Báo cáo khoa học: New insights into evolution of crustacean hyperglycaemic hormone in decapods – first characterization in Anomura pdf

New insights into evolution of crustacean hyperglycaemic
hormone in decapods – first characterization in Anomura
Nicolas Montagne
´
, Daniel Soyez, Dominique Gallois, Ce
´
line Ollivaux and Jean-Yves Toullec
Universite
´
Pierre et Marie Curie – Paris 6, FRE 2852 CNRS – Prote
´
ines: Biochimie Structurale et Fonctionnelle, Equipe Biogene
`
se des
Peptides Isome
`
res, Paris, France
The neurohormones of the crustacean hyperglycaemic
hormone (CHH) family are structurally related pep-
tides encoded by a multigene family that is specific to
arthropods. In decapods, these neurohormones are
mainly produced in the major neuroendocrine organ,
situated in the eyestalk: the X-organ ⁄ sinus gland
(XO ⁄ SG) system. They play important roles in metab-
olism, reproduction and development of the animals.
Their size ranges from 72 to 83 amino acid residues,
and their main structural signature is the conserved
spacing of six cysteyl residues, arranged in three disul-
fide bridges [1]. CHH family peptides are also present
in hexapods, as the ion transport peptide (ITP), a neu-
ropeptide characterized in several insect species [2–4]

shares the same structural signature.
With regard to crustaceans, two subtypes may be
distinguished when amino acid sequences of the
various CHH family peptides are aligned [5]. Type I
Keywords
Anomura; CHH; CPRP; molecular evolution;
neuropeptide
Correspondence
N. Montagne
´
, Equipe Biogene
`
se des
Peptides Isome
`
res – FRE 2852 CNRS,
Universite
´
Pierre et Marie Curie, 7 Quai
Saint-Bernard, 75252 Paris Cedex 05,
France
Fax: +33 1 44 27 23 61
Tel: +33 1 44 27 22 53
E-mail:
Database
Sequences for P. bernhardus CHH and
G. strigosa CHH have been submitted to
the GenBank database under the accession
numbers DQ450960 and EF492145, respec-
tively

(Received 11 October 2007, revised 4
December 2007, accepted 17 December
2007)
doi:10.1111/j.1742-4658.2007.06245.x
The neuropeptides of the crustacean hyperglycaemic hormone (CHH)
family are encoded by a multigene family and are involved in a wide spec-
trum of essential functions. In order to characterize CHH family peptides
in one of the last groups of decapods not yet investigated, CHH was stud-
ied in two anomurans: the hermit crab Pagurus bernhardus and the squat
lobster Galathea strigosa. Using RT-PCR and 3¢ and 5¢ RACE methods, a
preproCHH cDNA was cloned from the major neuroendocrine organs
(X-organs) of these two species. Hormone precursors deduced from these
cDNAs in P. bernhardus and G. strigosa are composed of signal peptides of
29 and 31 amino acids, respectively, and CHH precursor-related peptides
(CPRPs) of 50 and 40 amino acids, respectively, followed by a mature hor-
mone of 72 amino acids. The presence of these predicted CHHs and their
related CPRPs was confirmed by performing MALDI-TOF mass spectro-
metry on sinus glands, the main neurohaemal organs of decapods. These
analyses also suggest the presence, in sinus glands of both species, of a pep-
tide related to the moult-inhibiting hormone (MIH), another member of
the CHH family. Accordingly, immunostaining of the X-organ ⁄ sinus gland
complex of P. bernhardus with heterologous anti-CHH and anti-MIH sera
showed the presence of distinct cells producing CHH and MIH-like pro-
teins. A phylogenetic analysis of CHHs, including anomuran sequences,
based on maximum-likelihood methods, was performed. The phylogenetic
position of this taxon, as a sister group to Brachyura, is in agreement with
previously reported results, and confirms the utility of CHH as a molecular
model for understanding inter-taxa relationships. Finally, the paraphyly of
penaeid CHHs and the structural diversity of CPRPs are discussed.
Abbreviations

CHH, crustacean hyperglycemic hormone; CPRP, CHH precursor-related peptide; IPRP, ITP precursor-related peptide; ITP, ion transport
peptide; MIH, moult-inhibiting hormone; MOIH, mandibular organ-inhibiting hormone; SG, sinus gland; VIH, vitellogenesis-inhibiting hormone;
XO, X-organ.
FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works 1039
peptides, the CHHs sensu stricto, are typically
72 amino acid residues long, and their protein precur-
sors contain, between the signal peptide and the CHH
progenitor sequence, a cryptic peptide called a CHH
precursor-related peptide (CPRP), which is removed
during precursor post-translational processing. CPRP
is co-released with CHH within the haemolymph, from
the SG nerve endings [6], but, to date, no function has
been assigned to it. In every decapod species investi-
gated, at least one CHH form was found, but the pres-
ence of several isoforms in a single species has
frequently been reported: these isoforms may arise
from expression of different genes or from various
post-translational modifications such as N-terminal
cyclization or l ⁄ d isomerization of a specific residue
[7]. In addition, CHH-producing sites are located
outside eyestalks, and synthesize either an eyestalk-
like CHH [8] or a CHH-like peptide arising from the
same gene by tissue-specific alternative splicing, with
unknown function [9].
Historically, CHH was named so because of its most
prominent bioactivity upon injection within the ani-
mal, namely rapid and sustained hyperglycaemia.
However, a number of experimental studies have since
demonstrated that CHH is a pleiotropic hormone, but
its precise physiological roles are far from clear and

