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MBE Advance Access originally published online on May 4, 2009
Molecular Biology and Evolution 2009 26(8):1851-1864; doi:10.1093/molbev/msp092
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© The Author 2009. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Research Articles

Evolution of a Novel Carotenoid-Binding Protein Responsible for Crustacean Shell Color

Nicholas M. Wade*,{dagger}, Alina Tollenaere*, Mike R. Hall{dagger} and Bernard M. Degnan*

* School of Integrative Biology, University of Queensland, Brisbane, Queensland, Australia
{dagger} Australian Institute of Marine Science, Townsville, Queensland, Australia

E-mail: b.degnan{at}uq.edu.au.


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Carotenoids are commonly used by disparate metazoans to produce external coloration, often in direct association with specific proteins. In one such example, crustacyanin (CRCN) and the carotenoid astaxanthin combine to form a multimeric protein complex that is critical for the array of external shell colors in clawed lobsters. Through a combined biochemical, molecular genetic, and bioinformatic survey of the distribution of CRCN across the animal kingdom, we have found that CRCNs are restricted to, but widespread among, malacostracan crustaceans. These crustacean-specific genes separate into two distinct clades within the lipocalin protein superfamily. We show that CRCN differentially localizes to colored shell territories and the underlying epithelium in panulirid lobsters. Given the paramount importance of CRCN in crustacean shell colors and patterns and the critical role these play in survival, reproduction, and communication, we submit that the origin of the CRCN gene family early in the evolution of malacostracan crustaceans significantly contributed to the success of this group of arthropods.

Key Words: apolipoprotein D • crustacean • crustacyanin • coloration • lipocalin • neofunctionalization


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The color of many metazoans can be attributed to the presence of carotenoids that, on the most part, are unable to be synthesized by the animal themselves and must be obtained through their diets or in symbiotic relationship with photosynthetic organisms (Britton and Goodwin 1982Go; Garson et al. 1988Go; Matsuno 2001Go). These highly reactive molecules are stabilized when bound with proteins to form carotenoprotein complexes (Lee 1977Go; Britton et al. 1982Go). Such complexes have been identified in a wide range of invertebrate phyla, from Porifera and Cnidaria through to Mollusca and Arthropoda (Cheesman et al. 1967Go; Zagalsky 1976Go; Lee 1977Go; Elgsaeter et al. 1978Go; Ronnenberg et al. 1979Go; Litchfield and Liaaen-Jensen 1980Go; Czeczuga et al. 1999Go). The first of two broad categories of carotenoproteins, the lipovitellins, form weak nonspecific associations between a carotenoid and a lipoprotein and are responsible for coloration of such tissues as the blood, epithelium, eggs and ovaries (Cheesman et al. 1967Go; Ghidalia 1985Go; Zagalsky 1985Go). True carotenoprotein complexes, as found in crustacean carapaces and echinoderm epithelia, however, show a direct and well-defined stoichiometric relationship between the carotenoid and a simple apoprotein (Cheesman et al. 1967Go; Zagalsky 1985Go). With the exception of the clawed lobster Homarus gammarus, these carotenoprotein complexes have not been investigated in detail.

Crustaceans are particularly noted for their wide range of species-specific shell colors and patterns, which are used for protection through cryptic coloration, reproduction, and communication (Rao 1985Go; Bliss 1990Go; Horst and Freeman 1993Go). The nature of the components of shell color for all crustaceans has been inferred from the extensively studied multimeric protein complex known as {alpha}-crustacyanin ({alpha}-CRCN) isolated from the exoskeleton of the clawed lobster H. gammarus (Wald et al. 1948Go). This large molecular weight complex is composed of an octomer of dimeric β-crustacyanin (β-CRCN) subunits, with this dimer formed by two types of crustacyanin (CRCN) subunits (A and C) in association with two astaxanthin molecules (reviewed in Chayen et al. 2003Go). These CRCN complexes are known to cause a bathochromic shift in the emission spectrum of astaxanthin from red ({lambda}max = 472 nm in hexane) to purple as seen in β-CRCN ({lambda}max = 580–590 nm) or blue in the case of {alpha}-CRCN ({lambda}max = 632 nm) (Buchwald and Jencks 1968Go). The full protein sequences for the A2 and C1 CRCN subunits have since been identified (Keen et al. 1991aGo, 1991bGo) and the protein crystal structure of β-CRCN resolved (Cianci et al. 2002Go). Proteins with similar biochemical attributes to crustacean CRCN have been putatively identified in a range of invertebrate phyla, although specific gene or protein sequences have not been isolated (for reviews see Cheesman et al. 1967Go; Zagalsky 1985Go). Very little is known about the distribution of CRCN in other crustacean species or whether this protein is central to the colored carotenoprotein complexes identified in other phyla.

CRCN forms part of the lipocalin superfamily of proteins, a very large and widespread family of functionally diverse proteins that bind small hydrophobic molecules such as steroid hormones, carotenoids, odorants, and pheromones (Flower 1996Go, 2000Go). Representatives of this family include apolipoprotein D (ApoD), the insect cuticle coloration proteins insecticyanin and bilin-binding protein. Each member of this family displays a remarkable structural similarity despite low sequence similarity at the protein level. Most lipocalins share three structurally conserved regions (SCRs) and are known as kernel lipocalins (Flower 1996Go, 2000Go).

