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BMC Evolutionary Biology

, 18:107 | Cite as

Diversity, evolution, and function of myriapod hemocyanins

  • Samantha Scherbaum
  • Nadja Hellmann
  • Rosa Fernández
  • Christian Pick
  • Thorsten Burmester
Open Access
Research article
Part of the following topical collections:
  1. Phylogenetics and phylogeography

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 

Abbreviations

Hc

Hemocyanin

MYA

Million years ago

P50

Half-saturation pressure

PPO

Prophenoloxidase

RPKM

Reads Per Kilobase exon model per Million reads; see Additional file 2: Table S2 for abbreviations of the hemocyanin subunits

Background

Oxygen (O2) is required for efficient generation of metabolic energy. In many animals, O2 is transported in the body fluid (blood or hemolymph) by specific binding-proteins. Such respiratory proteins have long been considered unnecessary in Myriapoda, which appeared to be well equipped for diffusive O2 transport by the tracheal system [1]. In the past years, it has become evident that at least some myriapods possess hemocyanin (Hc) for O2 transport [2, 3].

Hc is the respiratory copper-protein typically found in many arthropod and molluscan species [4, 5, 6, 7, 8, 9, 10]. Molluscan and arthropod Hcs do not share significant sequence similarities and are most likely the result of convergent evolution from different types of tyrosinases [7, 11]. Arthropod Hcs form hexamers or oligohexamers (up to 8 x 6mers), which are composed of similar or identical subunits of ~ 75 kDa [4, 5, 6, 7, 9]. In each subunit, O2 binds to a pair of Cu+-ions, which are coordinated by six histidine residues of the protein chain (type III copper-center). In addition to its prominent role in O2 -transport and -storage, arthropod Hc may carry out other functions, such as a source for antimicrobial peptides [12], as phenoloxidase [13] or as storage protein that provides amino acids and metabolic energy [14].

Arthropod Hcs are members of a protein superfamily that additionally includes arthropod phenoloxidases (POs), which are copper-containing enzymes involved in various immunological functions and cuticle building [10, 15], and various proteins that are involved in energy and amino acid storage, such as pseudo-hemocyanins (cryptocyanins) of some decapod crustaceans, hexapod hexamerins, and dipteran hexamerin receptors [6, 9, 10, 15, 16, 17, 18]. Sequences that share significant similarities with the arthropod proteins have been found in tunicates, hemichordates, sponges, and fungi [19, 20, 21]. Although functional analyses are missing, these proteins probably act as phenoloxidases.

Hc structure, function, and subunit evolution have been thoroughly studied in Chelicerata and Crustacea [5, 6, 7, 10, 22, 23, 24, 25, 26]. In the past about 15 years, Hc has also been identified in Hexapoda [27, 28] as well as in Onychophora [29], which are the sister taxon of the (eu-)arthropods. The first evidence for the presence of Hc in Myriapoda was found in the centipede Scutigera longicornis (Scutigeromorpha, Chilopoda) [30]. Mangum and colleagues [3] demonstrated that the Hc from Scutigera coleoptrata is a 6×6-mer, which is composed of four distinct subunit types [31, 32]. The presence of Hc in Scutigera was attributed to the high activity of this species and its blind-ending tracheal system [3]. However, a structurally similar Hc, which occurs as a mixture of 6×6-mers and 3×6-mers, was identified in the hemolymph of Spirostreptus sp. (Spirostreptida, Diplopoda) [2] and Archispirostreptus gigas [33]. In Polydesmus angustus (Polydesmida, Diplopoda) Hc occurs as a 3×6-mer [34].

Here, we present a thorough survey of Hc in 56 myriapod species, which has been made possible by the recent availability of transcriptome and genome data [35, 36, 37, 38, 39]. Employing an RNA-seq approach, we found large differences in mRNA levels across species. By specifically analyzing the Hc of the centipede Scolopendra dehaani, we demonstrated that Hc can contribute to O2 transport in the hemolymph even at low expression levels and that Hc may have a specific role in early myriapod development.

Methods

Databases and hemocyanin sequences

Short read data (Illumina or 454) of transcriptomes from 54 myriapod species were obtained from the SRA database at NCBI (https://www.ncbi.nlm.nih.gov/sra/) (Table 1). The transcriptomes were assembled with Trinity [40] using the standard settings. Contigs with > = 500 bp were kept and searched for Hc and PO cDNA sequences using a locally installed BLAST tool [41]. The derived sequences were verified by back-mapping of the reads from the corresponding species employing the CLC Genomics Workbench 11.0.0 (https://www.qiagenbioinformatics.com/). This approach was also used to extend partial sequences. Few incomplete or ambiguous Hc sequences that could not be resolved were discarded. The genome of Strigamia maritima Leach, 1817 [38] was searched and analyzed for Hc and PPO sequences at http://www.ensembl.org/. Genomic sequences of Trigoniulus corallinus were obtained from [39]. Scaffolds and contigs that cover the single Hc gene were assembled by hand. The Hc coding sequences were predicted by AUGUSTUS (http://bioinf.uni-greifswald.de/webaugustus/) [42] and GENSCAN (http://genes.mit.edu/GENSCAN.html) [43], and verified by the aid of a multiple sequence alignment with other myriapod Hc sequences. The cDNA sequences were translated into proteins with the translate tool at the ExPASy Server of the Swiss Institute of Bioinformatics (http://web.expasy.org/translate/).
Table 1

List of short read sequences used in this study

Species

Order

Class

SRA

Tissue

Sequencing

Hemocyanin

RPKM

Hanseniella sp.

