Molecular Characterization and Phylogenetic Relationships of a Protein
with Potential Oxygen-Binding Capabilities in the Grasshopper Embryo. A
Hemocyanin in Insects?
Diego Sánchez,* Marı́a D. Ganfornina,* Gabriel Gutiérrez,† and Michael J. Bastiani*
*Biology Department, University of Utah; and †Departamento de Genética, Universidad de Sevilla, Sevilla, Spain
Arthropodan hemocyanins, prophenoloxidases (PPOs), and insect hexamerins form a superfamily of hemolymph
proteins that we propose to call the AHPH superfamily. The evolutionary and functional relationships of these
proteins are illuminated by a new embryonic hemolymph protein (EHP) that is expressed during early stages of
development in the grasshopper embryo. EHP is a 78-kDa soluble protein present initially in the yolk sac content,
and later in the embryonic hemolymph. Protein purification and peptide sequencing were used to identify an embryonic cDNA clone coding for EHP. In situ hybridization identifies hemocytes as EHP-expressing cells. As deduced
from the cDNA clone, EHP is a secreted protein with two potential glycosylation sites. Sequence analysis defines
EHP as a member of the AHPH superfamily. Phylogenetic analyses with all the currently available AHPH proteins,
including EHP, were performed to ascertain the evolutionary history of this protein superfamily. We used both the
entire protein sequence and each of the three domains present in the AHPH proteins. The phylogenies inferred for
each of the domains suggest a mosaic evolution of these protein modules. Phylogenetic and multivariate analyses
consistently group EHP with crustacean hemocyanins and, less closely, with insect hexamerins, relative to cheliceratan hemocyanins and PPOs. The grasshopper protein rigorously preserves the residues involved in oxygen
binding, oligomerization, and allosteric regulation of the oxygen transport proteins. Although insects were thought
not to have hemocyanins, we propose that EHP functions as an oxygen transport or storage protein during embryonic
development.
Introduction
Some arthropods rely on proteins called hemocyanins to transport the molecular oxygen needed for respiration. In hemocyanins, oxygen binds to a pair of copper atoms located between four antiparallel a-helices.
Each copper atom is coordinated to three histidine residues and is surrounded by a strong hydrophobic environment. Arthropodan hemocyanins are formed by subunits of ;75 kDa arranged in hexamers or multihexamers (for a review, see Van Holde and Miller 1995),
with each subunit containing a dinuclear copper site able
to bind one oxygen molecule.
Arthropodan hemocyanins share significant global
sequence similarity with two other protein families: hexamerins and prophenoloxidases (PPOs). All these proteins are presumably related by common ancestry (Beintema et al. 1994; Burmester and Scheller 1996), and
they might be considered to form a superfamily that we
propose to name the AHPH superfamily, an acronym
from arthropodan hemocyanins, PPOs, and hexamerins.
Hexamerins are proteins used for amino acid storage in
larval stages of insects (Telfer and Kunkel 1991). Some
hexamerins can be grouped into separate classes according to their content of methionine or aromatic residues
and are therefore called methionine-rich proteins and arAbbreviations: DIC, differential interference optics; EHP, embryonic hemolymph protein of the grasshopper; LHP, larval hemolymph
protein of the locust; ORF, open reading frame; PPO, prophenoloxidase; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; UTR, untranslated region.
Key words: grasshopper, hemocyanin, hexamerin, prophenoloxidase.
Address for correspondence and reprints: Diego Sánchez, Biology
Department, 229 South Biology, University of Utah, Salt Lake City,
Utah 84112. E-mail: [email protected].
Mol. Biol. Evol. 15(4):415–426. 1998
q 1998 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
ylphorins, respectively. They are hexamers with subunits in the 80-kDa range synthesized in the fat body
and secreted to the hemolymph, where they reach high
concentrations just before metamorphosis (Telfer and
Kunkel 1991). Hexamerins do not bind oxygen, since
the copper-binding histidine residues have been replaced. They are very common and abundant in insects
(Scheller, Fischer, and Schenkel 1990), and hexamerinlike proteins might exist in crustaceans (Markl et al.
1979a). However, no copper-free hemocyanin-related
protein has been found in chelicerates (Markl et al.
1979b). Phenoloxidase activity is ubiquitously found in
invertebrates, and PPOs from a crustacean and several
insects have recently been molecularly characterized
(Aspán et al. 1995; Fujimoto et al. 1995; Hall et al.
1995; Kawabata et al. 1995). As copper-containing proteins, PPOs preserve the dinuclear copper sites of hemocyanins. PPOs are synthesized in the cytoplasms of
hemocytes without a signal peptide, released to the hemolymph upon cellular rupture, and activated by proteolysis. In arthropods, the PPO active form (PO) is involved in defense reactions, wound healing, and cuticular sclerotization (Johansson and Söderhäll 1996; Sugumaran et al. 1992).
Hemocyanins have been found in three arthropod
groups: crustaceans, chelicerates, and a single order of
myriapods. However, hemocyanin has not been reported
in insects. This finding has been explained by the existence of a tracheal respiratory system in this subphylum. In this paper, we describe the finding of a soluble
protein present in the hemolymph of the grasshopper
(Schistocerca americana) embryo that we call EHP (for
embryonic hemolymph protein), whose sequence shows
high similarity to hemocyanins, and particularly preserves the residues involved, both directly and indirectly, in oxygen binding. We also study the EHP expression
415
416
Sánchez et al.
pattern and the phylogenetic relationships of this insect
protein with the AHPH proteins. These data support a
monophyletic origin of crustacean hemocyanins and
hexamerins but also suggest that hexamerins have
evolved from hemocyanins present in the insect lineage.
Moreover, the separate phylogenetic analyses of the
AHPH domains suggest an independent evolution for
these protein domains. Our phylogenetic and multivariate analyses strongly suggest that EHP is an insect hemocyanin, likely functioning in oxygen transport or storage during embryonic development.
Materials and Methods
Protein Purification, Microsequencing, and Molecular
Cloning of EHP cDNA
The EHP protein was accidentally purified by affinity chromatography with the mAb 7D2 against the
protein Conulin (Sánchez, Ganfornina, and Bastiani
1996) from the soluble fraction of embryonic grasshopper (Schistocerca americana) lysates. After purification,
the EHP protein was either N-terminally sequenced by
automated Edman degradation (Applied Biosystems
477A) or digested with Endolysine-C (Lys-C; Boehringer-Mannheim). The resulting peptides were then separated by reverse-phase HPLC and sequenced.