In this study, we screened a range of animals from diverse phyla for the presence of CRCN, using a combined molecular, biochemical, and bioinformatic approach. Thirteen new CRCN gene sequences have been identified by degenerate polymerase chain reaction (PCR) all within class Crustacea. Where possible, these were confirmed using a highly specific polyclonal antibody against CRCN. There was no evidence of the CRCN gene existing outside the Crustacea, leading us to conclude that this gene is a lineage-specific novelty. We also show that CRCN is localized in patterns that are consistent with a role in configuring shell coloration. These observations allow us to infer that the origin of CRCN in the crustacean lineage was critical for the evolution of this highly successful group of metazoans.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Isolation of CRCN Genes
Marine invertebrates from a range of phyla were collected and sampled as outlined in table 1. Tissues for RNA extraction were stored in RNA Later (Applied Biosystems/Ambion, Foster City, CA) and extracted using published methods (Hinman et al. 2003Go). Full-length protein sequence for the A2 and C1 subunits of CRCN from H. gammarus (Genbank accession # S19534—C1; S15391—A2) were used incorporating amino acid redundancies to create degenerate primers to the A2 subunit (CRCN-A2.1 5'-GTNGCNAAYCARGAYAAYTTYGA-3' and CRCN A2.2 5'-AAYTCNACNCCRTTYTTRTTRAA-3'), C1 subunit (CRCN C1.1 5'-TAYCARYTNATHGARAARTGYGT-3' and CRCN C1.2 5'-CCRAARTTRTARTCDATRCA-3'), or conserved regions of both A2 and C1 subunits (CRCN C1A2.1 5'-TTYGTNACNGCNGGNAARTGYGC-3' and CRCN C1A2.2 5'-GTYTCNADNAYNWCNWRNGGNGC-3'). Fragments were isolated by PCR from cDNA prepared from each species using combinations of the above primers under low stringency conditions (Hinman et al. 2003Go). Appropriately sized amplicons were purified, cloned, and sequenced using the pGEM-T Easy cloning system (Promega, Madison, WI).


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  		Table 1 Summary of Taxonomy, Species, Location, Tissue Type, Color, and Analysis Method Used for Each Animal in This Study

 
Full-length cDNA sequences were obtained for Panulirus cygnus CRCN A1 and A2 genes using the SMART random amplification of cDNA ends (RACE) cDNA Amplification Kit (BD Biosciences, Franklin Lakes, NJ). Tissue-specific CRCN gene expression was determined by reverse transcription polymerase chain reaction (RT-PCR) using primers specific for the P. cygnus CRCN A1 or A2 genes, respectively: PcCRCN A1.1 5'-GATTGAGATCCGATTTACACATGCC-3'; PcCRCN A1.2 5'-ACTTGT-AGTTGTCGGTACCG-3'; PcCRCN A2.1 5'-GGGCAGATGGTATCAGAGC-3'; and PcCRCN A2.2 5'-CCCTGTAGCCGCCCATCG-3'. The P. cygnus16s ribosomal gene was used as an amplification control using the primers Pc16s.1 5'-GCATGACCGTGCTAAGGTAG-3' and Pc16s.2 5'-GATTACGCTGTTATCCCTAAAC-3'.

Sequences identified in this study have the following Genbank accession numbers: P. cygnus CRCN-A1—FJ498893 [GenBank] ; P. cygnus CRCN-A2—FJ498894 [GenBank] ; Panulirus versicolor CRCN-A—FJ498899 [GenBank] ; P. versicolor CRCN-C—FJ498905 [GenBank] ; Penaeus monodon CRCN-A—898; Pe. monodon CRCN-C—904; Marsupenaeus japonicus CRCN-A—FJ498897 [GenBank] ; M. japonicus CRCN-C—FJ498903 [GenBank] ; Cherax quadricarinatus CRCN-A—FJ498895 [GenBank] ; C. quadricarinatus CRCN-C—FJ498902 [GenBank] ; Dardanus megistus CRCN-A—FJ498896 [GenBank] ; Gonodactylus smithii CRCN-A—FJ498900 [GenBank] ; Alpheus sp. CRCN-C—FJ498901. The expressed sequence tag (EST) sequence CV480162 from the blue crab Callinectes sipidus, listed as similar to CRCN-C1, was too short and could not be used for alignments, although this sequence showed very strong similarity to CRCN. The large numbers of ESTs from the freshwater shrimp Gammarus pulex, listed as CRCN, were largely redundant; hence, only the longest ESTs EH269222 and EH270832 were used in gene phylogenies. Similarly, a large number of EST sequences from Litopenaeus vannamei similar to ApoD were redundant, and hence, only two ESTs (BF023907 and DQ858916 [GenBank] ) were included in phylogenies.