vary greatly according to species. For example, CHH
is involved in the control of female reproduction,
either positively in the lobster Homarus americanus [10]
or negatively in the green tiger prawn Penaeus semi-
sulcatus [11]. CHH inhibits the synthesis of methyl
farnesoate – a juvenile hormone-like compound that
mainly acts on gonad growth – in the spider crab Libi-
nia emarginata, acts on lipid metabolism and is impli-
cated in osmoregulation in several decapod species
[12]. In the shore crab Carcinus maenas, CHH may be
involved in the control of moulting, together with a
neuropeptide from the type II subfamily: the moult-
inhibiting hormone (MIH) [13].
Type II subfamily peptides include moult-inhibiting
hormone (MIH), vitellogenesis-inhibiting hormone
(VIH) and mandibular organ-inhibiting hormone
(MOIH). At the preprohormone level, no cryptic pep-
tide such as CPRP is associated with type II peptides.
A paradigm in crustacean endocrinology is that, dur-
ing the intermoult stage, MIH inhibits ecdysteroid bio-
synthesis by the moulting glands, i.e. the Y-organs
[14]. MIHs have been described in all decapod groups
investigated, except the Homarida, in which a CHH
isoform has a moult-inhibiting function [15]. In this
taxon, another type II peptide has been described: the
VIH, which controls ovarian development by inhibit-
ing vitellogenesis in female lobsters [10]. This function
seems to be performed by CHH in other decapods, as
mentioned above. The last type II peptide known to
date is MOIH. It inhibits methyl farnesoate synthesis

by the mandibular organ, and has only been character-
ized in the crab Cancer pagurus [16]. It may have
arisen from an MIH gene duplication in the genus
Cancer only [17].
Since the first elucidation of a CHH sequence, in the
shore crab Carcinus maenas [18], about 100 CHH
family peptides have been characterized in 35 decapod
species, but some taxa remain relatively poorly investi-
gated. For example, over 70% of all CHHs known
have been described in brachyurans and penaeids, and
a few decapod groups remain unexplored. In order to
better comprehend the diversity of this peptide family,
we decided to focus our studies on one of these unin-
vestigated groups: Anomura. Two species found in the
French littoral were selected for this work: the hermit
crab Pagurus bernhardus and the squat lobster Gala-
thea strigosa. We cloned full-length CHH precursor
cDNAs from X-organs of the two species, and analy-
sed the peptide content of the sinus glands by
MALDI-TOF mass spectrometry to check for the
presence of the peptides predicted from molecular
cloning. Furthermore, immunochemistry was per-
formed on P. bernhardus eyestalks using heterologous
anti-CHH and anti-MIH sera. The CPRP and CHH
sequences identified in this work were included in sepa-
rate alignments, and a phylogenetic tree of decapod
CHHs was estimated by maximum-likelihood recon-
struction.
Results
Molecular cloning of CHH precursor cDNAs from

P. bernhardus and G. strigosa
After RT-PCR and 3¢ and 5¢ RACE steps, a complete
preproCHH cDNA sequence was obtained from the
total RNA extract of XO cells of P. bernhardus. This
796 bp sequence contains an open reading frame of
468 bp, encoding a 155 amino acid prepropeptide
(Fig. 1). The most likely signal peptide cleavage site,
based on the neural network and the hidden Markov
model prediction methods, is between residues Ser29
and Arg30. The 126 amino acid residue propeptide
resulting from signal peptide excision contains a typi-
cal di-basic processing site at residues Lys80–Arg81,
which is the cleavage site between the 50 amino acid
CPRP and the 74 amino acid CHH. The propeptide
ends with a C-terminal Gly154–Lys155, a typical
amidation site. This results in the production of a
72 residue amidated mature hormone. In the 3¢ UTR,
CHH characterization in Anomura N. Montagne
´
et al.
1040 FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works
a putative polyadenylation signal (AATAAA) is pres-
ent 12 bp upstream of the poly(A) tail.
As for the hermit crab, a complete preproCHH
cDNA was also sequenced from G. strigosa XO mate-
rial. This cDNA is 889 bp in length and contains an
open reading frame of 444 bp, corresponding to a
preprohormone of 147 amino acid residues (Fig. 2).
This precursor can be divided as follows: a signal pep-
tide of 31 residues (the most probable signal peptide

cleavage site predicted by both methods is between
Ala31 and Arg32), a CPRP of 40 residues, a di-basic
cleavage site for potential prohormone convertase mat-
uration, and a 74 amino acid residue CHH, with two
final residues (Gly146–Lys147) that may be removed
during C-terminal amidation of the peptide. The only
putative polyadenylation signal found (ATTAAA) is
69 bp upstream of the poly(A) tail.
Mass spectrometry analyses on P. bernhardus
and G. strigosa sinus gland extracts
Mass spectra obtained by analysis of a small amount
of SG extract (0.03 SG equivalent) are presented in
Fig. 3. In the spectrum from P. bernhardus SG extract
(Fig. 3A), several ions were observed in the 2000–
12 000 m ⁄ z range. The ion with an m ⁄ z at 8345 very
likely corresponds to CHH, as this mass value is very
close to the M + H
+
value of 8345.6 Da calculated
from the cDNA sequence and taking into account the
predicted post-translational maturation steps (forma-
tion of three disulfide bridges, cyclization of the N-ter-
minal glutaminyl residue and C-terminal amidation).
In addition, the spectrum shows an ion at m ⁄ z 9392
and three prominent ions with m ⁄ z values at
4787, 4844 and 5001, respectively. The latter probably
Fig. 1. Nucleotide sequence of the
P. bernhardus CHH precursor cDNA, with
the complete open reading frame in capital
letters. The deduced amino acid sequence