 

Symphyla

SRX1734405, SRX3326582

whole body, body tissue

Illumina

1 Hc

328.1

Scutigerella sp.

 

Symphyla

SRX1734406

whole body

Illumina

1 Hc

997.7

Symphylella vulgaris

 

Symphyla

SRX246919

whole body

454

n.d.

0

Symphylella sp.

 

Symphyla

SRX3256428

trunk

Illumina

n.d.

0

Pauropus huxleyi

 

Pauropoda

SRX3257465

whole body

Illumina

n.d.

0

Eudigraphis taiwaniensis

Polyxenida

Diplopoda

SRX1734393

body tissue

Illumina

n.d.

0

Polyxenus lagurus

Polyxenida

Diplopoda

SRX390592

whole body

454

n.d.

0

Cyliosoma sp

Sphaerotheriida

Diplopoda

SRX1734403

body tissue

Illumina

n.d.

0

Glomeris marginata

Glomerida

Diplopoda

SRX1638914

body tissue

Illumina

n.d.

0

Glomeris pustulata

Glomerida

Diplopoda

SRX246920

whole body

454

n.d.

0

Glomeridesmus sp.

Glomeridesmida

Diplopoda

SRX326775

n.a.

Illumina

n.d.

0

Brachycybe lecontii

Playtdesmida

Diplopoda

SRX326776

n.a.

Illumina

n.d.

0

Petaserpes sp.

Polyzoniida

Diplopoda

SRX326777

n.a.

Illumina

n.d.

0

Cleidogona sp.

Chordeumatida

Diplopoda

SRX326780

n.a.

Illumina

5 Hc

549.3

Abacion magnum

Callipodida

Diplopoda

SRX326781

n.a.

Illumina

4 Hc

232.2

Chamberlinius hualienensis

Polydesmida

Diplopoda

DRX028808

animals without gut

454

3 Hc

2145.4

Polydesmus angustus

Polydesmida

Diplopoda

SRX390267

whole body

454

3 Hc

195.8

Pseudopolydesmus sp.

Polydesmida

Diplopoda

SRX326779

n.a.

Illumina

3 Hc

460.6

Prostemmiulus sp.

Stemmiulida

Diplopoda

SRX326782

n.a.

Illumina

4 Hc

25,587.7

Narceus americanus

Spirobolida

Diplopoda

SRX1638916

body tissue

Illumina

n.d.

0

Trigoniulus corallinus

Spirobolida

Diplopoda

SRX700727

whole organism

Illumina (gemome)

1 Hc

n.a.

Cambala annulata

Spirostreptida

Diplopoda

SRX326783

n.a.

Illumina

4 Hc

1183.5

Cylindroiulus punctatus

Julida

Diplopoda

SRX1734404

body tissue

Illumina

n.d.

0

Scutigera coleoptrata

Scutigeromorpha

Chilopoda

SRX462011

n.a.

Illumina

5 Hc

22,719.8

Scutigerina weberi

Scutigeromorpha

Chilopoda

SRX1637773

body tissue

Illumina

4 Hc

1904.3

Sphendononema guildingii

Scutigeromorpha

Chilopoda

SRX1637754

body tissue

Illumina

4 Hc

3238.3

Thereuopoda cf. longicornis

Scutigeromorpha

Chilopoda

SRX450808

venom gland

454

n.d.

0

Craterostigmus tasmanianus

Craterostigmomorpha

Chilopoda

SRX461877

n.a.

Illumina

n.d.

0

Craterostigmus crabilli

Craterostigmomorpha

Chilopoda

SRX1638413

body tissue

Illumina

n.d.

0

Anopsobius giribeti

Lithobiomorpha

Chilopoda

SRX1638351

body tissue

Illumina

n.d.

0

Eupolybothrus cavernicolus

Lithobiomorpha

Chilopoda

ERX311347

whole organism

Illumina

n.d.

0

Lithobius forficatus

Lithobiomorpha

Chilopoda

SRX462145, SRX270896

n.a., whole organism

Illumina + 454

n.d.

0

Lithobius sp.

Lithobiomorpha

Chilopoda

SRX326784

n.a.

Illumina

n.d.

0

Paralamyctes validus

Lithobiomorpha

Chilopoda

SRX1638281

body tissue

Illumina

n.d.

0

Notiphilides grandis

Geophilomorpha

Chilopoda

SRX1734365

body tissue

Illumina

n.d.

0

Henia brevis

Geophilomorpha

Chilopoda

SRX1734368

body tissue

Illumina

n.d.