Bioinformatics and Phylogenetics
Similarity searches of published databases were performed using the full-length P. cygnus CRCN A1 and CRCN A2 sequences and the published H. gammarus CRCN A and C protein sequences using the BlastP or TBlastX algorithms (Altschul et al. 1997Go). The published genomes surveyed are listed as part of table 1. Multiple protein alignments were created using ClustalX v 1.83.1 (Chenna et al. 2003Go), edited with Seaview 2.4 (Galtier et al. 1996Go). Maximum likelihood (ML) trees rooted with bacterial lipocalin sequences were constructed using the Whelan and Goldman protein distance matrix model (Whelan and Goldman 2001Go) and 100 bootstrap replicates. ML trees rooted with bacterial lipocalin sequences were constructed using the web-based RAxML interface (http://phylobench.vital-it.ch/raxml-bb/index.php) (Stamatakis 2006Go). The best-fit model of amino acid substitution for each data set analyzed under ML was determined by Modeltest 3.7 (Posada and Crandall 1998Go). Bootstrap support was evaluated using 100 pseudoreplicates (random resampling to produce simulated replicates).

Purification of Exoskeleton Proteins
Purification of proteins from the exoskeleton of decapod crustaceans followed previously published methods (Zagalsky and Cheesman 1963Go). Briefly, washed exoskeletons were ground to a fine powder and decalcified overnight at 4 °C in 20 mM sodium phosphate buffer containing 0.8 M ethylene glycol tetraacetic acid (EGTA). After filtration using No. 1 Whatman paper, proteins were ammonium sulfate precipitated and equilibrated into 20 mM sodium phosphate buffer using Microcon YM-10 size exclusion filters (Millipore, Billerica, MA). Total protein samples from other noncalcareous organisms were prepared by homogenization in 0.2 M phosphate buffer in the presence of protease inhibitors. Protein concentrations were estimated using the bicinchoninic acid Protein Assay Kit (Pierce, Rockford, IL) according to the manufacturer's instructions.

Optimal chromatographic separation of western rock lobster shell extracts was achieved using a Gilson Binary System fitted with a HiTrap Q-Sepharose Fast Flow ion exchange column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and proteins were eluted using a linear gradient of 20 mM sodium phosphate buffer (pH 7.0) with 1 M NaCl over 30 min at a flow rate of 1 ml per min. Eluted compounds were detected at dual wavelength: 490 nm for colored proteins and 280 nm for total protein. Alternatively, shell extract was separated on a Superdex 75 fast protein liquid chromatography 10-mm x 300-mm gel filtration column (GE Healthcare Bio-Sciences AB cat #17-5174-01) using a 20 mM sodium phosphate buffer (pH 7.0) with a flow rate of 1 ml per min. Irrespective of the chromatographic method used, fractions were collected at 1-min intervals and then concentrated using Microcon YM-10 size exclusion filters (Millipore).

Denaturing or sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed using the Mini-Protean II electrophoresis system (Biorad, Hercules, CA) with 10% bis–tris gels. In general, 10 µg of total protein was heated at 95 °C for 10 min in Laemmli sample buffer containing 100 mM dithiothreitol. Prestained Precision Protein Standards (Biorad) were used to estimate protein molecular weights and total proteins were visualized using Coomassie Blue stain. In order to maintain tertiary and quaternary protein structure, shell extracts were separated under nonreducing conditions using NuPAGE Novex 4–12% gradient bis–tris precast gels (Invitrogen, Carlsbad, CA) run in NuPAGE 2-(N-morpholino) ethanesulfonic acid buffer (Invitrogen) and the XCell SureLock Mini-Cell system (Invitrogen) according to the manufacturer's instructions. In each lane, 10 µg of total protein was mixed with NuPAGE lithiium dodecyl sulphate sample buffer (Invitrogen) and heated to 65 °C for 5 min before loading. Candidate bands were excised from the membrane and N-terminal protein fragments directly sequenced by Edman degradation using a Procise cLC automated sequencer (Applied Biosystems).

Antibody Production, Western Blotting, and Immunocytochemisty
Protein bands identified as candidates for shell color formation in the western rock lobster (P. cygnus) were identified from Coomassie stained gels. One of the peptide sequences obtained from gel purification—NKIPSFVVPGKC—was used to generate a rabbit polyclonal antibody using a synthetic peptide that corresponded to this sequence. Replicate gels to the Coomassie stained gels outlined above were transferred to Immobilon-P polyvinylidene flouride membrane (Millipore) using the Mini-Protean II electrophoresis system (Biorad). For western blotting, the primary antibody rabbit serum was diluted 1:2,000, the anti-rabbit horse radish peroxidase (HRP)-conjugated secondary antibody (Sigma–Aldrich, St Louis, MO) was diluted 1:10,000, and blots were visualized using a SuperSignal West Pico Chemiluminescent Detection Kit (Pierce).

Fixed and decalcified P. cygnus epithelium and exoskeleton were paraffin embedded and 8-µm sections used for immunocytochemistry. Sections were deparaffinized and rehydrated through a series of ethanol washes then in water, followed by boiling for 1 min in antigen unmasking solution (VectorLabs, Burlingame, CA) and blocking of endogenous peroxidases using a 3% H2O2 solution. Sections were blocked for 30 min in 4% normal horse serum (NHS)/1% BSA/phosphate buffered saline (PBS), then incubated overnight with primary antibody rabbit serum diluted 1:1,000 in 4% NHS/1% PBS/0.1% Triton X-100. Sections were washed in 1% BSA/PBS several times, before the addition of a goat anti-rabbit biotinylated secondary antibody (Molecular Probes, Invitrogen) for 1 h at room temperature. Further washes preceded a 30-min incubation in an avidin–HRP conjugate (VectorLabs) before specific staining was visualized using diaminobenzine (VectorLabs).