is indicated below (the asterisk indicates the
stop codon). Both nucleotide and amino acid
numbers are indicated at the end of the
lines. The putative polyadenylation signal
(AATAAA) is indicated in bold italic letters.
The locations of upstream (PabCHH-U1 and
PabCHH-U2) and downstream (PabCHH-D1
and PabCHH-D2) primers, used for 5¢ RACE
and 3¢ RACE, respectively, are indicated by
grey arrows.
N. Montagne
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et al. CHH characterization in Anomura
FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works 1041
corresponds to the CPRP, as calculation of the mass
of the putative CPRP present in the CHH precursor
gives a theoretical M + H
+
value of 5002.6 Da. In
addition, several ions with masses below 3000 were
observed. Calculations were performed to check
whether these ions could correspond to multi-charged
ions of other observed peaks, with no result.
Similar conclusions may be drawn upon examination
of the mass spectrum from G. strigosa SG extract
(Fig. 3B): an ion with an m ⁄ z at 8315 was present,
which may correspond to CHH, as the calculated
M+H
+
value from cloning data is 8316.5 Da. Also,

as with P. bernhardus spectrum, an ion with high-
er m ⁄ z (9175) was present in the spectrum, and the ion
with an m ⁄ z at 4197 very likely corresponds to the
CPRP, the theoretical mass deduced from the cDNA
being 4198.7 Da. The major ion mass observed in
G. strigosa SG extract was at m⁄ z 7605: this value
perfectly fits with the predicted mass of the CHH trun-
cated by seven amino acid residues on the C-terminus
side, which may result in an ion with a calculated mass
of 7604.6 Da.
Anti-CHH and anti-MIH immunoreactivities in
P. bernhardus eyestalks
Immunocytochemistry experiments were conducted on
whole mounts of eyestalks of P. bernhardus using anti-
Homarus americanus CHH and anti-Cancer pagurus
MIH sera. Confocal micrographs revealed that both
the anti-CHH and anti-MIH antisera produced intense
and homogenous labelling all along the neurons of the
XO ⁄ SG system (Fig. 4A). Classically, the X-organ (the
grouping of perikarya in which the neuropeptides are
synthesized) is located inside the medulla terminalis of
the eyestalk, whereas neuronal endings constituting the
Fig. 2. Nucleotide sequence of the
G. strigosa CHH precursor cDNA, with the
complete open reading frame in capital
letters. The deduced amino acid sequence
is indicated below (the asterisk indicates the
stop codon). Both nucleotide and amino acid
numbers are indicated at the end of the
lines. The putative polyadenylation signal

(ATTAAA) is indicated in bold italic letters.
The locations of upstream (GasCHH-U1 and
GasCHH-U2) and downstream (GasCHH-D1
and GasCHH-D2) primers, used for 5¢ RACE
and 3¢ RACE, respectively, are indicated by
grey arrows.
CHH characterization in Anomura N. Montagne
´
et al.
1042 FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works
sinus gland are located on the periphery of the upper
medulla interna. About 30 CHH-immunoreactive
(CHH-IR) and 10 or so MIH-immunoreactive (MIH-
IR) perikarya were observed in the XO (27 CHH-IR
and nine MIH-IR cells in Fig. 4B). Labelling was
cytoplasmic and granular, and co-localization of the
two labels (which should result in an orange coloration
on merged images) was never observed. With regard to
morphological criteria, the two types of perikarya were
indistinguishable: they displayed an ovoid shape a
mean size of 30 · 35 lm, with the exception of one
larger CHH-IR cell body that was found in every
preparation examined (arrow on Fig. 4B). CHH-IR
and MIH-IR cells were not located in separate areas
of the XO, as a cluster of five MIH-IR cell bodies was
observed near the start of the axonal tract, whereas
the others were dispersed among CHH-IR structures
at the periphery of the XO. Axons were grouped
in a tract of approximately 1.5 mm long and
40 lm wide, in which MIH-IR and CHH-IR axons

were well separated (Fig. 4C). More varicosities were
seen in the MIH-IR axons than in the CHH-IR
axons. The SG measured around 600 lm long by
300 lm wide. MIH-IR neuronal endings were grouped
in a central area of the SG, whereas CHH-IR endings
were distributed all over the neurohaemal structure
(Fig. 4D).
Discussion
Early studies on CHH in Anomura mainly focused on
the group specificity of these peptides, based on cross-
injection of eyestalk extracts [19,20]. These experiments
revealed a weak cross-reactivity between Anomura and
closely related taxa (Brachyura and Astacida): eyestalk
extracts of P. bernhardus caused hyperglycaemia in the
crab Carcinus maenas, but not in the crayfish Asta-
cus leptodactylus or Orconectes limosus, and extracts
from any of these species failed to increase glycaemia
in the hermit crab. More intriguing is the fact that, in
the squat lobster Munida rugosa, even injection of its
own eyestalk extract did not trigger hyperglycaemia.
Similarly, in a more recent study, the effect of lipo-
polysaccharide injection was examined in various crus-
tacean groups [21]. Strong hyperglycaemia was elicited
in most of the species studied (in intact but not in
eyestalk-ablated animals), including the hermit crab
Paguristes oculatus, but not in M. rugosa [21]. There-
fore, the question of the presence or not of CHH in
anomurans, especially in squat lobsters, had remained
open until now. Given these data, we chose one species
each of the Paguridae and Galatheidae families to