0

Himantarium gabrielis

Geophilomorpha

Chilopoda

SRX461787

n.a.

Illumina

n.d.

0

Hydroschendyla submarina

Geophilomorpha

Chilopoda

SRX1638908

body tissue

Illumina

n.d.

0

Mecistocephalus guildingii

Geophilomorpha

Chilopoda

SRX1638910

body tissue

Illumina

n.d.

0

Stenotaenia linearis

Geophilomorpha

Chilopoda

SRX1638912

body tissue

Illumina

n.d.

0

Strigamia maritima

Geophilomorpha

Chilopoda

SRX530370-SRX530372

mixed

Illumina (gemome)

n.d.

0

Tygarrup javanicus

Geophilomorpha

Chilopoda

SRX1638906

body tissue

Illumina

n.d.

0

Akymnopellis chilensis

Scolopendromorpha

Chilopoda

SRX1638823

body tissue

Illumina

n.d.

0

Alipes grandidieri

Scolopendromorpha

Chilopoda

SRX205685

n.a.

Illumina

2 Hc

19.6

Cormocephalus westwoodi

Scolopendromorpha

Chilopoda

SRX273033

venom gland

454

n.d.

0

Cryptops hortensis

Scolopendromorpha

Chilopoda

SRX457664

n.a.

Illumina

1 Hc

338.0

Ethmostigmus rubripes

Scolopendromorpha

Chilopoda

SRX275322, SRX423979, SRX272980, SRX275340

venom gland + epidermis

454

n.d.

0

Newportia adisi

Scolopendromorpha

Chilopoda

SRX1638658

body tissue

Illumina

3 Hc

573.4

Rhysida longipes

Scolopendromorpha

Chilopoda

SRX1638865

body tissue

Illumina

n.d.

0

Scolopendra alternans

Scolopendromorpha

Chilopoda

SRX275341

venom gland

454

n.d.

0

Scolopendra morsitans

Scolopendromorpha

Chilopoda

SRX273031

venom gland

454

n.d.

0

Scolopendra mutilans

Scolopendromorpha

Chilopoda

SRX286707, SRX286708

whole organism

Illumina

3 Hc

589.7

Scolopendropsis bahiensis

Scolopendromorpha

Chilopoda

SRX1734364

body tissue

body tissue

2 Hc

273.2

Scolopocryptops sexspinosus

Scolopendromorpha

Chilopoda

SRX1638758

body tissue

Illumina

fragments

n.a.

Scolopendra dehaani

Scolopendromorpha

Chilopoda

SRX390596

body tissue (trunk)

454

2 Hc

74.1

Theatops spinicaudus

Scolopendromorpha

Chilopoda

SRX1734363

body tissue

Illumina

1 Hc

6

n.a. not available, n.d. not detected, RPKM cumulative RPKM

Cloning and sequencing of S. dehaani hemocyanins and phenoloxidase cDNAs

An adult female centipede S. dehaani Brandt, 1840 (Scolopendromorpha, Chilopoda) was obtained from a local pet shop. Selected tissues of S. dehaani were dissected, shock-frozen in liquid N2 and kept at − 80 °C until use. Total RNA was extracted either according to the method by Holmes and Bonner [44] or with the RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. The quality of the RNA was checked by measuring the OD 260/280 ratio and by gel electrophoresis. cDNA was obtained using the SuperScriptTM III RNase H-Reverse Transcriptase Kit (Invitrogen, Karlsruhe, Germany), employing an oligo(dT)-primer. Partial S. dehaani Hc and PO sequences were obtained with PCR using sets of primers that had been generated on the basis of partial cDNAs [35] (Additional file 1: Table S1). Missing 5′ and 3′ ends were completed by the RACE technique using the GeneRacer™ kit (Invitrogen) according to the manufacturer’s instructions. The PCR products were cloned into the pGEM-T vector (Promega, Mannheim, Germany) and sequenced by a commercial service (GATC, Konstanz, Germany).

Sequence analyses and phylogenetic studies

N-terminal signal sequences required for export into the extracellular space were predicted with SignalP 4.1 [45]. An alignment of the amino acid sequences of myriapod Hcs and prophenoloxidases (PPOs) (Additional file 2: Table S2) was constructed with MAFFT 7 [46] with the G-INS-i method and the BLOSUM 62 matrix. The final alignment covered 57 Hc and 16 PPO sequences and 868 characters (Additional file 3: Figure S1). An additional alignment was generated by eliminating poorly aligned sections by Gblocks v0.91b [47], employing the options for a less stringent selection (smaller final blocks, gap positions within the final blocks, less strict flanking positions). This approach resulted in an alignment of 531 amino acids. The best-fitting models of amino acid sequence evolution, LG [48] and WAG [49], were selected with ProtTest [50] under the Akaike Information Criterion. MrBayes 3.2.6 [51] was used for Bayesian phylogenetic analysis. The LG model was coded as GTR model with fixed priors using the prset command of MrBayes by specifying the parameters aarevmatpr and statefreqpr. Metropolis-coupled Markov chain Monte Carlo sampling was performed with one cold and three heated chains in two independent runs for 5 million generations on the CIPRES web portal [52]. Prior probabilities were equal, starting trees were random, and tree sampling was performed every 1000th generation. The final average standard deviation of split frequencies was < 0.005, suggesting convergence of the chains. Posterior probability densities of the nodes were estimated after discarding the initial 25% of the trees as burnin.