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Identification of CRCN Genes
A degenerate PCR approach identified one or more CRCN gene sequences in 9 of the 14 crustaceans where RNA was available, comprising a total of 13 new sequences (fig. 1, indicated by A or C). Using this approach, there was no evidence supporting the existence of the gene in any of the noncrustacean metazoans surveyed. Analysis of sequenced metazoan genomes (table 1) is consistent with CRCN gene being restricted to crustaceans.


Figure 1
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  		FIG. 1.— Detection of CRCN in representatives of disparate phyla using RT-PCR (RT) and western blotting (WB). Species reference numbers relate to the species listed in table 1 and broad phylogenetic relationships between taxa are shown above. Dashes indicate that material was unavailable for use by a particular method; crosses indicate species where no CRCN gene could be found by degenerate RT-PCR; A and C indicate the presence of individual CRCN subunits. Species used exclusively for EST and whole-genome searches are not shown.

 
In some crustacean species, both A and C subunits were identified, whereas in others, only a single A or C subunit could be amplified. Brachyuran crabs was the only crustacean clade surveyed where a CRCN gene was not successfully identified; however, this does not preclude the possibility that these species have CRCN genes that could not be detected using the degenerate oligonucleotide primers employed in this study. The identification of a CRCN-A ortholog in the stomatopod, G. smithii, extended the last common ancestor to possess a CRCN gene beyond an ancestral decapod to at least an ancestral malacostracan.

Two genes most similar to the CRCN-A subunit were identified in the western rock lobster, P. cygnus (PcCRCN-A1 and PcCRCN-A2); no corresponding C subunit was detected. Isolation of the full-length mRNA sequences by RACE PCR showed that both genes encoded 175 amino acid proteins with 74% similarity to each other and both contained an additional 16 amino acid signal sequence. PcCRCN-A1 was a single exon gene (fig. 2A). The PcCRCN-A2 gene was found to contain two small introns of 108 and 164 bp along with a shorter 3' un-translated region sequence (fig. 2A). Although the tissue distribution of the transcripts of these genes was similar, with predominant expression in the epithelial tissues, the relative expression of PcCRCN-A2 was observed to be less than that of PcCRCN-A1 (fig. 2B).


Figure 2
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  		FIG. 2.— Genomic structure and expression of the Panulirus cygnus CRCN-A1 and CRCN-A2 genes. (A) Both CRCN genes encoded for 175 amino acid proteins that shared 74% similarity to each other, and 86% and 78% similarity, respectively, with the known CRCN-A2 subunit from Homarus gammarus. Numbering indicates the location of various features within the mRNA sequence. (B) CRCN-A1 and CRCN-A2 were expressed predominantly in the epithelial tissue underlying the shell but, in the case of CRCN-A1, could also be detected in other tissues such as muscle, eyestalk, and gonads. The 16s rRNA gene is used as an amplification control.

 
CRCN Phylogeny
Additional CRCN genes were identified in other crustacean species by using similarity searches of EST databases for crustaceans and whole genome databases for the range of currently available species (table 1). The relatedness of all putative sequences was resolved using an ML phylogeny, which included selected members of the lipocalin family with bacterial lipocalins as an outgroup (fig. 3). Each of the crustacean sequences identified in this study strongly clustered into a distinct clade within the ApoD subfamily of the lipocalin family. This clade did not contain any genes that were not derived from crustaceans.


Figure 3
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  		FIG. 3.— Consensus ML phylogeny showing the relationship of CRCNs. The CRCN gene segregated into two distinct clades, A and C, with a series of crustacean ESTs of unknown function forming a sister clade to the CRCNs and another cluster of putative crustacean ApoD genes. These clades combined were nested within a small selection of recognized bilaterian ApoD representatives and was distantly related to the insect cuticle coloration genes. These data support the notion that the crustacean CRCN gene evolved from an ancient malacostracan proto-ApoD gene via ancestral gene duplications and divergence events. The tree is rooted to three bacterial lipocalin sequences and bootstrap values of 100 replicates are shown above each node. Genbank or EST accession numbers are shown for each gene following the three-letter species identifier code shown in table 1, with the exception of the following abbreviations: Aco—Agrius convolvuli; Blc—bacterial lipocalin; Bmo—Bombyx mori; Cuq—Culex quinquefaciatus; Dre—Danio rerio; Gme—Galleria mellonella; Hsa—Homo sapiens; Mmu—Mus musculus; Mse—Manduca sexta; Nvi—Nasonia vitripennis; Ocu—Oryctolagus cuniculus; Pbr—Pieris brassicae; and Sam—Schistocerca americana.

 
Most closely related to the CRCNs was a group of crustacean ESTs. These may represent a third subclade of crustacean CRCNs, although while these maintained the core lipocalin motif SCR-2, they were clearly divergent from the recognized CRCNs. The clawed lobster, Homarus americanus, and the kuruma prawn, M. japonicus, were the only species that contained representatives from each of these gene clusters, although a targeted search for transcripts similar to this unknown EST cluster was not undertaken in any other species. Likewise, the remaining species grouped in this cluster were not specifically targeted for the presence of CRCN genes.