Degenerate oligonucleotides were designed from
the peptide sequences to amplify fragments from grasshopper cDNA using PCR with Taq DNA polymerase
(Saiki et al. 1988). Embryos at 45% of development
(staged by percentage of embryonic development;
hatching in 20 days at 308C) were used to obtain the
cDNA. A nested PCR strategy was used, and only the
bands that reamplified with the expected size were studied further. PCR was conducted in a thermal cycler (Perkin Elmer Cetus), and cycling conditions were as follows: one cycle of 948C for 2 min; 35 cycles of 948C
for 30 s, 478C for 30 s, and 728C for 45 s; and a final
cycle of 728C for 5 min. A PCR product of 550 bp was
amplified, cloned into the pCR-II vector using the TA
system (Invitrogen), and sequenced.
The 550-bp fragment was radiolabeled by random
priming (Prime-It II kit, Stratagene) and used as a probe
to screen a cDNA library made from nerve cords dissected from 55% embryos. This library was constructed
using cDNA primed with oligo(dT) and directionally
cloned using the lZAP system (Stratagene). We
screened 0.5 3 106 plaque-forming units from the amplified cDNA library. Several positive clones were obtained, and two were studied further. The clone EHP-8
contains an insert of 2,310 bp and codes for the fulllength EHP protein. The clone EHP-1 contains an insert
of 2,067 bp and is identical to EHP-8, but is truncated
at the 59 end.
Both strands of the cDNA inserts were sequenced
using Sequenase (version 2.0, U.S. Biochemicals) and
custom primers. DNA and protein sequences were analyzed with the BLAST service (Altschul et al. 1990)
and the GCG programs (Devereux, Haeberli, and Smithies 1984). To study the codon preference in the EHP-8
open reading frame (ORF), a codon usage table was
made from available Schistocerca and Locusta coding
sequences.
Analysis of EHP RNA Expression
In situ hybridizations were carried out according to
a protocol for grasshopper whole-mount embryos (Ganfornina, Sánchez, and Bastiani 1995). A digoxigenin-11dUTP labeled (Genius-4 kit, Boehringer-Mannheim)
RNA probe was synthesized using the EHP 550-bp fragment as template. Embryos were dissected and fixed in
PEM-formaldehyde (37% formaldehyde 1:9 in 0.1 M
PIPES, 2 mM EGTA, 1 mM MgSO4, pH 6.9) for 50
min. After washing, they were incubated in hybridization solution (50% deionized formamide, 4 3 SSC, 250
mg/ml yeast tRNA, 500 mg/ml salmon sperm DNA, 50
mg/ml heparin, 0.1% Tween-20, 1 3 Denhardt’s solution, 5% dextran sulfate) at 558C. The labeled RNA
probe was added at 0.5 mg/ml, and incubation proceeded
for 36–48 h. After washes, the labeled RNA was detected with an alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehringer-Mannheim).
Phylogenetic Analysis
Sequences were retrieved from the databases
SwissProt, PIR, PDB, EMBL, and GenBank at their releases by February 13, 1997. The most recent entry was
considered when several sequences were available for a
given protein. Only the mature protein sequences were
used, either deduced from the known mature N-terminal
amino acid or predicted by von Heijne’s (1990) method.
Partial sequence entries were excluded. According to
Beintema et al. (1994), proteins were named using an
abbreviated species name followed by a functional label.
Protein sequences were aligned with CLUSTAL W (1.6)
(Thompson, Higgins, and Gibson 1994) using a PAM
series scoring matrix and a gap penalty mask based on
the aligned secondary structures of Pint.HcA and
Lpol.HcII. The alignment has been deposited in EMBL
(accession number DS32507). Phylogenetic analyses
were carried out using the PHYLIP (3.5) (Felsenstein
1993) and PAUP (3.0) (Swofford 1991) software packages. Alignment gaps were considered missing data and
not used for phylogenetic inference. Corrected distances
were calculated with the program PROTDIST using the
PAM 001 matrix. The neighbor-joining (Saitou and Nei
1987) method was used to reconstruct distance trees.
Parsimony analyses were performed with the PROTPARS program of the PHYLIP package, and with the
PAUP software under a heuristic search option. Analysis
with PAUP was performed for comparison but is not
shown in figures.
Multivariate Analysis
Principal-component analysis (PCA) on centered
variables was performed with the program NetMul
(Thioulouse and Chevenet 1996). The input for this
analysis consisted of the mole percentage amino acid
compositions of the mature AHPH proteins used in the
phylogenetic analyses, along with the compositions obtained by amino acid analysis of the locust LHP (de Kort
and Koopmanschap 1987), Pint.HcA, Ecal.HcD, and
EHP, a Putative Hemocyanin in the Grasshopper Embryo
FIG. 1.—Expression pattern of grasshopper EHP. In situ hybridizations in whole-mount grasshopper embryos with an antisense (A) or
sense (B) RNA probe made from a 550-bp DNA fragment of the EHP
cDNA. View from the dorsal surface with DIC optics. Anterior is up.
Hemocytes attached to the basal membrane appear labeled, while no
labeling is observed in muscle (arrow) and neural (asterisk) cells. Scale
bar 25 mm.
two hemocyanins from centipedes (Mangun et al. 1985).
Given the difficulty for the amino acid analysis to distinguish between residues Q-E, and N-D, the mole percentage values of these pairs estimated from the protein
sequences were combined. Seventeen variables, including 15 residues and the combinations Q 1 E and N 1
D, were considered for the analysis.
Results and Discussion
Identification of Grasshopper EHP as a Soluble Protein
Synthesized by Hemocytes During Embryonic
Development
The grasshopper EHP was identified during purification of the neural protein Conulin (Sánchez, Ganfornina, and Bastiani 1996). After purification and LysC digestion, the sequences of three EHP peptides were
obtained. These sequences were used to design degenerate oligonucleotides to PCR-amplify the embryonic
cDNA coding for the protein. A 550-bp DNA fragment
was obtained that shows a unique ORF and contained
regions coding for the N-terminal peptide and an internal peptide. We studied the spatial localization of EHP
mRNA using in situ hybridization to whole-mount
grasshopper embryos. Sense and antisense digoxigeninlabeled RNA probes were generated using the 550-bp
PCR fragment. EHP mRNA expression is detected at
45% of development in sessile hemocytes, shown in figure 1A over the basal membrane of the ventral nerve
cord. Neurons and muscle cells are highlighted with asterisks and arrows, respectively, to show the absence of
labeling in those cell types. No labeling was observed
in the cells of the fat body (not shown), the place of
synthesis of insect storage proteins (Telfer and Kunkel
1991). Hybridization is absent in embryos exposed to
the sense RNA probe (fig. 1B), indicating that the signal
observed with the antisense probe is specific for the endogenous mRNA. These results indicate that EHP
mRNA is present during embryonic development and
that EHP is synthesized by hemocytes.