search for anomuran CHHs.
In the present study, two complete CHH precursor
cDNA sequences were obtained from the X-organs
of P. bernhardus and G. strigosa. All the structural fea-
tures of the CHHs (type I peptides) are present in the
deduced amino acid sequences: the presence of a
CPRP, the di-basic cleavage site (Lys–Arg) between
the CPRP and the mature hormone sequence, the posi-
tion of the six cysteyl residues in the sequence, the size
of the putative mature peptide (72 amino acid resi-
dues), and presence of the amidation signal (Gly–Lys)
at the C-terminal end. Occurrence of the predicted
CHHs in the sinus glands of the two species was con-
firmed by comparison of the calculated molecular
masses and those measured by mass spectrometry anal-
yses performed on crude SG extracts. For each species,
MALDI-TOF mass spectrometry generated an ion
with an average m ⁄ z value that was in agreement with
the masses deduced from predicted sequences. In
G. strigosa SG extract, the major ion at 7605 m ⁄ z cor-
responds to a truncated form of CHH in which the
seven last residues have been removed by a proteolytic
process. Such degradation at the C-terminal side of the
CHH has been noted previously [15,22], and this may
be due to C-terminal proteolysis during preparation of
SG extracts.
Fig. 3. MALDI-TOF mass spectra of sinus gland extracts. (A) Analy-
sis of tissue equivalent to 0.03 SG from P. bernhardus. (B) Analysis
of 0.03 SG equivalents from G. strigosa.
N. Montagne

´
et al. CHH characterization in Anomura
FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works 1043
The presence in the mass spectra of a single ion
between m ⁄ z 8000 and 9000 (which is the mass range
for all CHHs characterized so far) suggests that a sin-
gle CHH form is present in the sinus glands of
P. bernhardus and G. strigosa. However, the presence
of a single ion does not preclude the existence of ste-
reoisomers, which are not distinguishable by mass
spectrometry, as seen in various astacidean species
[23,24]. On the other hand, a single peptide may origi-
nate from several CHH genes that differ at the level of
the CPRP, the signal peptide or the untranslated
region (UTR) but encode identical mature hormones.
Such a situation has been described in Brachyura
[9,25], Homarida [26] and Astacida [27]. Indeed, in
the mass spectrum of P. bernhardus SG extract, in
addition to an ion corresponding to the mass of the
deduced CPRP (5001 Da), two others with close
m ⁄ z values (4844 and 4787) were detected that could
correspond to CPRPs from different CHH precursors.
The fact that the corresponding cDNAs were not
found in our study may be explained by a paucity of
their transcripts in the XO cells relative to the major
one that has been cloned. In G. strigosa SG, occur-
rence of the CPRP deduced from the nucleotide
sequence was also confirmed by mass spectroscopy,
but, unlike P. bernhardus, no other putative CPRPs
were detected.

In all decapod taxa, in addition to CHH, type II
hormones (MIH, VIH, MOIH) are present in the SG.
Our results strongly suggest that this is also true for
A
B
D
C
Fig. 4. Confocal micrographs showing the
distribution of CHH-immunoreactive (green)
and MIH-immunoreactive (red) structures in
the eyestalk of P. bernhardus. (A) Anti-CHH
and anti-MIH labelling of the whole X-organ ⁄
sinus gland system (assembled from
three projections, B, C and D, each
consisting of a series of confocal sections).
(B) Twenty-seven CHH-IR and nine MIH-IR
perikarya observed in the X-organ, with no
co-localization. (C) CHH-IR and MIH-IR
axons in the axonal tract. (D) CHH-IR and
MIH-IR neuronal endings in the sinus gland.
CHH characterization in Anomura N. Montagne
´
et al.
1044 FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works
Anomura. A first indication is given by mass spec-
trometry analysis: ions with m ⁄ z values of 9392 and
9175 were found in SG extracts of P. bernhardus and
G. strigosa, respectively, which fit with the mass range
of the type II peptides characterized so far. As for the
CHHs, only a single ion was detected in this mass

range, suggesting that only one mature type II peptide
is present in these species, as is the case in most deca-
pods. A second indication of the presence of type II
peptide(s) in Anomura is seen in our immunohisto-
chemical study. Immunostaining performed on hermit
crab eyestalks using both anti-Homarus americanus
CHH and anti-Cancer pagurus MIH sera revealed the
existence of two distinct groups of neurons: the major-
ity of them were stained only by the anti-CHH serum,
and therefore probably represent the CHH secretory
cells, whereas other neurons were only reactive to the
anti-MIH serum. This result indicates that the XO ⁄ SG
system of P. bernhardus synthesizes a peptide that
shares structural similarities with brachyuran MIHs.
However, it is not yet known whether this peptide is a
functional MIH or not. The organization of the dis-
tinct CHH and MIH-like production systems observed
in the hermit crab is similar in brachyurans [28], but
the number of immunoreactive perikarya in the
XO ⁄ SG system (about 40) is significantly lower in
P. bernhardus. This is especially true for MIH-IR cells,
which are three times less numerous than CHH-IR
ones in the hermit crab, compared with two times less
in crabs. During this study, we attempted to clone an
MIH-like peptide using degenerate primers deduced
from consensus sequences in the MIH subfamily, but
these attempts were not successful.
The tree presented in Fig. 5 was estimated by maxi-
mum likelihood from the updated CHH data set that
includes anomuran CHHs (Table 1). In this phylogeny,