Two genes, more distantly related to CRCN and termed putative ApoD genes, were identified in the genome of the crustacean, Daphnia pulex. These did not appear to be CRCN orthologs nor could a CRCN ortholog be identified in the D. pulex genome. This entire clade of crustacean lipocalin genes (CRCNs, unknown ESTs and putative ApoDs) was nested within the recognized the ApoD subfamily, which comprised a range of bilaterian representatives. Orthologs of the Daphnia ApoD–related genes were identified in two penaeid prawn species (Pe. monodon and L. vannamei) that also possessed recognizable CRCN genes, supporting the notion that the malacostracan crustacean CRCN gene evolved from within the ApoD subfamily via ancestral gene duplications and divergence events.

Specifically within the CRCN clade, the sequences clearly segregated into two subclades, the A and C subunits, indicating a second gene duplication. The phylogenetic relationship of the protein sequences within these clades did not reflect the taxonomic classification of these species. Two CRCN-A subunits were identified in P. cygnus that may represent a further, relatively recent, gene duplication event. PcCRCN-A1 was most similar to the A subunit of a very closely related species, P. versicolor, whereas PcCRCN-A2 appeared distantly related to the genes of the clawed lobster lineage.

CRCN Protein Structure
Amino acid translations of all genes that clustered within the CRCN clade were aligned to the known homarid lobster CRCN-A2 protein sequence (fig. 4). The alignment showed strong conservation of the three lipocalin SCRs within both subunits. Some amino acids outside the core lipocalin SCRs were strictly conserved in both A and C sequences, specifically residues Y51 (Y56 in CRCN-C) and H90 (H92 in CRCN-C), which have been previously implicated in the binding of the carotenoid chromophore (Keen et al. 1991aGo, 1991bGo; Zagalsky 2003Go). Absolute conservation was also observed for cysteine residues known to form the three disulfide bridges in the CRCN-C crystal structure (C12–C121, C51–C173, and C117–C150), as well as the two known disulfide bridges in the CRCN-A crystal structure (C12–C119, C46–C170). Interestingly, the H. gammarus CRCN-A sequence was the only representative that contained a T residue at position 147, directly opposing the conserved C115 residue in the crystal structure. Sequences from all other species contained a C at this position, potentially allowing the C115–C147 disulfide bridge to be maintained within the CRCN-A tertiary structure, similar to the C117–C150 disulfide bridge in CRCN-C.


Figure 4
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  		FIG. 4.— Sequence alignment of the crustacean proteins that formed the CRCN A and C cluster, using the Homarus gammarus A sequence as a reference. All members of the CRCN clade were well conserved, with two regions in particular that showed consistent amino acid differences between the A or C subunit (boxed in red). Some were major changes in amino acid category, charge, or both (shaded in red) and were often adjacent to residues thought to interact directly with the carotenoid chromophore (shaded in purple). As such, these residues may play an important role in the bathochromic shift of the carotenoid. SCRs among lipocalin protein family members are boxed in blue. Residues that form β-strands within the crystal structure from H. gammarus are shaded in green on either the A or C subunits, whereas {alpha}-helix secondary structures are shaded in light blue. Predicted signal peptides are shaded in green. Dashes indicate gaps in alignments or unidentified residues, whereas dots indicate matches with the reference sequence. Genbank or EST accession numbers are labeled for all genes after their individual three-letter species code shown in table 1.

 
Returning to the sequence comparisons, two broad regions spanning residues 36–65 and 87–106 of the HaCRCN-A2 sequence were found to contain residues specific for either the A or C subunit. Many of these individual amino acid variations between the two subunits were changes in the charge, polarity, or both. Spanning the highly conserved functional Tyr and His residues outlined above were two regions (CRCN-A 48–54 and 85–91; CRCN-C 53–60 and 86–93) that showed species conserved and marked amino acid variation between the two subunits, outlining their potential importance for protein function or maintaining tertiary structure. Focusing on the residues that surround the carotenoid and form the hydrophobic calyx in the crystal structure, very strong conservation was also observed for aromatic residues spread along the length of the CRCN sequence. In particular, regions T122 to F133 (CRCN-A) and Y124 to F136 (CRCN-C) contained aromatic amino acids approximately every second residue that were interspersed with residues specific for either CRCN-A or CRCN-C. Either the aromatic residues were conserved in these locations or variants were conservative replacements with other aromatic amino acids. These residues have been shown to form part of the 3D structure surrounding the carotenoid backbone and deep in the calyx where astaxanthin is bound, with the side chain of one of the conserved aromatic residues, F99 (CRCN-A) and F101 (CRCN-C) having previously been found to undergo a large shift between monomeric and dimeric crystal structures of CRCN (Chayen et al. 2003Go). Combined, these data further confirm the functional importance of aromatic residues for carotenoid binding and the function of CRCN.