417
The purification of the protein from the soluble
fraction of embryonic lysates, its hemocyte origin, and
the presence of a signal sequence in the molecule (see
below) suggest that EHP is exported into the hemolymph. It was consequently named embryonic hemolymph protein. Intriguingly, EHP is present as a soluble
protein at 45% of development, long before dorsal closure and heart functioning (which occurs at 65%–70%
of development), and thus before a true hemolymph is
formed. Therefore, at early developmental stages, EHP
is probably free in the yolk sac.
EHP-8, the full-length cDNA coding for EHP, has
a 2,310-bp insert that includes the three sequenced peptides (underlined in fig. 2A). It comprises an ORF of
2,025 bp after a 59 untranslated region (UTR) of 27 bp.
The 550-bp probe hybridizes to bp 91–664. The first
methionine codon encountered in frame in the EHP-8
clone is in an appropriate context to be the translation
initiation site according to Cavener and Ray (1991) and
by comparison with the nucleotide flanking sequences
found in other grasshopper genes. The correctness of the
ORF defined above is further supported by the GC bias
at every third base and the codon preference analysis.
Two potential polyadenylation signals are found in the
257-bp 39 UTR, which ends with a poly-A sequence.
The N-terminal end of EHP predicted by this ORF
is highly hydrophobic (fig. 2B) and may represent a
leader signal peptide. The predicted cleavage site for the
signal peptide (von Heijne 1990) (arrowhead in fig. 2A)
agrees with the N-terminal protein sequence obtained.
This and the lack of other suspected membrane-anchoring regions indicates that EHP is a secreted protein. The
predicted mature EHP would have a molecular mass of
76.2 kDa and an isoelectric point of 6.38. EHP has two
potential N-glycosylation sites (residues N172 and
N317, circled in fig. 2A), and five cysteines (boxed in
fig. 2A) that may form disulfide bonds.
Similarity of EHP to Crustacean Hemocyanins;
Phylogenetic and Multivariate Analysis of the AHPH
Superfamily
When the sequence of the mature EHP is compared
with those of other known proteins, it shows the highest
similarity with subunit A of the lobster Panulirus interruptus hemocyanin (Pint.HcA; Bak and Beintema 1987).
The Pint.HcA and EHP sequences align with minor
gaps, and the overall pairwise similarity (43% identity;
64% when considering conservative substitutions) is
higher than that between crustacean and cheliceratan hemocyanins (31%–33% identity; Beintema et al. 1994).
The grasshopper EHP showed significant global similarity with hemocyanins, insect hexamerins, and PPOs.
Therefore, we studied the phylogenetic relationships of
these protein families (see fig. 3 and table 1), including
the grasshopper EHP. After aligning the amino acid sequences, two methods were used to find phylogenetic
relationships (corrected distances and maximum parsimony, see Materials and Methods). Trees inferred with
both methods showed identical topologies for the main
branches (fig. 3). Numbers at nodes indicate bootstrap
percentage from 100 replicates. A majority rule of 50%
418
Sánchez et al.
was established, unsupported nodes were excluded, and
their branches were forced to yield polytomies. We used
PPOs as an outgroup due to their assumed presence in
all arthropod groups and their closer relationship with
cheliceratan hemocyanins, which suggest a common ancestry with hemocyanins before the separation of the
main arthropod groups. Three main protein groups are
determined. The PPOs and cheliceratan hemocyanins
both appear as different clades, while a single monophyletic group is formed by crustacean hemocyanins
and insect hexamerins. The grasshopper EHP groups
with the crustacean hemocyanins with a well supported
node, and consistently separates from the monophyletic
hexamerins. The internal branching pattern of crustacean hemocyanins and PPOs is well supported, but that
of cheliceratan hemocyanins shows some unsupported
nodes. A number of nodes organizing the hexamerin tree
have low bootstrap values and vary when parsimony or
distance methods are considered, as previously reported
(Beintema et al. 1994; Burmester and Scheller 1996).
However, some groups, like the methionine-rich hexamerins (sequences 7, 13, 14, 16, and 17 of table 1),
and the dipteran (sequences 19–21 of table 1) and lepidopteran (sequences 8, 9, 11, and 12 of table 1) arylphorins consistently separate in distinct clusters. Other
hexamerins that group together are the juvenile hormone-suppressible proteins (sequences 10 and 15 of table 1), the hexamerins from two cockroaches (sequences
1 and 3 of table 1), and the Rcla.cyaA-B cyanoproteins
(sequences 4 and 5 of table 1). The poorly supported
topology and the longer overall branch length of the
hexamerin distance tree (not shown) imply a higher divergence in the sequences of these insect proteins, which
probably relates to a release of their sequences from
functional constraints, a conclusion also reached by Burmester and Scheller (1996).
A PCA of the amino acid content of the AHPH
proteins under study (fig. 4) was used as a different approach to establish groups based on biochemical features other than protein sequence. This analysis also allowed us to include the locust LHP (de Kort and Koopmanschap 1987) and two centipede hemocyanins (Mangun et al. 1985) whose amino acid sequences are not
available yet. PCA shows hemocyanins and PPOs as
compact and close groups, while hexamerins form a
scattered group with three to four main clusters. One of
these clusters (I in fig. 4A) clearly segregates the methionine-rich hexamerins, while others (II, III, and IV in
fig. 4A and C) group three dipteran arylphorins, lepidopteran arylphorins and the Bdis.Hex, and two juvenile
hormone-suppressible proteins, respectively. An independent PCA (fig. 4C) was carried out with the proteins
grouped in the lower left quadrant of the plot in figure
4A. PPOs and hemocyanins clearly become apart, and
EHP and the centipede proteins group with hemocyanins. LHP segregates between EHP and Lmig.JHBP (arrowhead in fig. 4C). Although EHP is recognized by a
serum anti-LHP (kindly provided by S. C. de Kort; data
not shown), PCA analysis questions an orthologous relationship between these two proteins. The validity of
PCA for including proteins with amino acid composition
obtained biochemically was demonstrated by the inclusion in the hemocyanin cluster of the experimental residue composition of Pint.HcA and Ecal.HcD (dotted circles in fig. 4A and C).
