Diversity, evolution, and function of myriapod hemocyanins

Abstract

Background: Hemocyanin transports O₂ in the hemolymph of many arthropod species. Such respiratory proteins have long been considered unnecessary in Myriapoda. As a result, the presence of hemocyanin in Myriapoda has long been overlooked. We analyzed transcriptome and genome sequences from all major myriapod taxa - Chilopoda, Diplopoda, Symphyla, and Pauropoda - with the aim of identifying hemocyanin-like proteins.

Results: We investigated the genomes and transcriptomes of 56 myriapod species and identified 46 novel full-length hemocyanin subunit sequences in 20 species of Chilopoda, Diplopoda, and Symphyla, but not Pauropoda. We found in Cleidogona sp. (Diplopoda, Chordeumatida) a hemocyanin-like sequence with mutated copper-binding centers, which cannot bind O₂. An RNA-seq approach showed markedly different hemocyanin mRNA levels from ~ 6 to 25,000 reads per kilobase per million reads. To evaluate the contribution of hemocyanin to O₂ transport, we specifically studied the hemocyanin of the centipede Scolopendra dehaani. This species harbors two distinct hemocyanin subunits with low expression levels. We showed cooperative O₂ binding in the S. dehaani hemolymph, indicating that hemocyanin supports O₂ transport even at low concentration. Further, we demonstrated that hemocyanin is > 1500-fold more highly expressed in the fertilized egg than in the adult.

Conclusion: Hemocyanin was most likely the respiratory protein in the myriapod stem-lineage, but multiple taxa may have independently lost hemocyanin and thus the ability of efficient O₂ transport. In myriapods, hemocyanin is much more widespread than initially appreciated. Some myriapods express hemocyanin only at low levels, which are, nevertheless, sufficient for O₂ supply. Notably, also in myriapods, a non-respiratory protein similar to insect storage hexamerins evolved from the hemocyanin.

Keywords: Arthropoda; Evolution; Hemocyanin; Myriapoda; Phenoloxidase; Subunit diversity.

Fig. 1

The occurrence of hemocyanin in myriapods. The phylogenetic interrelationships of the myriapod classes and orders were taken from [37]; the position of the Pauropoda was added according to a recent study [36]

Fig. 2

Copper-binding sites A and B of hemocyanin subunits and phenoloxidase of Cleidogona sp. Conserved residues are shaded in grey. The six copper-binding histidines are shaded in black and denoted by asterisks. Note the replacements of three histidines in CleHcBI2, which cannot bind copper

Fig. 3

Phylogenetic analysis of the myriapod hemocyanin subunits. A Bayesian phylogenetic tree was inferred from an alignment of myriapod Hc and PPO amino acid sequences (Additional file 5: Figure S2). The displayed tree derived from the analysis of the full 868 amino acid (aa) alignment assuming the WAG model. The numbers at the nodes indicate the Bayesian support values according to the 868 aa alignment/WAG model, 531 aa alignment /WAG model, 868 aa alignment/LG model, 531 aa alignment/LG model. The black dots indicate support values > 0.98 in all four analyses. The bar represents 0.3 PAM distance. Chilopod Hc subunits and PPO are shaded in light grey; the symphylan proteins are shaded in dark grey; the different Hc subunit types are boxed and indicated on the right-hand side. See Additional file 2: Table S2 for the abbreviations

Fig. 4

Hemocyanin expression in myriapods. The mRNA levels of Hc subunits were determined by RNA-Seq based on the transcriptomes (Table 1) and displayed as RPKM values. RPKM numbers are given Additional file 6: Table S3, the abbreviations of the subunits in Additional file 2: Table S2. A figure displaying the log-values is provided as Additional file 5: Figure S2

Fig. 5

Oxygen-binding properties of the S. dehaani hemocyanin. Oxygen-binding curves were determined at pH 6.8 (grey) and 7.7 (black) by a polarographic-fluorometric method. Protein concentration was 0.2 mg/ml; the temperature was 20 °C

Fig. 6

Quantification of the S. dehaani hemocyanin and phenoloxidase. a Levels of mRNA of Hc subunit and PPO in hepatopancreas and egg, as estimated by qRT-PCR. b About 5 μg total protein of hemolymph, egg and hepatopancreas of S. dehaani were separated by SDS-PAGE, and Hc proteins were detected using an antibody directed against S. coleoptrata Hc