Quantitative real-time reverse transcription-PCR

Reverse transcription was performed with 775 ng total RNA from eggs and fat body of S. dehaani employing the SuperScriptTM III RNase H-Reverse Transcriptase Kit (Invitrogen) and oligo(dT)20 primer according to the manufacturer’s instructions. Quantitative real-time reverse transcription PCR (qRT-PCR) was performed with primer sets specific for Hc subunits, PO, β-actin and RPLP0 (Additional file 1: Table S1) using the Power SYBR Green PCR Master Mix and the 7500 Real-Time PCR System (Applied Biosystems, Darmstadt). qRT-PCR reactions were performed in technical triplicates. Amplification was carried out using a standard PCR protocol (95 °C for 15 s, 58 °C for 15 s, and 72 °C for 30 s; 40 cycles) and fluorescence was measured at the last step of each cycle. The specificity of the amplification reactions was validated by analysis of the dissociation curve. The mRNA copy numbers were calculated with the standard curve approach, which employs a dilution series with plasmids carrying the respective cDNA sequences [53]. Calculations were performed with the 7500 Software 2.0.6 (Applied Biosystems).

RNA-Seq

RNA-Seq analyses were performed with the CLC Genomics Workbench. The reads of the individual transcriptomes were mapped to the Hc cDNA sequences. The following parameters were applied: Masking mode = No masking, Update contigs = No, Match score = 1, Mismatch cost = 2, Cost of insertions and deletions = Linear gap cost, Insertion cost = 3, Deletion cost = 3, Length fraction = 0.5, Similarity fraction = 0.95, Global alignment = No, Auto-detect paired distances = Yes, Non-specific match handling = Map randomly. The mRNA levels of the Hc subunit were calculated as RPKM (Reads Per Kilobase exon model per Million reads).

SDS-PAGE and western blotting

Proteins concentrations from total hemolymph, egg and fat body extracts were determined photometrically. The proteins were separated by SDS-PAGE on a 10% gel with standard conditions [54]. The gels were stained with 0.1% Coomassie Brilliant Blue dissolved in 10% acetic acid/ 25% isopropanol. For Western blotting, proteins were transferred onto nitrocellulose. Non-specific binding sites were blocked with 4% non-fat dry milk in TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl). An antiserum raised in rabbits against S. coleoptrata Hc [32] was diluted 1:10,000 in 5% non-fat dry milk in TBS and used for detection overnight at 4 °C. After four successive washing steps with 0.1% Tween-20 in TBS, the secondary antibody (goat α-rabbit Fab; Dianova, Hamburg) was applied in a 1:10,000 dilution for 1 h at room temperature. After four additional washing steps, the Hc bands were detected with nitroblue tetrazolium and 5-Bromo-4-chloro-3-indolyl phosphate in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, in the dark. The intensity of the Hc bands was estimated with the ImageJ program (https://imagej.nih.gov/ij/).

Oxygen binding curves

Oxygen-binding curves were determined by the polarographic-fluorometric method [55], which bases on the fluorescence of deoxygenated Hc upon excitation with light with a wavelength of 290 nm. The intensity of this fluorescence linearly decreases with increasing O2 saturation of Hc. The fluorescence was measured with Hitachi F4500 (Binninger Analytic, Germany) at 338 nm, while the oxygen concentration of the Hc solution was determined simultaneously with an oxygen electrode (Microelectrodes. Inc., Bedford, USA) equipped with a home-built amplifier. Hemolymph was diluted twofold with 100 mM Tris-HCl, 20 mM MgCl2, 20 mM CaCl2 directly after sampling. The diluted hemolymph was centrifuged (10 min, 12,000 x g) and the supernatant was further diluted into Ringer solution (500 mM NaCl, 12 mM KCl, 12 mM CaCl2, 20 mM MgCl2, 10 mM Tris/HCl) at the indicated pH. The resulting O2 binding curves were used to determine the half-saturation pressure P50 and, after conversion by a Hill plot, cooperativity.

