Institute of Zoology, University of Mainz, Mainz, Germany
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Abstract |
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Introduction |
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While hemocyanins have been studied in detail in many chelicerate and crustacean species (e.g., Markl 1986
; Markl and Decker 1992
; van Holde and Miller 1995
), specialized oxygen transport proteins have been poorly known in the Myriapoda, and their existence is still ignored by the textbooks. Respiratory proteins had been considered unnecessary in this taxon because Chilopoda (centipedes) and Diplopoda (millipedes) possess, similar to the insects, a typical tracheal system that was thought to be sufficient to supply the internal organs with an adequate amount of oxygen (Brusca and Brusca 1990
; Hilken 1998
). Nevertheless, a 36mer (6 x 6) oxygen-carrying protein had been described in the genus Scutigera (Chilopoda) that closely resembled the other arthropod hemocyanins (Mangum et al. 1985
; Boisset, Taveau, and Lamy 1990
; Gebauer and Markl 1999
). The exceptional presence of a hemocyanin in this taxon was attributed to the high activity of the Scutigeramorpha and their peculiar blind-ending tracheal system (Mangum and Godette 1986
). However, it is now evident that hemocyanins also occur in at least one family of the Diplopoda, i.e., the Spirostreptidae, suggesting that such oxygen-carrying proteins are much more widespread within the Myriapoda than previously thought (Jaenicke et al. 1999
). Here we report the cDNA-cloning and analysis of a hemocyanin subunit from the diplopod Spirostreptus sp., which is the first myriapod hemocyanin sequence, and discuss its implications for hemocyanin evolution and arthropod phylogeny.
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Materials and Methods |
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Cloning of Spirostreptus Hemocyanin
RNA was extracted from the complete animal excluding the cuticle. Poly(A)+RNA was purified from total RNA using the PolyATract kit (Promega). Five milligrams of poly(A)+RNA were used for the construction of a directionally cloned cDNA expression library applying the Lambda ZAP-cDNA synthesis kit (Stratagene). The library was amplified once and screened with the anti-Spirostreptus hemocyanin antibodies. Positive phage clones were converted to plasmid vectors using the material provided in the cDNA synthesis kit. The hemocyanin cDNAs inserted in the pBK-CMV vector were sequenced on both strands by a commercial sequencing service (Genterprise, Mainz, Germany). The incomplete clones were extended by RT-PCR using a set of specific oligonucleotide primers and a degenerate primer designed according to the highly conserved amino acid sequence of the arthropod hemocyanin CuA site. The missing 5' end was obtained by two successive 5' rapid amplification of cDNA ends (RACE) assays (Gibco-BRL kit) with a series of nested oligonucleotide primers according to the manufacturer's instructions. The sequences were obtained after the cloning of the PCR products into the pGEMTeasy vector (Promega).
Sequence Data Analysis and Phylogenetic Studies
The Genetics Computer Group (GCG) Sequence Analysis Software Package 8.0 and the tools provided by the ExPASy Molecular Biology Server of the Swiss Institute of Bioinformatics (http://www.expasy.ch) were used for sequence analyses. Multiple-sequence alignments were carried out with the aid of GeneDoc 2.6 (Nicholas and Nicholas 1997
). The Spirostreptus hemocyanin DNA and amino acid sequences were added to previously published alignments of the hemocyanin superfamily (Burmester 2001
) (see Supplementary Material). This alignment was constructed using CLUSTALX (Thompson et al. 1997
) and corrected considering the crystallographic structure of Limulus polyphemus (Hazes et al. 1993
) and Panulirus interruptus (Gaykema et al. 1984). Long gap regions and highly divergent regions were deleted from the final data set. The nucleotide alignment follows that of the proteins. Because saturation of silent sites was assumed, only the first and second codon positions of the DNA sequences were used. The program packages PHYLIP 3.6 (Felsenstein 2000
) and TREE-PUZZLE 5.0 (Strimmer and von Haeseler 1996
) were applied for phylogenetic inference. Gamma-corrected distances were calculated using the PAM (Dayhoff, Schwartz, and Orcutt 1978
) and JTT (Jones, Taylor, and Thornton 1992
) models with eight rate categories. Nucleotide distances were calculated according to the HKY model (Hasegawa, Kishino, and Yano 1985
). Tree constructions were performed by the neighbor-joining and maximum-parsimony methods available in the PHYLIP package. The reliability of the trees was tested by nonparametric bootstrap analysis (Felsenstein 1985
) with 100 replications using PUZZLEBOOT (shell script by M. Holder and A. Roger). Maximum-likelihood and likelihood mapping analyses were carried out using a reduced data set of 38 and 32 sequences, respectively, containing a maximum of 10 representatives of each protein family. TREE-PUZZLE 5.0 was used to test alternative topologies using the Dayhoff (Dayhoff, Schwartz, and Orcutt 1978
), JTT, and VT (Müller and Vingron 2000
) models of amino acid substitution and the HKY model for nucleotide replacement, each under the assumption of a rate heterogeneity with eight gamma categories (Strimmer and von Haeseler 1996, 1997
). Likelihood ratio tests were carried out according to Kishino and Hasegawa (1989)
. Z-values of >3.29 are significant with P < 0.001.