the Anomura appear to be clearly monophyletic, as
P. bernhardus CHH and G. strigosa CHH form a clade
supported by a bootstrap value of 98. Monophyly is
also well supported for Brachyura, Astacida and
Homarida, thus enlightening the high group specificity
of CHHs. The Anomura are a sister group to Brachy-
ura, which is consistent with their currently recognized
phylogenetic position, i.e. grouping in a clade named
Meiura. Taking this analysis further, relationships
between the various groups of Reptantia deduced from
our results are identical to those proposed in a recent
phylogeny established on the basis of both molecular
and morphological data [29], but it should be noted
that these inter-group relationships are not significantly
supported by the bootstrap scores (e.g. 45 for Meiura,
or 25 for the entire Reptantia). These low bootstrap
values could be explained by the short length of CHH
sequences compared to the number of taxa included in
the data set (72 residues and 29 taxa), and also by the
relatively fast evolution of CHH among crustaceans.
Unlike the taxa cited above, the Penaeidea are para-
phyletic, and the fact that two types of genes (contain-
ing either three or four exons) can encode penaeid
CHHs may explain this paraphyly. Indeed, all CHH
genes formerly described in Penaeidea contained three
exons, whereas those described in other decapods
(Pleocyemata) contained four exons [30]. However,
recently, two genes with four exons have been
described in the white shrimp Litopenaeus vannamei
(one of which encodes CHH2) in addition to a three-

exon one (encoding the so-called CHH) [31,32]. Addi-
tionally, we have deduced a CHH sequence from an
EST of Marsupenaeus japonicus eyestalk (see Experi-
mental procedures for detail), and, based on the
structure of the mRNA, this CHH also arises
from transcription of a four-exon gene. In Fig. 5,
L. vannamei CHH2 and Ma. japonicus CHH cluster
together with Pleocyemata CHHs – also arising from
four-exon genes – whereas the other penaeid sequences
form a separate branch, in which only peptides arising
from three-exon genes are present. This separation at
the base of the tree between the ‘three-exon’ and ‘four-
exon’ clades, which is well supported by the bootstrap
values, probably represents a duplication event associ-
ated with an exon deletion that may have occurred
only in Penaeidea, leading to the presence of two types
of CHH gene in this taxon. According to this hypothe-
sis, the ancestral CHH gene would exhibit four exons,
which agrees with the presence of a similar four-exon
pattern in genes encoding insect ITPs [30]. To be
definitively established, such a scheme requires eluci-
dation of CHH gene structure in taxa other than
decapods.
Among decapods, the size of CPRPs is not as well
conserved as that of CHHs. Until the present study,
the number of amino acid residues in CPRPs was
known to range from 4, in the giant tiger prawn
Penaeus monodon [33], to 43, in the euryhaline crab
Pachygrapsus marmoratus [25]. With 50 residues, the
CPRP deduced from the preproCHH sequence of

P. bernhardus is the longest ever reported. Associated
with the fact that G. strigosa CPRP is ten residues
shorter, this illustrates well the variability of these
cryptic peptides, even within the same decapod
group. As seen in the alignment of CPRP sequences
(Fig. 6A), the first 15 residues at the N-terminal
end are relatively well conserved in Pleocyemata.
The only CPRP that does not exhibit this 15-residue
stretch is that from G. strigosa in which the three
residues at positions 9–11 are missing. Consensus
N. Montagne
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et al. CHH characterization in Anomura
FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works 1045
sequences of this domain in each taxon are shown in
Fig. 6B. They are represented separately for each
taxon rather than for all decapods to avoid the bias
generated by over-representation of brachyuran
CPRPs compared with other taxa. Indeed, there are
only two available CPRP sequences in Anomura
(present study) and in Caridea, for which both
sequences are from Macrobrachium sp. only. With
regard to Penaeidea, the CPRPs deduced from
L. vannamei CHH2 [32] and Ma. japonicus CHH
precursors (arising from four-exon genes) are similar
to those of Pleocyemata and do possess the 15-resi-
due N-terminal domain (Table 1). On the other hand,
the other penaeid CPRPs, in which this region is
either reduced to the first 4–6 residues or is entirely
absent, seem to be related to CHH precursors

encoded by three-exon genes, as demonstrated for
Metapenaeus ensis CHHs A and B [34,35]. The CPRP
Fig. 5. Phylogeny of decapod CHHs based on maximum-likelihood analysis of the CHH amino acid data set (29 taxa, 72 residues) using a
JTT + G model of protein evolution. Schistocerca gregaria ITP was assigned as the out-group. Sequence accession numbers or references
are given in Table 1. Numbers at nodes are bootstrap values based on 100 replicates. The Anomuran sequences that were determined in
this study are shown in bold. For taxa in which the CHH genes have been sequenced, the number of exons (three or four) is indicated in a
black circle after the name of the species. A four-exon pattern was also assigned for taxa in which two peptides arising by alternative splic-
ing have been described.
CHH characterization in Anomura N. Montagne
´
et al.
1046 FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works
C-terminal sequence seems to be much less conserved,
except for a histidine or a glutamine located four res-
idues before the C-terminal end. However, the vari-
ability is less significant when comparisons are made
within each taxon, indicating the possibility of an
intra-taxon signature (Fig. 6B). In penaeid CPRPs
obtained from three-exon genes, the lack of such a
signature emphasizes the high variability of these
genes compared with four-exon ones. Interestingly,
an histidyl residue is also present at an identical posi-
tion in the precursors of the only hexapod peptide
known to belong to the CHH family, the ion trans-
port peptide (ITP), in which a short sequence (7–
10 amino acid residues) is present between the signal
peptide and the mature ITP (Fig. 6A). To date, these
ITP precursor-related peptides (IPRPs) are only
Table 1. Amino acid sequences used in this study. CHH, crustacean hyperglycemic hormone; CPRP, CHH precursor-related peptide; IPRP,
ITP precursor-related peptide; ITP, ion transport peptide; MOIH, mandibular organ-inhibiting hormone; SG, sinus gland.