Analysis of the Evolutionary History and Functions of
the Three AHPH Protein Domains
Arthropodan hemocyanins are structurally composed of three domains (Volbeda and Hol 1989a; Hazes
et al. 1993), with domain 2 being directly involved in
oxygen transport. This oxygen-binding domain, in particular the pair of helices forming each copper site, has
been proposed to be the ancestral structural character
shared by otherwise unrelated oxygen-binding proteins
(Volbeda and Hol 1989b). Arthropodan hemocyanins inherited this domain, but two new protein domains were
apparently added during evolution. Domain 1 is very
important for the allosteric regulation of oxygen binding
(Magnus et al. 1994) and seems to determine the extent
of the oligomerization (Hazes et al. 1993). Domain 3 is
structurally very different from the others, as it contains
a seven-stranded Greek key b barrel. It contains a putative calcium-binding site that can play a structural and
regulatory role (Hazes et al. 1993).
The overall sequence conservation exhibited by
AHPH proteins suggests a preserved tertiary structure
with three distinct domains that were probably present
in the ancestor of these proteins. This prompted us to
investigate the existence of different evolutionary trends
in each individual domain. If they have followed similar
pathways, one may expect congruency between the topology of the tree inferred from the whole sequence (fig.
3) and that of those reconstructed from the separate domains. The initial alignment was subdivided according
to the domains of Pint.HcA (Volbeda and Hol 1989a).
Corrected distances were used to infer phylogenetic
trees of the three domains separately (fig. 5). The overall
tree topologies of the different domains are similar to
those inferred from the entire sequence (separating the
clades of PPOs, cheliceratan hemocyanins, and a group
that joins crustacean hemocyanins, grasshopper EHP,
and insect hexamerins), as was observed in previous reports (Beintema et al. 1994; Burmester and Scheller
1996). However, important differences emerge from our
analysis.
→
FIG. 2.—A, cDNA and deduced protein sequence of EHP. Nucleotide numbers are on the left, and amino acid numbers are on the right
(referred to the first methionine). The cleavage site of the signal peptide is marked by an arrowhead. Two potential N-linked glycosylation sites
are circled. Cysteine residues are boxed. The N-terminal peptide and the peptides obtained by Lys-C digestion are underlined. The polyadenylation
sites are shown in italics. B, Hydropathy plot of the predicted protein sequence determined by Kyte and Doolittle’s (1982) method using a
window of nine residues. The hydrophobic domain at the N-terminal region of the protein represents the signal peptide.
EHP, a Putative Hemocyanin in the Grasshopper Embryo
419
420
Sánchez et al.
FIG. 3.—Phylogenetic analysis of the AHPH proteins listed in table 1 derived from a protein sequence alignment conducted with CLUSTAL
W. The secondary structures of two hemocyanins (Lpol.HcII and Pint.HcA) were used to guide the alignment. The topologies of trees inferred
with distance matrix (left side) or maximum parsimony (right side) methods are shown. The trees were rooted with PPOs as outgroup. Branch
lengths are not shown. Numbers at nodes are bootstrap values from 100 replicates. A 50% majority rule was used to yield polytomies.
Position of Grasshopper EHP in the Domain Trees
The grasshopper EHP and crustacean hemocyanins
form a monophyletic group for domains 1 and 2. These
results add support to the hypothesis that EHP is an
insect hemocyanin. However, EHP becomes grouped
with hexamerins when analyzing domain 3. Both topologies support the existence of a common ancestor for
hexamerins and crustacean hemocyanins as proposed by
Burmester and Scheller (1996), but the existence of a
monophyletic association of EHP and the hexamerin
clade for domain 3 refines this proposal. This result suggests that unless convergence is invoked, hexamerins
have evolved from insect hemocyanins, with which they
still share higher similarity in domain 3, and not directly
from crustacean hemocyanins. Taken together, these data
reinforce the current view of a monophyletic origin of
insects and crustacea (Averof and Akam 1995).
Domainal Analysis of Different AHPH Clades
The tree topologies observed in the PPO clade appear to be well maintained. The branch lengths are longer for domain 1, which might be explained by the pro-
teolytic separation of domain 1 from the active PO form
(Aspán et al. 1995). This would liberate domain 1 from
functional constraints, enabling this domain to evolve
with higher rates of change in different arthropods.
The branching patterns of the cheliceratan hemocyanins broadly vary depending on the domain considered, but are generally weakly supported. This variation
can be due to the presence of hemocyanins from several
classes and orders of chelicerates in the sample of sequences available. These hemocyanins show differences
in their quaternary structures (Van Holde and Miller
1995) that could be reflected in their sequences. The
branch lengths in this clade also differ depending on the
domain under study. The longest are those of domain 1,
and the shortest are those of the oxygen-binding domain 2.
Crustacean hemocyanins show a well-supported
branching pattern with only a slight change in domain
2. The branch lengths show the same tendency as in
other hemocyanins and PPOs. The shorter distances observed in domain 2 suggest a higher divergence in the
domains not directly related to the oxygen-binding func-
EHP, a Putative Hemocyanin in the Grasshopper Embryo
Table 1
List of Proteins Used for Amino Acid Sequence Alignments
Protein
Taxon and Species
Abbreviation
No.
Accession No.
Hexamerins
Hexamerin subunit precursor. . . . . . . . . . . . . .
Juvenile hormone (JH) binding protein . . . . .
Allergen (clone (C12) . . . . . . . . . . . . . . . . . . .
Cyanoprotein a subunit precursor. . . . . . . . . .
Cyanoprotein b subunit precursor. . . . . . . . . .
Diapause protein 1 . . . . . . . . . . . . . . . . . . . . . .
Sex-specific storage protein 1 . . . . . . . . . . . . .
Sex-specific storage protein 2 . . . . . . . . . . . . .
Arylphorin precursor . . . . . . . . . . . . . . . . . . . .
JH-suppressible protein . . . . . . . . . . . . . . . . . .
Arylphorin a subunit precursor. . . . . . . . . . . .
Arylphorin b subunit precursor. . . . . . . . . . . .
Methionine-rich storage protein 2 . . . . . . . . . .
Storage protein 1 . . . . . . . . . . . . . . . . . . . . . . .
Acidic JH-suppressible protein . . . . . . . . . . . .
Basic JH-suppressible protein 1 . . . . . . . . . . .
Basic JH-suppressible protein 2 . . . . . . . . . . .
Insecticidal toxin. . . . . . . . . . . . . . . . . . . . . . . .
Hexamerin . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Arylphorin subunit A4 . . . . . . . . . . . . . . . . . . .
Larval serum protein 1 b subunit . . . . . . . . . .
Larval serum protein 2. . . . . . . . . . . . . . . . . . .
Hemocyanins
Embryonic hemolymph protein. . . . . . . . . . . .