Results

Occurrence of hemocyanin sequences in Myriapoda

We screened the available transcriptomes of 54 myriapod species [35, 36, 37] for the presence of Hc or PPO (Table 1). We found putative Hc sequences in the transcriptomes of 20 species belonging to Chilopoda (Scutigeromorpha and Scolopendromorpha), Diplopoda (Chordeumatida, Callipodida, Polydesmida, Stemmiulida, Spirobolida, and Spirostreptida) and Symphyla (Fig. 1). We included in the survey the transcriptomes of the venom glands of several scolopendromorphs and scutigeromorphs, which were found devoid of Hc mRNA. A Hc gene could also be assembled from the genome of T. corallinus (Spirobolida) [39]. By contrast, no Hc gene was found in the genome of the geophilomorph S. maritima [38]. Therefore, a total of 21 of the 56 (54 transcriptomes + two genomes) investigated myriapod species harbor at least one putative Hc genes.
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]

The contigs that included Hc were extracted and the Hc cDNA sequences were deduced (Additional file 4: Data S1). In some cases, contigs were re-assembled or completed by back-mapping of the original Illumina reads. By this approach, we obtained 50 full-length Hc subunits sequences from the genomes and transcriptomes of 20 myriapod species. In the transcriptome of Scolopocryptops sexspinosus (Scolopendromorpha), only few Hc fragments were found, which could not be assembled. This species was ignored in our analyses. The sequences of the Hc subunits A – D of S. coleoptrata obtained before [32] were identical to that in the transcriptomes, except three isofunctional replacements in subunit B (ScoHcB). For ScoHcX, a highly divergent subunit that is not included in the native Hc [32], only a cDNA fragment was found, which displays multiple substitutions. In summary, this study revealed 46 novel Hc cDNA-sequences. The transcriptome of S. dehaani showed the presence of Hc-containing contigs, which could be assigned to two distinct subunits. The full-length sequences were obtained by 5′ and 3’ RACE methods.

The cDNAs were translated in silico into proteins, which were included in an alignment that contained all other available myriapod Hc sequences [32, 33, 34, 56], resulting in a dataset of 57 putative myriapod Hc subunit sequences. These were assigned to specific subunit types on the basis of phylogenetic analyses (see below). We also added 16 PPO sequences identified in this or a previous study [34]. In all Hc subunits but HcBI2 (CleHcBI2) of Cleidogona sp. (Chordeumatida) the six histidines required for copper-binding and thus O2 transport are conserved (Additional file 3: Figure S1; Fig. 2). In both copper-binding sites (CuA and CuB) of CleHcBI2, one or two copper-coordinating histidines are replaced by another amino acid. Except for a low content of histidines (22 vs. 47 in CleHcBI1), no peculiarities in amino acid composition could be found in CleHcBI2.
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

Molecular phylogeny of myriapod hemocyanin subunits

The multiple sequence alignment of the myriapod Hc and PPO sequences were subject to Bayesian phylogenetic reconstructions. As previous studies demonstrated the monophyly of the myriapod Hc subunits in respect to the Hc subunits of other arthropod subphyla [32, 34], we restricted the present phylogenetic analyses to the sequences of the Myriapoda. We employed a full alignment and, also, an alignment in which poorly aligned sections have been removed by Gblocks [47], and two different models of amino acid evolution (WAG and LG). The PPOs were considered as the outgroup.

The four resulting trees were largely similar (Fig. 3). In all trees, we found five different types of subunits, which we named according to the subunit types of the centipede S. coleoptrata A-D [31, 32]. In phylogenetic analyses, A and B-type subunits on the one hand and C and D-type subunits on the other hand, form two distinct clades (Fig. 3). Within the C and D-type subunits, chilopod and diplopod Hc subunits are strictly separated. Symphylans only have B-type subunits, which associated with the chilopod B-type subunits, albeit with poor support (0.45 to 0.96 Bayesian posterior probability; BPP). In the Diplopoda, we did not find subunit type A, but two well-supported clades of distinct B-type subunits, which were thus named BI and BII. It should be noted that B-type subunits were found in all investigated myriapod species that have Hc. The copper-free Hc subunit HcBI2 of Cleidogona sp. is associated with the “typical” subunit HcBI1 of the same species.
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

Estimating Hc subunit mRNA levels by RNA-seq

We studied the expression of Hc subunit mRNA by RNA-Seq, employing the publically available transcriptomes of 19 species (Table 1). The origin of the RNA used for RNA-Seq varied, although in most cases whole organisms or mixed body tissue have been used. We found highly divergent expression levels, which varied several orders of magnitude (Fig. 4; Additional file 5: Figure S2; Additional file 6: Table S3). The lowest Hc mRNA level was observed in the scolopendromorph Theatops spinicaudus, which has a single Hc subunit that displays 6 RPKM. Prostemmiulus sp. (Stemmiulida) had a cumulative RPKM of to 25,587.7. High Hc expression levels were also found in the Scutigeromorpha, with S. coleoptrata having the highest Hc expression levels (cumulative RPKM 22719.8). In most species, the RPKM of the different Hc subunits were in the same range, except the HcX-subunit of S. coleoptrata, which has a more than 1000-fold lower mRNA level than the other Hc subunits of this species. However, HcX is not a component of the native Hc and has an unknown function [32].
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

