encodes for a protein of 641 aa (73.6 kDa). Phylogenetic analyses of the arthropod hemocyanin sequences show that the Onychophora form a robust sister-group of the Euarthropoda, whereas the monophyly of the Tracheata is not supported.

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Hemocyanins are large oligomeric copper‐containing proteins that serve for the transport of oxygen in many arthropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnecessary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilopoda), as deduced from 2D‐gel electrophoresis, MALDI‐TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemocyanin to be fully sequenced, and allows the investigation of hemocyanin structure–function relationship and evolution. S. coleoptrata hemocyanin is a 6 × 6‐mer composed of four distinct subunit types that occur in an approximate 2 : 2 : 1 : 1 ratio and are 49.5–55.5% identical. The cDNA of a fifth, highly diverged, putative hemocyanin was identified that is not included in the native 6 × 6‐mer hemocyanin. Phylogenetic analyses show that myriapod hemocyanins are monophyletic, but at least three distinct subunit types evolved before the separation of the Chilopoda and Diplopoda more than 420 million years ago. In contrast to the situation in the Crustacea and Chelicerata, the substitution rates among the myriapod hemocyanin subunits are highly variable. Phylogenetic analyses do not support a common clade of Myriapoda and Hexapoda, whereas there is evidence in favor of monophyletic Mandibulata.

Arthropod hemocyanins are large respiratory proteins that are composed of up to 48 subunits (8 £ 6-mer) in the 75 kDa range. A 3D reconstruction of the 1 £ 6-mer hemocyanin from the European spiny lobster Palinurus elephas has been performed from 9970 single particles using cryoelectron microscopy. An 8 Å resolution of the hemocyanin 3D reconstruction has been obtained from about 600 final class averages. Visualisation of structural elements such as a-helices has been achieved. An amino acid sequence alignment shows the high sequence identity (. 80%) of the hemocyanin subunits from the European spiny lobster P. elephas and the American spiny lobster Panulirus interruptus. Comparison of the P. elephas hemocyanin electron microscopy (EM) density map with the known P. interruptus X-ray structure shows a close structural correlation, demonstrating the reliability of both methods for reconstructing proteins. By molecular modelling, we have found the putative locations for the amino acid sequence (597 -605) and the C-terminal end (654 -657), which are absent in the available P. interruptus X-ray data.