Hemocyanin
Hemocyanin
Hemocyanin
Hemocyanin
chain a. . . . . . . . . . . . . . . . . . . . .
chain b. . . . . . . . . . . . . . . . . . . . .
chain c. . . . . . . . . . . . . . . . . . . . .
precursor . . . . . . . . . . . . . . . . . . .
Hemocyanin AA6 chain. . . . . . . . . . . . . . . . . .
Hemocyanin chain a. . . . . . . . . . . . . . . . . . . . .
Hemocyanin chain d. . . . . . . . . . . . . . . . . . . . .
Hemocyanin chain e. . . . . . . . . . . . . . . . . . . . .
Hemocyanin subunit II. . . . . . . . . . . . . . . . . . .
Hemocyanin a subunit . . . . . . . . . . . . . . . . . . .
Prophenoloxidases
Prophenoloxidase subunit 1 . . . . . . . . . . . . . . .
Prophenoloxidase subunit 2 . . . . . . . . . . . . . . .
Prophenoloxidase . . . . . . . . . . . . . . . . . . . . . . .
Prophenoloxidase A1 . . . . . . . . . . . . . . . . . . . .
Prophenoloxidase . . . . . . . . . . . . . . . . . . . . . . .
Arylphorin receptors
Arylphorin receptor . . . . . . . . . . . . . . . . . . . . .
Fat-body protein 1 precursor . . . . . . . . . . . . . .
Storage protein-binding protein. . . . . . . . . . . .
Insecta
Orthoptera
Blaberus discoidalis
Locusta migratoria
Periplaneta americana
Hemiptera
Riptortus clavatus
Riptortus clavatus
Coleoptera
Leptinotarsa decemlineata
Lepidoptera
Bombyx mori
Bombyx mori
Galleria mellonella
Galleria mellonella
Manduca sexta
Manduca sexta
Manduca sexta
Hyphantria cunea
Trichoplusia ni
Trichoplusia ni
Trichoplusia ni
Hymenoptera
Bracon hebetor
Diptera
Anopheles gambiae
Calliphora vicina
Drosophila melanogaster
Drosophila melanogaster
Insecta
Schistocerca americana
Crustacea
Panulirus interruptus
Panulirus interruptus
Panulirus interruptus
Penaeus vanameii
Chelicerata
Androctonus australis
Eurypelma californica
Eurypelma californica
Eurypelma californica
Limulus polyphemus
Tachypleus tridentatus
Insecta
Lepidoptera
Bombyx mori
Bombyx mori
Manduca sexta
Diptera
Drosophila melanogaster
Crustacea
Pacifastacus leniusculus
Insecta
Diptera
Calliphora vicina
Drosophila melanogaster
Sarcophaga peregrina
Bdis.Hex
Lmig.JHBP
Pame.Aller
1
2
3
U31328
U74469
L40818
Rcla.cyaA
Rcla.cyaB
4
5
D87272
D87273
Ldec.DP1
6
X76080
X86074
Bmor.SSP1
Bmor.SSP2
Gmel.Ary
Gmel.JHSP
Msex.AryA
Msex.AryB
Msex.MRSP
Hcun.SP1
Tni.AJHSP
Tni.BJHSP1
Tni.BJHSP2
7
8
9
10
11
12
13
14
15
16
17
X12978
P20613
A61619
L21997
P14296
P14297
L07610
U60988
M57443
Q06342
Q06343
Bheb.Tox
18
1612710
Agam.Hex
Cvic.Ary
Dmel.LSP1
Dmel.LSP2
19
20
21
22
U51225
X59391
U63556
X97770
Same.EHP
23
AF038569
Pint.HcA
Pint.HcB
Pint.HcC
Pvan.Hc
24
25
26
27
A24183
S02707
S21221
S55387
Aaus.Hc6
Ecal.HcA
Ecal.HcD
Ecal.HcE
Lpol.HcII
Ttri.HcA
28
29
30
31
32
33
P80476
A37975
P02241
S06701
A26713
Linzen et al. 1985
Bmor.PPO1
Bmor.PPO2
Msex.PPO
34
35
36
D49370
D49371
L42556
Dmel.PPO
37
D45835
Plen.PPO
38
X83494
Cvic.AryR
Dmel.FBP1
Sper.ABP
39
40
41
S46948
Q04691
D29741
421
422
Sánchez et al.
With respect to domain 3, we did separate distance
and parsimony analyses (not shown) including three arylphorin receptors that seem to be phylogenetically related to their arylphorin ligands (Burmester and Scheller
1996). Only domain 3 was considered, as the other two
domains show nonsignificant sequence similarities when
all the AHPH proteins are included in the analysis. The
arylphorin receptors form a monophyletic clade within
the hexamerins in distance and parsimony phylogenies,
in contrast to a previous report (Burmester and Scheller
1996) in which the arylphorin receptors and hexamerins
separate in different clades. The different grouping cannot be explained by substantial alignment differences,
but instead might be the result of the larger number of
sequences included in our analysis.
Functional and Structural Conservation of Domain 3
FIG. 4.—Principal-component analyses on centered variables of
the amino acid compositions of selected AHPH proteins. A, Projection
of the two most significant components (1 5 36% of the total variance;
2 5 19% of the total variance) of all the AHPH proteins (names detailed in table 1). C, Projection of the two most significant components
(1 5 28% of the total variance; 2 5 18% of the total variance) when
only the proteins segregated in the lower left quadrant of plot A were
considered. B and D, Amino acid (single-letter code) contribution to
the two first components obtained in A and C, respectively. Symbol
identification is shown on the right. Hollow symbols show amino acid
compositions calculated from the mature protein sequence. Crossed
hollow circles are the Pint.HcA and Ecal.HcD compositions calculated
from the mature protein sequences. Dotted hollow circles are the
Pint.HcA and Ecal.HcD compositions obtained by amino acid analysis.
These and the centipede hemocyanin compositions are reported in
(Mangun et al. 1985). Locust LHP composition obtained from (de Kort
and Koopmanschap 1987). Arrows point at the juvenile hormone-suppressible hexamerins. Arrowhead indicates Lmig.JHBP. The clusters of
hemocyanins, PPOs, and hexamerins are enclosed by continuous, short
dashed, and long dashed lines, respectively. Roman numbers represent
different hexamerin clades (see Results for details).
tion. However, we might expect this clade topology to
change substantially when hemocyanins from other
crustacean classes are described.