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Results |
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Phylogenetic analyses were carried out with maximum-parsimony, distance matrix, and maximum-likelihood methods using both amino acid and nucleotide sequence alignments. Highly diverged regions were excluded from the calculation. The arthropod phenoloxidases most likely constitute the most ancient branch of the arthropod hemocyanin superfamily and thus were considered as the outgroup (Burmester and Scheller 1996
; Sánchez et al. 1998
; Burmester 2001
). Except for the position of the Spirostreptus hemocyanin, the general topologies of the resulting trees are identical (fig. 3
). While the parsimony methods consistently support a sister group position of the myriapod and the chelicerate hemocyanins with high bootstrap values (99% and 90%, respectively; table 1
), distance matrix methods consider, with lower support values (65% and 78%; table 1
), the myriapod hemocyanin as basal to all members of the hemocyanin superfamily except the phenoloxidases.
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Discussion |
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Spirostreptus Hemocyanin
The Spirostreptus hemocyanin sequence contains all amino acids that are required for the function of a true oxygen carrier, particularly the six conserved copper-binding histidines. It is clearly distinct in sequence, structure, and biochemical properties from the phenoloxidases and the various nonrespiratory hemocyanin-like proteins. While the latter proteins are most likely absent in the Myriapoda, the phenoloxidase of Spirostreptus is a distinct protein with different antigenic and physiological characteristics (unpublished data). The Spirostreptus hemocyanin includes a typical N-terminal signal peptide necessary for the intracellular transfer into the endoplasmic reticulum and subsequent release into the hemolymph. While the chelicerate (arachnid) hemocyanins do not contain any signal sequences (Voit et al. 2000)
and are released from cyanocytes by cell rupture (Markl and Decker 1992
), the crustacean and insect hemocyanins have such signal peptides and are synthesized by the hepatopancreas or other organs (Sellos, Lemoine, and Van Wormhoudt 1997
; Sánchez et al. 1998
; Kusche and Burmester 2001
). Thus, the mode of hemocyanin synthesis in the Myriapoda resembles that of the Crustacea and insects.
Hemocyanin Evolution
Hemocyanins are present in all euarthropod subphyla, including the Myriapoda and at least one insect. While the role of the insect protein is not well understood (Sánchez et al. 1998
), the myriapod hemocyanins closely resemble their crustacean and chelicerate cognates in physiological and structural properties. The phylogenetic analyses including the Spirostreptus sequence show a monophyletic origin of all arthropod hemocyanins (fig. 3 ), most likely from an enzymatic phenoloxidase-like ancestor. Thus, the hemocyanins must have evolved in the arthropod stemline before the radiation of the major extant subphyla more than half a billion years ago. Their emergence might be correlated with the increase in body size of the animal and the formation of a hard cuticle. Both events were crucial in the evolution of the Arthropoda but made simple diffusion inefficient to supply the internal organs with sufficient oxygen. The hemocyanins fulfilled the upcoming need for an efficient oxygen carrier (cf. Burmester 2001
).
The basic structure of a hexamer is conserved among all hemocyanins; therefore, a 1 x 6 molecule was the most likely design of the last common ancestor of the arthropod hemocyanins. However, the number and arrangement of the hexamers essentially differ among the crustacean, chelicerate, and myriapod hemocyanins (Markl and Decker 1992
; van Holde and Miller 1995
), and the formation of these multimers clearly occurred independently in these subphyla. This multimerization is most likely correlated with an enhanced oxygen transport capacity with less osmotic impact. While among the Crustacea and Chelicerata the structures of these multimers vary, ranging from 1 x 6 to 8 x 6 subunits, the 36mer hemocyanin appears to be unique to the myriapod hemocyanins. This structure was apparently conserved since the separation of the Diplopoda and Chilopoda, at least 400 MYA if not much earlier (Robinson 1990
; Friedrich and Tautz 1995
; Shear 1997
). The unique hemocyanin structure can be considered an additional synapomorphy that favors a monophyly of the Myriapoda.