Sequence
Mature peptide
included in the CHH
data set
Precursor-related
peptide included in
the CPRP data set
UniProt acc.
number or
reference
Aedes aegypti ITP + Q1XAU4
Astacus leptodactylus CHH + + Q1RN83
Bythograea thermydron CHH + + Q9BKJ5
Callinectes sapidus CHH + + Q6QJL8
Cancer pagurus CHH + + P81032
Cancer productus CPRP I + [37]
Carcinus maenas CHH + + P14944
Cherax destructor CHH B + P83486
Gecarcinus lateralis CHH A + A0EVE7
Gecarcoidea natalis CHH + + A1E290
Homarus americanus CHH A + P19806
Homarus gammarus CHH A + Q3HXZ6
Homarus gammarus CHH B + Q3HXZ5
Jasus lalandii CHH I + P56687
Jasus lalandii CHH II + [38]
Libinia emarginata MOIH
a
+ + P56688
Litopenaeus schmitti CHH + P59685
Litopenaeus vannamei CHH + Q26181

Litopenaeus vannamei CHH 2 + + [32]
Locusta migratoria ITP + Q9XYF9
Macrobrachium lanchesteri CHH + O77220
Macrobrachium rosenbergii CHH + + Q9NHU3
Manduca sexta ITP + Q1XAU8
Marsupenaeus japonicus CHH
b
+ + Q2MGW2
Marsupenaeus japonicus SGP I
a
+ + O15980
Marsupenaeus japonicus SGP V
a
+ O15981
Marsupenaeus japonicus SGP VII
a
+ + O15982
Metapenaeus ensis CHH A + + O96688
Metapenaeus ensis CHH B + Q9NGP0
Nephrops norvegicus CHH A + + Q6WGR4
Orconectes limosus CHH A + + Q25589
Pachygrapsus marmoratus CHH A + + Q6Y5A6
Penaeus monodon SGP I
a
+ O97383
Penaeus monodon SGP II
a
+ O97384
Penaeus monodon SGP III
a

+ O97385
Penaeus monodon SGP IV
a
+ + O97386
Potamon ibericum CHH + + Q2VF26
Procambarus clarkii CHH + + Q25683
Schistocerca gregaria ITP + Q26491
Scylla olivacea CHH + + Q6UDR5
a
Not labelled as CHH in the database but undoubtedly a type I peptide based on its structure.
b
Deduced from the sequence of the alterna-
tively spliced product submitted to the database.
N. Montagne
´
et al. CHH characterization in Anomura
FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works 1047
A
B
Fig. 6. (A) Multiple-sequence alignment of 32 CHH precursor-related peptide (CPRP) sequences from 26 decapod species and three ITP pre-
cursor-related peptides (IPRP) sequences from three hexapod species, with the amino acid number indicated at the end of the line (see
Table 1 for sequence accession numbers or references). (B) Consensus sequences of N- and C-terminal domains of each taxon. The one-let-
ter code and standard colouring are used ( The major amino acids are shown by large
letters; small fonts indicate minor amino acids in the sequences. Two letters of the same font size indicate equivalent occurrence.
CHH characterization in Anomura N. Montagne
´
et al.
1048 FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works
known from in silico prediction, and it remains to be
determined whether they are stored in neuroendocrine

structures of hexapods and co-released with ITPs,
like CPRPs are co-released with CHH, or not.
In conclusion, this work has focused on Anomura
because it remained a ‘black hole’ in our understand-
ing of CHH family diversity in decapods. The phylo-
genetic position of Anomura noted in our study, as a
sister group to Brachyura, is in agreement with those
previously described. Furthermore, this effort to
extend CHH membership leads to a tree in which the
inter-taxa relationships are congruent with those of
recent decapod phylogenies. This confirms the possi-
bility of using CHHs as molecular data in phyloge-
netic analyses, in spite of their short sequence. In
addition, the ever-increasing number of complete
insect genomes available in databanks increases our
knowledge regarding CHH family peptides in hexa-
pods. It would be interesting to carry out similar
comparative studies on taxa that appeared before
decapods and hexapods during arthropod evolution.
This will help to establish a relevant evolutionary
scenario for this neuropeptide family, and could also
produce valuable information about disputed phyloge-
netic relationships within Arthropoda and even
Ecdysozoa.
Experimental procedures
Animals and dissection
Both hermit crabs P. bernhardus and squat lobsters
G. strigosa were collected on the Channel littoral of
Roscoff, France. They were kept in aquaria with running
seawater at 13 °C. Animals were anaesthetized in ice