The same difficulties in assigning hexamerin relationships obtained from the entire protein sequences are
observed when individual domains are considered. Although the internal branching patterns of hexamerins are
disparate, several clades are relatively well supported,
such as the methionine-rich hexamerins, the juvenile
hormone-suppressible proteins, and the two groups of
arylphorins. It is important to emphasize the lightly sustained topology in domain 1, where even the monophyletic nature of hexamerins is challenged with a 38%
bootstrap node. Distances are larger for domains 2 and
1, which can be explained by the missing oxygen-binding function reported in these insect proteins. In contrast, domain 3 displays shorter and more homogeneous
distances.
The divergence among the distinct clades of AHPH
proteins, as derived from the corrected distances of the
main branches of the trees, also indicate that domain 1
is the most variable, while domains 2 and 3 are well
preserved. The conservation of domain 2 in hemocyanins and PPOs is not surprising, given the functional
similarities between those proteins. However, the preservation of domain 3, not only in hemocyanins and
PPOs, where it could play an important role in the regulation of copper-binding, but also in the highly divergent hexamerins and their receptors, is interesting.
The secondary structure of domain 3 appears to
have convergently evolved in a number of other proteins
(Hazes and Hol 1992), and it could be argued that its
preservation is merely due to a structural requirement
for the AHPH proteins. However, it is clear from other
protein families (e.g., the lipocalins) that a strongly conserved b barrel topology can be achieved without the
necessity of a well-preserved primary structure. Our
analysis shows a highly conserved sequence for the domain 3 of the AHPH proteins and leads us to propose
its further preservation for functional reasons. Given its
outer location in the hexamer (Volbeda and Hol 1989a;
Hazes et al. 1993), we hypothesize a role in protein–
protein interactions such as those involving receptor-mediated endocytosis of hexamerins (Wang and Haunerland 1994) and those of PPOs with other proteins participating in cellular defense reactions (Aspán et al.
1995). The inner cavity of the barrel could also be used
for binding ligands such as biliverdin (found in the hexamerins Rcla.cyaA-B [Chinzei et al. 1990] and in a locust hexamerin [De Bruyn, Koopmanschap, and de Kort
1986]) or riboflavin (found in several hexamerins; Miller and Silhacek 1995). Moreover, domain 3 has been
proposed to be conserved between the AHPH proteins
and the yeast and vertebrate tyrosinases (Kupper et al.
1989), where this domain is proteolytically separated
from the active form of the enzyme. The tyrosinases and
molluscan hemocyanins are thought to be ancestrally related to some AHPH proteins due to the similarity exhibited in the pair of a-helices of copper site B (Van
Holde and Miller 1995). We propose as a working hypothesis that domain 3 was part, together with these a-
EHP, a Putative Hemocyanin in the Grasshopper Embryo
423
FIG. 5.—Phylogenetic relationships of the three separate domains of the AHPH proteins estimated by a distance matrix method. Domain
boundaries are taken from Pint.HcA (Volbeda and Hol 1989a). Protein sequences are numbered as shown in table 1. Boxes, circles, and diamonds
represent hexamerins, hemocyanins, and PPOs, respectively. EHP is represented as a black circle. Bootstrap values (100 replicates) are shown
with a 50% majority rule.
helices, of the ancestral protein that originated hemocyanins and tyrosinases.
In summary, our results support the view that the
three domains present in the AHPH proteins are evolving as independent modules, under different selective
pressures and functional constraints.
Sequence Analysis Strongly Supports a Functional
Relationship of EHP with Hemocyanins
The results above suggest a well-supported relationship of EHP with crustacean hemocyanins and,
therefore, the existence of hemocyanins in insects. To
better substantiate this proposal, we studied in detail the
residues conserved between EHP and Pint.HcA. The
overall similarity between these two proteins becomes
even more significant when the oxygen-binding domain
2 (56% identity) is considered separately. An alignment
of this domain of EHP with two hemocyanins and se-
lected PPOs and hexamerins is shown in figure 6. EHP
shares with hemocyanins and PPOs the six copper-binding histidine residues. Moreover, the grasshopper protein
preserves every residue involved in the hydrogen-bonding network of the dinuclear copper site and 80% of the
residues forming the hydrophobic core close to the copper atoms of Pint.HcA. The residue F49 in domain 1
(not shown), key in the allosteric regulation of oxygen
binding, and E309, which controls the entrance of oxygen to the coppers, are also conserved in EHP (Hazes
et al. 1993; Magnus et al. 1994). Finally, two out of the
five cysteine residues of EHP (C562, C611) are shared
with Pint.HcA, where they form a disulfide bond involved in the formation of cavities surrounding the copper sites. This disulfide link is conserved in hemocyanins and PPOs but is present only in some hexamerins.
The extensive sequence similarity suggests that EHP
probably contains copper that would allow it to bind
424
Sánchez et al.
FIG. 6.—Alignment of the oxygen-binding domain of hemocyanins with EHP and selected hexamerins and prophenoloxidases (see table 1
for protein identification). Domain 2 boundaries are taken from Pint.HcA (Volbeda and Hol 1989a). The sequences were aligned with CLUSTAL
W. Gray boxes show residue identities of EHP either with Pint.HcA or with more than three of the selected proteins. Important residues involved
in oxygen binding and oligomerization of Pint.HcA are highlighted as detailed in the figure.
molecular oxygen. Moreover, residues involved in the
quaternary structure of Pint.HcA (dimer and trimer contact sites in the hexamer) are also significantly conserved in EHP (fig. 6). These residues are also conserved in hexamerins and suggest a common hexameric
structure for the whole clade.
Several other features of EHP appear to be related
to this evolutionary history. EHP is a secreted protein,
a character shared with hexamerins and crustacean hemocyanins, and not with cheliceratan hemocyanins and
PPOs, which lack a signal peptide. The secreted nature
of crustacean hemocyanins is based on the signal peptide present in Pvan.Hc (Sellos, Lemoine, and Van
Wormhoudt 1997). However, EHP is synthesized by hemocytes, while crustacean hemocyanins and insect hexamerins are synthesized by the hepatopancreas (Van
Holde and Miller 1995) and the fat body (Telfer and
Kunkel 1991), respectively. Like EHP, PPOs are synthesized by hemocytes (Kawabata et al. 1995), and these
enzymes are expected to be present in grasshoppers.
However, the sequence similarity values, the numerous
and distinctly situated gaps present in the alignment of
EHP with insect PPOs, and the differences in their biosynthetic pathways and posttranslational modifications
of these proteins do not support a functional relationship
between EHP and PPOs.