O2 binding properties of S. dehaani hemocyanin

Equilibrium O2 binding curves of S. dahaani Hc were determined by measuring the O2-dependent fluorescence of the hemolymph of this species. Because there is no other respiratory protein in the hemolymph of this species and oxygen-dependent changes of the tryptophan fluorescence at this low oxygen concentration is specific for Hc, this approach is valid. We employed two different pH values in our measurements: At pH 7.7, the P50 was ~ 19 Torr (2.6 kPa), at pH 6.8 ~ 27 Torr (3.6 kPa) (Fig. 5). The reduced O2 affinity at low pH indicates that S. dahaani Hc displays the typical Bohr effect observed in hemocyanins. The sigmoidal O2-binding curve indicates cooperative O2-binding and a Hill coefficient (h) of 1.9 (pH 7.7) and 1.6 (pH 6.8) was calculated.
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

mRNA and protein expression of S. dehaani hemocyanin

Here, we investigated a female specimen of S. dehaani that carried fertilized eggs. Relative expression of Hc and PPO mRNA levels was determined using qRT-PCR in the hepatopancreas and the eggs. Notably, we found 3400-fold (SdeHcA) and 1500-fold (SdeHcB) higher mRNA levels in the oocyte compared to the hepatopancreas (Fig. 6a). The differences in the levels of PPOs were less pronounced, with factors ranging from 0.4 to 237. In addition, proteins were isolated from hemolymph, eggs and hepatopancreas, and analyzed by Western blotting. A single band with a mass of 80 kDa was detected (Fig. 6b). The relative band intensities were quantified with the software ImageJ, showing six-fold higher Hc protein levels in the egg than in the hepatopancreas.
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

Discussion

For a long time, it has been assumed that – regardless of species and/or developmental stage – myriapods do not have Hc or any other respiratory protein. Only the Scutigeromorpha were considered as exceptions [3, 30]. However, more recent data have suggested that Hc is also present in other myriapods [2, 33, 34, 56]. A thorough survey on the occurrence, subunit diversity, and expression pattern was missing. Also, it was unknown why Hc was lost in some myriapod taxa, and whether Hc can significantly contribute to O2 supply.

Widespread occurrence, losses and divergent expression levels of hemocyanins in Myriapoda

The examination of the available transcriptomes and genomes of the myriapods [35, 36, 37, 38, 39] showed that Hc genes are present in three of the four myriapod classes, i.e., Chilopoda, Diplopoda, and Symphyla (Table 1; Fig. 1). No evidence for Hc was found in the transcriptome of Pauropoda. These findings confirm previous notions that Hc was present in the myriapod stem-lineage. Mapping of the occurrence of Hc onto the myriapod phylogenetic tree (Fig. 1) did not reveal a clear pattern. However, we must consider that a Hc gene may only be expressed in some developmental stages or certain tissues (see below), and might have been missed in this survey because the corresponding samples were not available. Thus, conclusive evidence for the absence of a Hc gene could only be derived from the genome of S. maritima. In those other species, in which no Hc mRNA was identified in the transcriptomes, at least a role of Hc in O2 supply in the adult can be excluded.

The phylogenetic pattern of the presence/absence suggests that multiple independent losses of the Hc gene occurred during myriapod evolution. Losses of Hc genes were also observed in certain taxa of Chelicerata, Crustacea and Hexapoda [5, 22, 28, 57]. Within Diplopoda, Hc appears to be restricted to the Eugnatha (except Julida), while Hc is absent in the early diverging diplopod orders. In the chilopods, Hc is present in Scutigeromorpha and Scolopendromorpha. While Scutigeromorpha split from the other Chilopoda early in evolution, Scolopendromorpha occupy a terminal position as the sister group to Geophilomorpha. Thus, there is no true correlation between the presence of Hc and myriapod phylogeny (Fig. 1). With the exception of Scutigeromorpha (see below), there is also no apparent morphological, physiological or ecological explanation for the presence of Hc, as the species with Hc have quite different body plans, behavior, and habitats. Thus, there must be others – yet unknown – features that have made a respiratory protein redundant in some species and explain its retention in others.

We also observed divergent expression rates across the myriapod taxa. Also in this context, we must consider that some variations in the RPKM may be due to differences in tissues or developmental stages used for transcriptome generations. There is no clear pattern across the taxa (Fig. 4), although in chilopods there is a tendency of high Hc mRNA levels in Scutigeromorpha and low Hc mRNA levels in Scolopendromorpha. The high mRNA Hc levels in Scutigeromorpha agrees with previous observations [3, 30]. A requirement of an effective O2 transporting system in the Scutigeromorpha can be explained by the high activity of this species, which is also reflected by the presence of a peculiar, highly branched circulatory system and tracheal lungs [3, 58]. The highest Hc mRNA levels were found in the diplopod Prostemmiulus sp. (Stemmiulida). To the best of our knowledge, there is at present no specific characteristics that could explain the high expression of Hc mRNA in this species. Notably, S. coleoptrata and Prostemmiulus sp. display approximately tenfold higher Hc mRNA levels (> 20,000 RPKM) than any other myriapod species. The very low Hc mRNA in several Scolopendromorpha (S. dehaani, Alipes grandidieri and Theatops spinicaudus) with RPKM < 100 raised the question whether Hc contributes to O2 transport, or whether this protein may have another function in those species (see below).