before dissection. Eyestalks were removed, then medullae
terminalii (containing XO) and the SG were dissected in
sterile cold seawater, except for those used for immuno-
chemistry, which were removed from their cuticle and
immediately dipped into fixative solution. Dissected XO
were immediately placed in liquid nitrogen and stored
at )80 °C until use.
RNA extraction and RT-PCR
Total RNA from 60 medullae terminalii of P. bernhardus
and 20 medullae terminalii of G. strigosa was isolated using
an SV Total RNA Isolation System (Promega, Madison,
WI, USA). Single-stranded cDNA was synthesized
from 2 l g of total RNA using 200 units of SuperScriptÔ II
reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and
an oligo(dT) primer. The mixture was incubated at 42 °C
for 50 min, then the reaction was stopped by heating
at 70 °C for 15 min. PCR amplifications were performed
in 25 lL total volume with 1.5 units of Taq DNA polymer-
ase (Promega), using two degenerate oligonucleotide prim-
ers (0.4 lm each) that were designed based on conserved
C-terminal and N-terminal parts of CHH sequences of
Brachyura: 5¢-CAGAT(C ⁄ T)TACGA(C ⁄ T)(A ⁄ T)(C ⁄ G)GT
C(C ⁄ T)TGCAAGGG-3¢ (sense), corresponding to amino
acid sequence QIYD(R ⁄ S ⁄ T)SCKG(1–9), and 5¢-CTTC
(C ⁄ T)TGCCAACCATCTG-3¢ (antisense), corresponding to
QMVG(K ⁄ R)K(70–75). After a denaturation step at 94 °C
for 3 min, 35 amplification cycles were carried out
(94 °C for 30 s, 58 °C for 30 s, 72 ° C for 30 s), followed by
a final extension step at 72 °C for 10 min.
After purification from 2% agarose gel (GENE-

CLEANÒ III kit, Qbiogene, Irvine, CA, USA), the PCR
products were cloned using the pGEMÒ-T Easy Vector
and JM109-competent Escherichia coli cells (Promega).
Recombinant plasmids were purified using a WizardÒ
Plus SV Miniprep kit (Promega). Sequencing was per-
formed using Genome Express (Meylan, France) by the
dideoxy chain termination method.
3¢ and 5¢ RACE
Specific primers were designed from the sequences obtained
by RT-PCR in order to perform RACE experiments. Pri-
mer sequences and localization in the CHH sequences are
indicated in Figs 1 and 2. For the 3¢ and 5¢ RACE reac-
tions, 500 ng of total RNA from medullae terminalii of
each species were reverse-transcribed using the SMAR-
TÔ RACE cDNA amplification kit (Clontech, Mountain
View, CA, USA) according to the manufacturer’s instruc-
tions. cDNA was then amplified using AdvantageÔ 2 poly-
merase mix (Clontech), using the Universal Primer
Mix supplied in the kit and the gene-specific primers
Pab ⁄ GasCHH-U1 for 3¢ RACE and Pab ⁄ GasCHH-D1
for 5¢ RACE under the following conditions: five cycles
of 94 °C for 30 s and 72 °C for 2 min, five cycles of
94 °C for 30 s, 70 °C for 30 s and 72 °C for 2 min,
25 cycles of 94 °C for 30 s, 68 °C for 30 s and 72 °C
for 2 min, and 72 °C for 10 min as a final extension step.
Nested PCR was performed realized on 1 ⁄ 150th of
the volume of the first PCRs, using the nested
universal primer from the kit and the primers Pab ⁄
GasCHH-U2 for 3¢ RACE and Pab ⁄ GasCHH-D2 for
5¢ RACE, under the following conditions: 25 cycles of

94 °C for 30 s, 68 °C for 30 s and 72 °C for 2 min, fol-
lowed by 72 ° C for 10 min. PCR products were cloned and
sequenced as described above.
Both neural networks and hidden Markov model algo-
rithms were used to examine the presence and location of
signal peptide cleavage sites in the sequences [36] on the
SignalP 3.0 server web page ( />services/SignalP/).
N. Montagne
´
et al. CHH characterization in Anomura
FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works 1049
Mass spectrometry
Peptides were extracted from 20 SG from each species
in 500 lL of a 10% acetic acid solution at 80 °C, as
described previously [23]. Then 5 lL aliquots of the SG
extract were vacuum dried. The residue was redissolved in
5 lL of 50% acetonitrile in water with 0.1% trifluoroacetic
acid, and 0.5 lL of the solution (i.e. tissue equivelant of
0.03 SG) was mixed with an equal volume of saturated
matrix solution (a-cyano-4-hydroxycinnamic acid) and
spotted onto the target plate of a mass spectrometer
MALDI-TOF Voyager-DEÔ (Applied Biosystems, Foster
City, CA, USA). Spectra were acquired in linear positive
mode from m ⁄ z 2000 to 12 000, and the masses were calcu-
lated, after external calibration, using Data ExplorerÒ
(Applied Biosystems). moverz software (Genomic Solutions,
Ann Arbor, MI, USA) was used to draw the spectra.
Immunohistochemistry
After dissection, P. bernhardus eyestalks were fixed over-
night at 4 °C in an 8% paraformaldehyde solution in

0.01 m NaCl ⁄ P
i
, and washed for 6 h with PBS. All subse-
quent incubations were performed at room temperature
under constant agitation. Before application of antibodies,
non-specific binding was blocked by incubation of tissues
for 4 h in buffer A (0.01 m NaCl ⁄ P
i
containing 0.05% Tri-
ton X-100 and 5% normal goat serum). Primary antisera
were anti-HoaCHH (polyclonal IgG raised in guinea pig
against Homarus americanus CHH), and anti-CapMIH
(antiserum raised in rabbit against Cancer pagurus MIH,
supplied by S. G. Webster, University of Wales, Bangor,
UK), both used at 1 : 500 dilution in buffer A overnight.
After three washes in buffer A, the eyestalks were incubated
overnight with secondary fluorescent antibodies (Molecular
Probes, Eugene, OR, USA) diluted in buffer A: goat anti-
rabbit IgG conjugated to fluorochrome Alexa FluorÒ 568
(red), and goat anti-guinea pig IgG conjugated to fluoro-
chrome Alexa FluorÒ 488 (green), both at 1 : 500 dilution.
After three washes in 0.01 m NaCl ⁄ P
i
, tissues were mounted
on slides and cover-slipped using VectaShieldÒ mounting
medium (Vector, Burlingame, CA, USA). Preparations were
stored at 4 °C until use.
Samples were observed using a Leica TCS SP5 confocal
microscope (Leica Microsystems, Heidelberg, Germany)
equipped with a 20 · objective (NA = 0.7) and an argon–