Characterization of the P. cygnus CRCN Protein
To further study the function of the CRCN gene family, soluble shell proteins extracted from P. cygnus (with a {lambda}max of 490 nm) were separated using ion exchange chromatography and N-terminal sequences were identified for four proteins potentially responsible for this color peak (fig. 5A). One of the peptides showed high sequence similarity to CRCN from H. gammarus (table 2), whereas the other sequences showed some similarity to unrelated bacterial proteins with varying functions and were not analyzed further. Using a synthetic peptide derived from this partial P. cygnus CRCN sequence, polyclonal antisera was generated that strongly and specifically detected a single band of approximately 20 kDa in the extracts of P. cygnus lobster shells but not other control cell line extracts (fig. 5B). This band also corresponded in size with the CRCN-A2 and C1 subunits from H. gammarus (Quarmby et al. 1977Go). Further characterization of the P. cygnus CRCN protein quarternary structure under nonreducing conditions showed a predominant protein band approximately 20 kDa in size (fig. 5C), in addition to a number of bands at regular size increments (fig. 5C arrowed). Under denaturing conditions, these high molecular weight bands were reduced to a single 20-kDa subunit (fig. 5D). Although this antibody did not appear to be able to recognize the multimeric subunits in a quantitative manner, these bands potentially corresponded to the known dimeric and multimeric quaternary structures of CRCN (Quarmby et al. 1977Go) and demonstrates that CRCN also forms large macromolecular structures in other crustacean species, similar to those complexes observed previously in H. gammarus.


Figure 5
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  		FIG. 5.— Analysis of CRCN in Panulirus cygnus. (A) Size separation of EGTA soluble shell proteins fractionated by ion exchange chromatography (corresponding fractions shown above). N-terminal sequencing of bands potentially responsible for shell color (numbered 1–4) showed that Band 1 had high sequence similarity to CRCN from the European lobster Homarus gammarus. (B) By western blot, the anti-CRCN antibody specifically detected a single 20-kDa band from EGTA soluble lobster shell protein extracts, with no band in control Human Embryonic Fibroblast (HEF) and Baby Hamster Kidney (BHK) cell lysates. (C) Soluble shell proteins were separated on the basis of size using gel filtration chromatography (corresponding fractions shown above) on nondenaturing gels. Total proteins were stained with Coomassie Blue staining (left) and the corresponding western blot detection of CRCN (right). A number of bands were recognized by the anti-CRCN antibody, specifically a predominant 20-kDa band corresponded to the monomeric protein and bands possibly corresponding to the dimeric, tetrameric and octomeric quaternary structures (indicated by arrows). (D) Corresponding Coomassie stained and western blot gels of samples run under denaturing conditions, demonstrated these higher molecular weight complexes were reduced to a single 20-kDa monomeric form of CRCN. Marker sizes for all gels are indicated in kDa.

 

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  		Table 2 Protein Sequences Identified from Panulirus cygnus Shells

 
CRCN Protein Prevalence
The presence of other possible CRCN homologues was then analyzed using this antibody in total protein extracts from a range of crustacean species as well as other more distantly related phyla. A band was detected exclusively in a number of crustaceans; however, it was absent from 20 protein extracts from 9 other phyla (fig. 1). In some shell extracts, a doublet band was detected that potentially showed the slight differences in electrophoretic mobility between the two CRCN subunits, as has been observed previously (Zagalsky et al. 1995Go). This would also indicate that two subunit genes are present in the G. smithii genome, although only one gene sequence could be identified by sequencing RT-PCR amplicons. Interestingly, using this approach, no CRCN band was identified in the same five brachyuran crabs outlined in a previous section. Although perhaps reflective of the poor sequence conservation of the antibody recognition epitope—an inherent drawback of this type of analysis—strong biochemical evidence suggests that colored carotenoproteins exist in exoskeletal extracts of a number of other brachyuran crustaceans including the green crab Carcinus maenus (Garate et al. 1984Go), the snow crab Chionoecetes opilio (Manutawiah and Haard 1987Go), and the portunid crab Macropipus puber (Gomez et al. 1984Go). Further evidence exists in the literature of a CRCN-like colored protein from a range of decapod crustaceans; however, these proteins have rarely been characterized in any detail. Such evidence is particularly compelling where high molecular weight colored proteins composed of small ~20-kDa subunits have been biochemically purified directly from the exoskeleton, including the crayfish Procambarus clarkii (Milicua et al. 1985Go; Garate, Barbon, et al. 1986Go; Garate, Gomez, et al. 1986Go; Gomez et al. 1986Go) and Astacus leptodactylus (Milicua et al. 1986Go; Rivas et al. 1988Go).

Shell Patterning and CRCN Expression
Using P. cygnus as a model, we investigated the distribution of CRCN protein within the epithelium and the three distinct shell layers: the epicuticle, exocuticle, and endocuticle. CRCN immunostaining of shell sections showed that the protein was localized to a specific 10 µm region of the exocuticle approximately 7–8 µm beneath the outer waxy epicuticle and also accumulated in the tissue of the outer epithelium immediately beneath the cuticle (fig. 6A, inset [i] and [ii] arrowed). Preabsorption using a 20-fold excess of the synthetic CRCN antigen completely removed this specific staining, and a control monoclonal antibody against mouse Keratin 14 detected low levels of protein in only hypodermal cells (data not shown). Panulirus cygnus has a number of patterned regions on the shell, in particular finely detailed red and white patches and a lateral white stripe visible along the length of the carapace. Such red regions corresponded with distinctive coloration in the outer shell layer just beneath the surface of untreated shell samples, whereas white regions were devoid of color (fig. 6B arrowed). Immunohistochemical detection of CRCN across the lateral white stripe showed a direct overlap of CRCN staining with the observed exoskeletal color pattern (fig. 6C), with expression restricted to the tissue underlying the red sections of the shell. Combined, these data indicate the shell color and associated pattern is correlated with the deposition and accumulation of CRCN within the exoskeleton, and not merely by the presence or absence of the carotenoid chromophore.