Myriapod hemocyanin subunit diversity and evolution

Previous studies have demonstrated that distinct Hc subunit types occur in chelicerates, crustaceans, and hexapods [5, 10, 22, 28, 59]. Up to eight distinct subunit-types may occur in a single Hc molecule [25]. The presence of distinct subunit types is a common feature of arthropod Hc; they emerged independently in the different subphyla, and thus the subunit-types may have a long and independent evolutionary history of several hundred million years [10, 22, 28, 60, 61]. The presence of multiple distinct subunits has been associated with the controlled assembly to distinct quaternary structure, containing up to 48 subunits, and thus allowing establishment and regulation of high cooperativity in Hc or – if differentially expressed – may reflect specific needs during development and in response to environmental changes.

Most chelicerates have conserved 4 × 6-mer or 8 × 6-mer Hcs with seven distinct subunit types that emerged ~ 540 million years ago (MYA) [22, 25, 62]. The pattern in crustaceans is more complex and further complicated by the paraphyletic nature of crustacean and hexapod Hc subunits [60, 61, 63].

Myriapod species have between one and five distinct Hc subunits. Because the subunit HcX of S. coleoptrata is not present in the native Hc protein [32] and the copper-free subunit HcBI2 of Cleidogona sp. is also probably not part of the respiratory protein, the maximum number of distinct subunits in the native (oligo-)hexameric Hc is probably four. Notably, a B-type subunit was found in all myriapod species investigated here, suggesting that this polypeptide may be the central building block of the native Hc. The phylogenetic studies showed that orthologous C- and D-type subunits occur in both Diplopoda and Chilopoda. A-type subunits appear to be restricted to Chilopoda; the HcX subunit of S. coleoptrata is a B-type variant. In Diplopoda, two paralogous HcB subunits are present (HcBI and HcBII). Most likely, the duplication of the HcB gene was the response to the loss of the HcA subunit in this taxon. In a previous study [34], we identified two clades of Hc subunits in the myriapods, one which are built by the subunit types A and B and one which contains only C and D subunits. We could confirm this result in the current study. Further, the tree topology implies that the four distinct subunit types diverged before the Diplopoda, Symphyla and Chilopoda separated more than 500 MYA ago [35, 37, 64]. In case that multiple subunits occur within a single species, mRNA expression analyses showed that their RPKM values are similar within an order of magnitude. However, from the expression data, it is difficult to estimate the exact subunit composition of the (oligo-) hexameric Hc protein.

A myriapod hemocyanin-like protein that does not bind O2

Cleidogona sp. (Diplopoda:Chordeumatida) harbors a phenoloxidase and five sequences that share significant similarities with the typical diplopod Hc subunits. Notably, we found that the HcBI subunit is duplicated. Closer investigation showed that in one of these subunits (which we called CleHcBI2) three of the six copper-binding histidines are replaced by another amino acid (CuA: H→Y, H→N; CuB: H→L; Fig. 2). Therefore, CleHcBI2 cannot bind copper and thus O2, and is unlikely to be integrated into Hc, or has a PO-like function. Phylogenetic analyses (Fig. 3) showed an about 4.5-fold faster evolutionary rate of CleHcBI2 compared to CleHcBI1, which is most likely due to the relief of the constraints imposed by the function of O2 transport. We speculate that CleHcBI2 carries out a similar function as the insect hexamerins or decapod pseudo-hemocyanins (cryptocyanins), Hc-related proteins which are used mainly for the storage of energy and amino acids [10, 16, 17, 18, 65]. Many hexamerins accumulate specific amino acids [18, 66], a feature that was not observed in CleHcBI2. The only notable difference in amino acid composition between CleHcBI2 and CleHcBI1 was the reduced relative amount of histidines (3.38% vs. 7.14%) beyond the replacement of the copper-binding sites. The phylogenetic tree (Fig. 3) suggests that this copper-free protein is not a common feature of myriapods or diplopods, but rather evolved specifically within the Chordeumatida. Nevertheless, it is remarkable that Hc has lost three times independently in different arthropod subphyla its respiratory function and probably evolved into a storage protein. This suggests that Hc has particular structural advantages, which may be for example their high stability or the ability to accumulate many amino acids with low osmotic impact due to their large size [17].

Hemocyanin supports O2 supply even at low concentration

The low expression of Hc in many myriapod species raised the question whether this protein can in principle support O2 transport. We, therefore, analyzed the Hc of the centipede S. dehaani. This protein consists of two distinct subunits (SdeHcA and SdeHcB), and expression analyses showed RPKM values of 25.6 and 48.5, respectively (Fig. 4; Additional file 6: Table S3). We showed that the hemolymph and thus the Hc of S. dehaani does reversibly bind O2. Moreover, it displays a Bohr effect and cooperative O2-binding. These features strongly suggest a respiratory function of Hc in S. dehaani.