Implications for Arthropod Evolution
One of the most intensively debated issues in animal systematics focuses on the relative relationships within the arthropod phylum, i.e., how the Chelicerata, Crustacea, Myriapoda, and Hexapoda are related (e.g., Fortey and Thomas 1997
; Giribet and Ribera 2000
) (fig. 5
). In the textbooks, the Myriapoda have long been combined with the insects in a taxon named "Tracheata," "Antennata," or "Atelocerata" (e.g., Snodgrass 1938
; Brusca and Brusca 1990
; fig. 5A
). In recent years, evidence from both molecular and comparative developmental studies has suggested that the "Tracheata" do not exist, but rather that the Hexapoda are allied with the Crustacea (Turbeville et al. 1991
; Averof and Akam 1995
; Friedrich and Tautz 1995
; Dohle 1997
; Boore, Lavrov, and Brown 1998
; Giribet and Ribera 2000
; Shultz and Regier 2000
) or are even nested within a "pancrustacean" taxon (e.g., Zrzav
, Hyp
a, and Vlá
ková 1997
; García-Machado et al. 1999
; Wilson et al. 2000
; Burmester 2001
). However, in none of the molecular studies could the relationship of the Myriapoda to the other arthropods be resolved with sufficient confidence. Phylogenetic analyses of hemocyanins and related proteins had demonstrated a remarkably good resolution of the arthropod trees, at least at a higher taxonomic level (Beintema et al. 1994
; Burmester and Scheller 1996
; Burmester et al. 1998
; Burmester 2001
). Thus, we hoped to resolve the relative position of the Myriapoda with the help of the Spirostreptus hemocyanin. In fact, all phylogenetic approaches strongly reject the integrity of the Tracheata (fig. 4A
and table 1
). The significance levels are higher than those obtained from ribosomal DNA (Turbeville et al. 1991
; Friedrich and Tautz 1995
; Giribet and Ribera 2000
) or other molecular phylogenetic study (Regier and Shultz 1997; Shultz and Regier 2000
). This demonstrates the general usefulness of the hemocyanin superfamily in the reconstruction of arthropod phylogeny (Burmester 2001
). Nevertheless, the available methods still give conflicting results (figs. 3 and 4 and table 1
). Distance matrixbased methods support a basal position of the myriapod hemocyanin with respect to all other arthropod hemocyanins (figs. 3A and 5B
) and agree with the studies using arthropod mitochondrial DNA (Ballard et al. 1992
). Most studies using 18S and 28S ribosomal DNA tend to ally the Myriapoda with the Chelicerata (Turbeville et al. 1991
; Friedrich and Tautz 1995
; Giribet et al. 1996
; Giribet and Ribera 2000
; fig. 5C
). We found the highest degree of sequence similarity of the Spirostreptus hemocyanin with the chelicerate hemocyanin subunits. In addition, one of the most striking structural features of these sequences is the deletion of the second alpha helix of domain 1 (alpha helix 1.2; fig. 2
), which is otherwise present in all other proteins of the hemocyanin superfamily, including the outgroup (i.e., phenoloxidases). This should be considered a strong molecular synapomorphy that would indeed combine the Chelicerata and Myriapoda, although we cannot rule out independent deletion events. Such grouping was also inferred by the maximum-parsimony methods (fig. 3B
). From a morphological point of view, the distinctiveness of the chelicerate mouth appendages would suggest a common clade of the Myriapoda, Insecta, and Crustacea (i.e., Mandibulata; Brusca and Brusca 1990) (fig. 5D
). Most surprisingly, the integrity of the Mandibulata is the least likely topology supported by the hemocyanin sequences, although the maximum-likelihood approaches do not reject this hypothesis (fig. 4B
and table 1
).
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Abbreviations: PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis.
2 Keywords: Arthropoda
Diplopoda
hemocyanin
hexamerin
Hexapoda
Myriapoda
Spirostreptus
3 Address for correspondence and reprints: Thorsten Burmester, Institute
of Zoology, University of Mainz, D-55099 Mainz, Germany. E-
mail: burmeste{at}mail.uni-mainz.de
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