Figure 6
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  		FIG. 6.— Localization of CRCN in the Panulirus cygnus shell and epithelium (A). A distinct layer approximately 10 µm thick was stained in the outer region of the exocuticle when shell sections were immunoreacted with the anti-CRCN antibody. Staining was also observed in outer epithelial cells underlying the shell. (B) Regions of the crustacean shell have intricate surface color patterns and this color was reflected in the presence or absence of color in the outer shell layer (arrowed). (C) Immunostaining of sections of the lateral white stripe along the length of the animal showed that CRCN was restricted to colored regions of the shell and corresponding epithelium. These results are compatible with shell color patterns being laid down by the expression and incorporation of CRCN into the exoskeleton, not simply the absence of the carotenoid chromophore.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
The combined genomic, proteomic, and bioinformatic results obtained in this study are consistent with the CRCN gene being a crustacean-specific genomic innovation. A number of CRCN orthologs have been identified in decapod crustacean species, suggesting a diversification of this gene within this arthropod lineage. The identification of a CRCN gene in stomatopods is compatible with this gene being present in the last common ancestor to the living Malacostraca. Within lobsters, CRCN forms multimeric complexes that have the potential to generate the diversity of colors observed in crustacean shells (Chayen et al. 2003Go, this study). Here, we show that CRCN accumulates in the outer layer of the hypodermis and is deposited in a discrete band in the outer margin of the exoskeleton only in the colored regions of the shell. In Macrobrachium rosenbergii, the expression of a lipocalin, which clustered in this study within the CRCN-C clade, was shown to be sharply activated immediately after molting (Wang et al. 2007Go). Combined, these observations, along with the presence of residues that appear to directly interact with the carotenoid chromophore, suggest that the spatial regulation of CRCN genes in the crustacean hypodermis is a major contributor to the diversity of colored patterns observed in many, if not most, malacostracan crustaceans.

A CRCN ortholog could not be identified outside the Crustacea using targeted genetic and proteomic approaches, and whole genome information from representative poriferans, cnidarians, nematodes, insects, molluscs, annelids, echinoderms, and chordates. Most compelling was the inability to detect this gene in searches of the only currently sequenced crustacean genome from D. pulex, which is consistent with the CRCN gene family evolving after the divergence of branchiopod and malacostracan crustaceans. An ApoD gene ortholog was present in the Daphnia genome and we cannot formally exclude the possibility the CRCN gene may have been lost from D. pulex.

Within the lipocalin family, and specifically within the ApoD subfamily, the CRCN genes form a distinct clade. The lipocalin protein family, which contains eubacterial and eukaryote representatives, has been noted for the strong conservation of tertiary structure of its members (Flower et al. 2000Go; Ganfornina et al. 2000Go). Despite showing very little sequence similarity, by definition the lipocalin family members are capable of binding small hydrophobic molecules such as steroid hormones or carotenoids (Flower 1996Go). Phylogenetic analyses indicate that the malacostracan crustacean CRCNs evolutionarily originated from an ApoD-like ancestor, most likely through gene duplication and divergence (fig. 7). Natural selection preserved and acted on this duplicate—the proto-CRCN gene—allowing eventual co-option into the skeletogenic program. This neofunctionalized duplicate subsequently evolved the capacity to bind astaxanthin and thus played a structural role in forming patterned shell colors. At this time, it is unclear if the related crustacean-specific clade of ApoD-like or unknown EST proteins also have the capacity to bind carotenoid chromophores, although certain species within classes Branchiopoda or Cephalocarida are known to have proteins that are associated with carotenoids (Krinsky 1965Go; Gilchrist 1968Go; Czeczuga and Czerpak 1969Go; Czeczuga 1973Go; Zagalsky and Gilchrist 1976Go).


Figure 7
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  		FIG. 7.— Schematic diagram of the evolution of the crustacean-specific CRCN gene. CRCNs have been identified in most branches of the Malacostraca (highlighted in dark gray) suggesting a radiation and duplication of this gene among the species in this class. Outside the Malacostraca, some evidence exists for the presence of carotenoprotein complexes; however, no CRCN sequence has been identified (highlighted in light gray). Most compelling is the absence of a CRCN ortholog in the Daphnia genome (Branchiopoda: Diplostraca) as well as any of the hexapod genomes (indicated where possible by an X). Members of a distinct cluster within the lipocalin family were identified in the branchiopod lineage that also had decapod crustacean orthologs; however, it is not known if this gene has carotenoid-binding properties. Presence of these proteins in other crustacean classes, or within certain unstudied malacostracan lineages, remains unknown (indicated by a ?).