S. dehaani Hc displays a low O2 affinity (P50 = 19 Torr [2.6 kPa] to 27 Torr [3.6 kPa]; Fig. 5), which is higher than that of S. coleoptrata (P50 = 55 Torr [7.32 kPa] at pH 7.5) [3] but lower than the affinities measured for the diplopod Hcs (Spirostreptus Hc P50 = 4.7 Torr [0.63 kPa] at pH 7.5; A. gigas Hc (P50 = 3.45 Torr [0.46 kPa] at pH 8.1) [2, 33]. A similar pattern was found for the cooperativity, with high Hill coefficient for of S. coleoptrata (h = 8.9 at pH 7.5), an intermediate value for S. dehaani (h = 1.6 to 1.9) and low cooperativity for (h = 1.3 ± 0.2) for Spirostreptus Hc. These values show the flexibility of the O2 binding behavior of myriapod Hc, which most likely reflect differences in habitat and lifestyle. For example, a low O2 affinity and high cooperativity of S. coleoptrata Hc may be adaptive in efficient O2 release in this highly active species, whereas the high O2 affinity of Spirostreptus Hc may be interpreted as an adaptation for O2 storage function or efficient extraction of O2 in the sub-terrestrial environment. S. dehaani Hc appears to be better adapted to O2 release.

The embryo may require high levels of Hc

We studied a female S. dehaani specimen with fertilized eggs. The exact developmental stage of the egg is unknown. Notably, we found much more Hc mRNA and also slightly enhanced Hc protein levels in the eggs than in the hepatopancreas, which is the principal site of Hc synthesis in adult crustaceans, hexapods and myriapods [27, 56, 67]. The relative Hc protein amount in the egg is probably underestimated due to the high concentration of yolk proteins. Our findings suggest that high levels of Hc mRNA are required for the early development of the S. dehaani. An embryo-specific role of Hc has also been demonstrated in the hexapods [68, 69, 70]. The accumulation of Hc mRNA in the fertilized egg may be required to meet the O2 requirements of the early development of the embryo and the restriction of O2 diffusion across the eggshell. In adult S. dehaani, the tracheal system additionally supports O2 supply, thus a lower concentration of Hc is required.

Conclusions

Our results demonstrated that Hc was most likely the standard respiratory protein in the myriapod stem-lineage. Although Hc has been lost in certain taxa, it is still much more widespread in myriapods than initially appreciated. In previous studies, the presence of Hc in some myriapod species may have been overlooked due to the restriction to early developmental stages. Furthermore, some myriapods express Hc only at very low levels. Nevertheless, it is likely that Hc’s function is to support O2 supply by the hemolymph since the cooperative binding behavior is retained. Notably, in myriapods a non-respiratory protein evolved from Hc, which may act as a storage protein similar to insect hexamerins.

Notes

Acknowledgments

We thank Andrej Fabrizius (Hamburg University, Germany) for his help with the bioinformatic software and Danilo Harms (Hamburg University, Germany) for his advice on centipede reproduction. We acknowledge the support by Gonzalo Giribet (Harvard University, USA).

Funding

This work is supported by the Deutsche Forschungsgemeinschaft (Bu956/9). The funding body had no role in the design of the study and collection, analysis, interpretation of data, or in writing the manuscript.

Availability of data and materials

All supporting data are available in the Additional files 1, 2, 3, 4, 5 and 6. The short read sequences listed in Table 1 are available from the SRA database at NCBI: https://www.ncbi.nlm.nih.gov/sra

Authors’ contributions

SS and CP obtained the sequences and biochemical data from S. dehaani. NH collected the O2-binding data. RF obtained the transcriptome sequences. TB analyzed the sequence and expression data, and wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

We followed the regulations of the German Animal Welfare Act (TierSchG).

Consent for publication

Does not apply.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary material

12862_2018_1221_MOESM1_ESM.pdf (65 kb)
Additional file 1 Table S1. List of primer sequences used in this study. (PDF 64 kb)
12862_2018_1221_MOESM2_ESM.docx (25 kb)
Additional file 2 Table S2. List of sequences used in this study. (DOCX 24 kb)
12862_2018_1221_MOESM3_ESM.pdf (101 kb)
Additional file 3: Figure S1. Multiple sequence alignment of myriapod hemocyanins and phenoloxidases in FASTA format. (PDF 101 kb)
12862_2018_1221_MOESM4_ESM.zip (82 kb)
Additional file 4: Data S1. Nucleotide sequences of the Hc and PPO cDNAs and gene identified in this study. (ZIP 81 kb)
12862_2018_1221_MOESM5_ESM.tif (3 mb)
Additional file 5: Figure S2. The mRNA levels of Hc subunits were determined by RNA-Seq based on the transcriptomes (Table 1) and displayed as log RPKM values. (TIF 3 mb)
12862_2018_1221_MOESM6_ESM.pdf (72 kb)
Additional file 6: Table S3. Expression of hemocyanin subunit mRNA in myriapods. (PDF 72 kb)

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.Institute of ZoologyUniversity of HamburgHamburgGermany
  2. 2.Institute for BiophysicsJohannes Gutenberg University of MainzMainzGermany
  3. 3.Museum of Comparative Zoology & Department of Organismic and Evolutionary BiologyHarvard UniversityCambridgeUSA
  4. 4.Bioinformatics & Genomics Unit, Center for Genomic RegulationBarcelonaSpain

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