How do we interpret the sequence relationships of
EHP with hemocyanins? One could consider EHP to be
a nonfunctional relic of hemocyanins in insects. We
think, however, that this is an unlikely hypothesis, given
the relative abundance of this embryonic protein and the
high divergence rates that it should have experienced
when released from a functional constraint, as is clear
for hexamerins (see above). A second alternative is that
EHP represents an ancestral state in the evolution of
hexamerins. Orthopteran hexamerins are expected to be
more closely related to crustacean hemocyanins, given
that orthopteroids are primitive insects with a long, independent evolution. This view is supported by the disposition of Lmig.JHBP, Bdis.Hex, and Pame.Aller close
to the base of the hexamerin clade in our trees. However,
EHP never groups with these orthopteran proteins,
which eliminates the possibility that EHP is an ancestral
insect hexamerin.
In summary, sequence similarity and other molecular features suggest a close relationship of EHP and
hemocyanins. This is surprising, given the apparent lack
of hemocyanins in arthropods equipped with an efficient
EHP, a Putative Hemocyanin in the Grasshopper Embryo
tracheal respiratory system. However, EHP is present
during developmental stages in which the tracheal system is not fully developed. We do not know whether
EHP expression is restricted to embryonic life, which
might be a reason why EHP-like proteins have not been
found in other insects in which only postembryonic development has been explored. Based on the currently
available data on grasshopper EHP, a question arises:
What is the role of this protein during the embryogenesis of this orthopteran? Given its presence as a soluble
protein in the yolk sac, we propose that EHP might be
transporting or storing molecular oxygen in the egg. Oxygen is presumably transported in the egg by simple
diffusion (Chapman 1971), but an ancillary pathway involving an oxygen transport protein might be a safety
mechanism during periods of low oxygen supply to the
egg or periods of accelerated embryonic growth with
high metabolic rates. A similar function has been demonstrated for hemoglobins found in solution in the hemolymph of some aquatic insects (Chapman 1971;
Markl and Decker 1992). In spite of the plausibility of
this hypothesis, a definitive demonstration for a role of
EHP as an oxygen transport protein and its prevalence
in insects will require further investigations. However,
the existence of insect proteins with strong sequence
similarity to hemocyanins raises interesting questions
about their physiological role and their evolutionary relationships within the AHPH protein superfamily.
During the review process of this article, a new
crustacean hemocyanin from the Dungeness crab Cancer magister was reported (Durstewitz and Terwilliger
1997), and its phylogenetic relationship with some
members of the AHPH family was analyzed. A parsimony analysis groups the crab hemocyanin with
Pint.HcC.
Acknowledgments
We want to thank S. C. de Kort for his gift of the
locust LHP anti-serum, and E. Ball, J. Law, R. Doolittle,
J. Seger, and two anonymous reviewers for helpful comments on the manuscript. We also thank M. Herrera for
help with protein purification. L. Giuli, a high school
student sponsored by a Howard Hughes fellowship, enthusiastically helped us with the molecular cloning. Peptide sequencing and synthesis of oligonucleotides were
performed at the Protein/DNA Core Facility of the Utah
Cancer Center under the direction of R. W. Schackmann.
This work was supported by an NIH grant to M.J.B.
(NS25387). G.G. held an HFSP fellowship (SF-474/96).
LITERATURE CITED
ALTSCHUL, S. F., W. GISH, W. MILLER, E. W. MYERS, and D.
J. LIPMAN. 1990. Basic local alignment search tool. J. Mol.
Biol. 215:403–410.
ASPÁN, A., T. HUANG, L. CERENIUS, and K. SÖDERHÄLL. 1995.
cDNA cloning of prophenoloxidase from the freshwater
crayfish Pacifastacus leniusculus and its activation. Proc.
Natl. Acad. Sci. USA 92:939–943.
AVEROF, M., and M. AKAM. 1995. Insect-crustacean relationships: insights from comparative developmental and molec-
425
ular studies. Philos. Trans. R. Soc. Lond. B Biol. Sci. 347:
293–303.
BAK, H. J., and J. J. BEINTEMA. 1987. Panulirus interruptus
hemocyanin: the elucidation of the complete amino acid
sequence of subunit A. Eur. J. Biochem. 169:333–348.
BEINTEMA, J. J., W. T. STAM, B. HAZES, and M. P. SMIDT.
1994. Evolution of arthropod hemocyanins and insect storage proteins (hexamerins). Mol. Biol. Evol. 11:493–503.
BURMESTER, T., and K. SCHELLER. 1996. Common origin of
arthropod tyrosinase, arthropod hemocyanin, insect hexamerin, and dipteran arylphorin receptor. J. Mol. Evol. 42:
713–728.
CAVENER, D. R., and S. C. RAY. 1991. Eukaryotic start and
stop translation sites. Nucleic Acids Res. 19:3185–3192.
CHAPMAN, R. F. 1971. The insects. Structure and function.
Elsevier, New York.
CHINZEI, Y., T. HARUNA, K. MIURA, H. NUMATA, and S. NAKAYAMA. 1990. Purification and characterization of biliverdin-associated cyanoprotein from eggs and hemolymph
of the bean bug, Riptortus clavatus (Heteroptera: Alydidae).
Insect Biochem. 20:545–555.
DE BRUYN, S. M., A. B. KOOPMANSCHAP, and C. A. D. DE
KORT. 1986. High-molecular-weight serum proteins from
Locusta migratoria: identification of a protein specifically
binding juvenile hormone III. Physiol. Entomol. 11:7–17.
DE KORT, C. A. D., and A. B. KOOPMANSCHAP. 1987. Isolation
and characterization of a larval hemolymph protein in Locusta migratoria. Arch. Insect Biochem. Physiol. 4:191–
202.
DEVEREUX, J., P. HAEBERLI, and O. SMITHIES. 1984. A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Res. 12:387–395.
DURSTEWITZ, G., and N. B. TERWILLIGER. 1997. cDNA cloning of a developmentally regulated hemocyanin subunit in
the crustacean Cancer magister and phylogenetic analysis
of the hemocyanin gene family. Mol. Biol. Evol. 14:266–
276.
FELSENSTEIN, J. 1993. PHYLIP: phylogeny inference package.
Distributed by the author, Department of Genetics, University of Washington, Seattle.
FUJIMOTO, K., N. OKINO, S. KAWABATA, S. IWANAGA, and E.
OHNISHI. 1995. Nucleotide sequence of the cDNA encoding the proenzyme of phenol oxidase A1 of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 92:7769–7773.
GANFORNINA, M. D., D. SÁNCHEZ, and M. J. BASTIANI. 1995.
Lazarillo, a new GPI-linked surface lipocalin, is restricted
to a subset of neurons in the grasshopper embryo. Development 121:123–134.