 
The CRCN gene class consists of two subclasses, A and C. In some decapod lineages, one of these CRCN genes may have been lost (e.g., in P. cygnus and Dardanus megistos). The presence of both the A and C subunits of CRCN has been shown to be important for reconstituting a β-CRCN complex capable of modifying the color of the carotenoid chromophore (Gomez et al. 1986Go). This concept is supported by differences in the resolved crystal structures of apo-CRCN homodimers composed of only one subunit (Wright et al. 1992Go; Chayen et al. 1996Go, 2000Go; Cianci et al. 2001Go; Gordon et al. 2001Go; Habash et al. 2003Go) compared with the structure of true β-CRCN heterodimers composed of one A and one C subunit (Chayen et al. 1996Go; Cianci et al. 2002Go; Habash et al. 2004Go). Therefore, the loss of one of these genes in a particular species may have direct implications on the ability to modify the emission spectrum of astaxanthin. For example, despite no evidence of a C subunit, we demonstrated the presence of dimeric β-CRCN, as well as larger tetra and octameric CRCNs in P. cygnus; however, the absorbance maximum of this protein was not significantly shifted from that of astaxanthin alone (protein bound {lambda}max of 490 nm compared with unbound astaxanthin {lambda}max of 470 nm). A comparative crystal structure of this molecule would provide greater insight into the binding of astaxanthin to CRCN. In the resolved β-CRCN crystal structure, some degree of distortion of the carotenoids has been observed to make them coplanar upon protein binding (Cianci et al. 2002Go). The measured and theoretical effects of this distortion have been shown to partially explain the shift in carotenoid wavelength in two independent studies (Durbeej and Eriksson 2004Go; Bartalucci et al. 2009Go). To explain the remaining shift in wavelength seen in {alpha}-CRCN or β-CRCN, a further color-tuning mechanism must be at play. Current theories include polarization of the carotenoid polyene chain caused by protonation of the keto O atoms or hydrogen bonding with the keto O atoms (Weesie et al. 1997Go; Durbeej and Eriksson 2003Go, 2004Go), or strong exciton coupling between the protein-bound astaxanthin molecules (Ilagan et al. 2005Go; van Wijk et al. 2005Go). Irrespective of the mechanism, subtle differences in primary sequence within and between CRCN subclasses, while maintaining overall structural integrity, may provide the environment in which these interactions or modifications can occur and therefore the ability of different members of this protein class to interact at the molecular level with astaxanthin. Differences in amino acid sequences in lipocalins have been attributed to the diversity of ligands this family binds (Ganfornina et al. 2000Go) and thus the functional diversity of this gene family. Likewise, changes in the primary sequence of CRCN, and the differences highlighted here between the A and C subunits, has the potential to change binding affinities with astaxanthin, the degree to which astaxanthin may be distorted, the forces exerted on the end rings, the effects on the dimeric and multimeric subunit formation, and conformation in functional {alpha}-CRCN. Independently or in combination, these influences can directly impact on the entire spectrum of possible crustacean shell colors and the intricate shell color patterning that accompanies it.

Based on current evidence, we infer that the evolution and adaptation of crustaceans into their cryptic environments may be linked to the sequence diversity, expression, and evolution of the CRCN gene. Cryptic coloration is often strongly involved in camouflage or in elements of behavioral characteristics such as communication and mate selection. Shell coloring and patterning of the fiddler crab Uca vomeris has been directly linked to degree of predation by birds (Hemmi et al. 2006Go). All fish, the most common predators of large crustaceans, possess the genetic basis for color vision and are often equipped with extremely complex color vision systems (Collin and Trezise 2004Go). Stomatopods have also evolved a highly complex visual and coloration system for use in camouflage, predation, and communication, incorporating aspects of external coloration in the color spectrum as well as ultraviolet wavelengths (Marshall et al. 1999Go, 2007Go; Chiao et al. 2000Go). Elements such as camouflage are directly related to fitness and survival of many crustacean species and hence the ability to produce or modify external color and, as suggested by these results, is perhaps intrinsically linked to the presence of CRCN protein and CRCN gene expression. Given that all current evidence points to the CRCN gene being a crustacean-specific innovation, probably evolving from a duplicated ApoD liopocalin ancestor, we infer the origin of the proto-CRCN gene and its co-option in the shell morphogenetic program significantly contributed to the success and diversification of crustaceans. The crustacean CRCN gene class provides a clear case of how the neofunctionalization of a duplicated gene can have profound influence on the ecology and evolution of a specific taxon.


   Acknowledgements
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors wish to thank Mr Chris Wood at the School of Molecular and Microbial Sciences, University of Queensland, Australia, for conducting the N-terminal protein sequencing; Dr Christelle Adolphe from the Institute for Molecular Bioscience, University of Queensland, Australia, for the use of the anti-keratin antibody; Dr Dan Jackson from the Geobiology Group, University of Göttingen, Germany, for helpful discussions and comments; and Dr Toby Johnson from the Department of Medical Genetics, University of Lausanne, Switzerland, for assistance with creation and interpretation of phylogenetic trees. This research was supported by a grant from the Australian Research Council to M.R.H. and B.M.D.


   Footnotes
 
Billie Swalla, Associate Editor


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Accepted for publication April 24, 2009.


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