HALL, M., T. SCOTT, M. SUGUMARAN, K. SÖDERHÄLL, and J.
H. LAW. 1995. Proenzyme of Manduca sexta phenol oxidase: purification, activation, substrate specificity of the active enzyme, and molecular cloning. Proc. Natl. Acad. Sci.
USA 92:7764–7768.
HAZES, B., and W. G. J. HOL. 1992. Comparison of the hemocyanin b-barrel with other Greek key b-barrels: possible
importance of the ‘‘b-zipper’’ in protein structure and folding. Proteins Struct. Funct. Genet. 12:278–298.
HAZES, B., K. A. MAGNUS, C. BONAVENTURA, J. BONAVENTURA, Z. DAUTER, K. H. KALK, and W. G. J. HOL. 1993.
Crystal structure of deoxygenated Limulus polyphemus
subunit II hemocyanin at 2.18 Å resolution: clues for a
mechanism for allosteric regulation. Protein Sci. 2:597–619.
JOHANSSON, M. W., and K. SÖDERHÄLL. 1996. The prophenoloxidase activating system and associated proteins in invertebrates. Prog. Mol. Subcell. Biol. 15:46–66.
KAWABATA, T., Y. YASUHARA, O. MASANORI, S. MATSUURA,
and M. ASHIDA. 1995. Molecular cloning of insect pro-
426
Sánchez et al.
phenol oxidase: a copper-containing protein homologous to
arthropod hemocyanin. Proc. Natl. Acad. Sci. USA 92:
7774–7778.
KUPPER, U., D. M. NIEDERMANN, G. TRAVAGLINI, and K.
LERCH. 1989. Isolation and characterization of the tyrosinase gene from Neurospora crassa. J. Biol. Chem. 264:
17250–17258.
KYTE, J., and R. F. DOOLITTLE. 1982. A simple method for
displaying the hydropatic character of a protein. J. Mol.
Biol. 157:105–132.
LINZEN, B., N. M. SOETER, A. F. RIGGS et al. (17 co-authors).
1985. The structure of arthropod hemocyanin. Science 229:
519–524.
MAGNUS, K. A., B. HAZES, H. TON-THAT, C. BONAVENTURA,
J. BONAVENTURA, and W. G. J. HOL. 1994. Crystallographic analysis of oxygenated states of arthropod hemocyanin
shows unusual differences. Proteins 19:302–309.
MANGUN, C. P., J. L. SCOTT, R. E. L. BLACK, K. I. MILLER,
and K. E. VAN HOLDE. 1985. Centipedal hemocyanin: its
structure and its implications for arthropod phylogeny. Proc.
Natl. Acad. Sci. USA 82:3721–3725.
MARKL, J., and H. DECKER. 1992. Molecular structure of the
arthropod hemocyanins. Adv. Comp. Environ. Physiol. 13:
325–376.
MARKL, J., A. HOFER, G. BAUER, A. MARKL, B. KEMPTNER,
M. BRENZINGER, and B. LINZEN. 1979a. Subunit heterogeneity in arthropod hemocyanins: II. Crustacea. J. Comp.
Physiol. 133:167–175.
MARKL, J., A. MARKL, W. SCHARTAU, and B. LINZEN. 1979b.
Subunit heterogeneity in arthropod hemocyanins: I. Chelicerata. J. Comp. Physiol. 130:283–292.
MILLER, S. G., and D. L. SILHACEK. 1995. Riboflavin binding
proteins and flavin assimilation in insects. Comp. Biochem.
Physiol. 110:446–475.
SAIKI, R. K., D. H. GELFAND, S. STOFFEL, S. J. SCHARF, R.
HIGUCHI, G. T. HORN, K. B. MULLIS, and H. A. EHRLICH.
1988. Primer-directed enzymatic amplification of DNA
with a thermostable DNA polymerase. Science 239:487–
494.
SAITOU, N., and M. NEI. 1987. The neighbor-joining method:
a new method for reconstructing phylogenetic trees. Mol.
Biol. Evol. 4:406–425.
SÁNCHEZ, D., M. D. GANFORNINA, and M. J. BASTIANI. 1996.
Developmental expression and biochemical analysis of
Conulin, a protein secreted from a subset of nerve growth
cones. J. Neurosci. 16:663–674.
SCHELLER, K., B. FISCHER, and H. SCHENKEL. 1990. Molecular properties, functions and developmentally regulated
biosynthesis of arylphorin in Calliphora vicina. Pp. 155–
162 in H. H. HAGEDORN, J. G. HILDEBRAND, M. G. KIDWELL, and J. H. LAW, eds. Molecular insect science. Plenum
Press, New York.
SELLOS, D. Y., S. LEMOINE, and A. VAN WORMHOUDT. 1997.
Molecular cloning of hemocyanin cDNA from Penaeus
vannamei (Crustacea, Decapoda): structure, evolution and
physiological aspects. FEBS Lett. 407:153–158.
SUGUMARAN, M., L. B. GIGLIO, H. KUNDZICZ, S. J. SAUL, and
V. SEMENSI. 1992. Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster.
Arch. Insect Biochem. 19:271–283.
SWOFFORD, D. L. 1991. PAUP: phylogenetic analysis using
parsimony. Version 3.0s. Illinois Natural History Survey,
Champaign.
TELFER, W. H., and J. G. KUNKEL. 1991. The function and
evolution of insect storage hexamers. Annu. Rev. Entomol.
36:205–228.
THIOULOUSE, J., and F. CHEVENET. 1996. NetMul, a WorldWide Web user interface for multivariate analysis software.
Comp. Stat. Data Anal. 21:369–372.
THOMPSON, J. D., D. G. HIGGINS, and T. J. GIBSON. 1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680.
VAN HOLDE, K. E., and K. I. MILLER. 1995. Hemocyanins.
Adv. Protein Chem. 47:1–81.
VOLBEDA, A., and W. G. J. HOL. 1989a. Crystal structure of
hexameric haemocyanin from Panulirus interruptus refined
at 3.2 Å resolution. J. Mol. Biol. 209:249–279.
. 1989b. Pseudo 2-fold symmetry in the copper-binding domain of arthropodan haemocyanins. J. Mol. Biol.
206:531–546.
VON HEIJNE, G. 1990. The signal peptide. J. Membr. Biol. 115:
195–201.
WANG, Z., and N. HAUNERLAND. 1994. Receptor-mediated endocytosis of storage proteins by the fat body of Helicoverpa
zea. Cell Tissue Res. 278:15–26.
MANOLO GOUY, reviewing editor
Accepted December 30, 1997