helium–neon laser used at 488 and 561 nm wavelengths.
Micrographs were processed and assembled using AdobeÒ
photoshopÒ CS2 (Adobe Systems Inc., San Jose, CA,
USA).
Sequence alignments and phylogeny
Two sequence data sets were created: a CHH data set com-
posed of mature hormone sequences and a CPRP data set
of precursor-related peptide sequences. The sequences
selected are summarized in Table 1. Alignments were per-
formed using mafft version 5.8 (shu-u.
ac.jp/mafft/online/server/), with default parameters (BLO-
SUM62 scoring matrix, gap open penalty of 1.53, and no
gap extension penalty), and then processed manually when
necessary.
For CPRPs, the sequences deduced from CHH precur-
sors were added only when the maximum cleavage site
probability between the signal peptide and the CPRP was
>0.7 using the hidden Markov model algorithm (in order
to avoid doubts about the N-terminal end of the putative
CPRPs). Only one sequence of each species was used
(except for Penaeidea), and when several CPRPs from
different species shared the same sequence, just one was
included in the alignment. Three putative IPRPs were
also deduced from ITP precursors and added to the
alignment.
For CHHs, 28 sequences representing all decapod taxa
in which CHHs have been described were aligned. Only
type I peptides (the CHHs sensu stricto) were included in
the alignment, and not the CHH-like peptides originating
from alternative splicing of CHH genes with four exons.

The putative Ma. japonicus CHH sequence included in our
data set is not the one submitted to the GenBank
database; it is the type I sequence that we deduced from
this using the alternative splicing rule. The ITP of the
locust Schistocerca gregaria was added as an out-group,
and 72 unambiguously aligned residues were used in the
alignment.
The phylogeny was created maximum-likelihood recon-
struction. The best-fit model for protein evolution was
determined using prottest 1.3 [39]: based on Akaike
Information Criterion (AIC), the JTT amino acid substitu-
tion model [40] was selected, with four substitution rate
categories and accounting for rate heterogeneity using an
estimated gamma distribution shape (JTT + G). Tree
reconstruction was performed using phyml software [41],
and node support was assessed using a bootstrap procedure
based on 100 replicates.
Acknowledgements
The authors are grateful to Professor Franc¸ ois Lallier
(Station Biologique de Roscoff, France) for animal
supply, and to Dr Yves Desdevises (Observatoire
Oce
´
anologique de Banyuls-sur-Mer, France) for his
advice and help on phylogenetics.
References
1 Keller R (1992) Crustacean neuropeptides: structures,
functions and comparative aspects. Experientia 48,
439–448.
CHH characterization in Anomura N. Montagne

´
et al.
1050 FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works
2 Meredith J, Ring M, Macins A, Marschall J, Cheng
NN, Theilmann D, Brock HW & Phillips JE (1996)
Locust ion transport peptide (ITP): primary structure,
cDNA and expression in a baculovirus system. J Exp
Biol 199, 1053–1061.
3 Endo H, Nagasawa H & Watanabe T (2000) Isolation
of a cDNA encoding a CHH-family peptide from the
silkworm Bombyx mori. Insect Biochem Mol Biol 30,
355–361.
4 Dai L, Zitnan D & Adams ME (2007) Strategic expres-
sion of ion transport peptide gene products in central
and peripheral neurons of insects. J Comp Neurol 500,
353–367.
5 Lacombe C, Greve P & Martin G (1999) Overview on
the sub-grouping of the crustacean hyperglycemic hor-
mone family. Neuropeptides 33, 71–80.
6 Wilcockson DC, Chung SJ & Webster SG (2002) Is
crustacean hyperglycaemic hormone precursor-related
peptide a circulating neurohormone in crabs? Cell
Tissue Res 307, 129–138.
7 Soyez D (2003) Recent data on the crustacean hypergly-
cemic hormone family. In Recent Advances in Marine
Biotechnology (Fingerman M & Nagabhushanam R,
eds), Vol. 10, pp. 279–301. Science Publishers,
Plymouth.
8 Chung JS, Dircksen H & Webster SG (1999) A remark-
able, precisely timed release of hyperglycemic hormone

from endocrine cells in the gut is associated with ecdysis
in the crab Carcinus maenas. Proc Natl Acad Sci USA
96, 13103–13107.
9 Dircksen H, Bo
¨
cking D, Heyn U, Mandel C, Chung JS,
Baggerman G, Verhaert P, Daufeldt S, Plosch T, Jaros
PP et al. (2001) Crustacean hyperglycaemic hormone
(CHH)-like peptides and CHH-precursor-related pep-
tides from pericardial organ neurosecretory cells in the
shore crab, Carcinus maenas, are putatively spliced and
modified products of multiple genes. Biochem J 356,
159–170.
10 De Kleijn DP, Janssen KP, Waddy SL, Hegeman R,
Lai WY, Martens GJ & Van Herp F (1998) Expression
of the crustacean hyperglycaemic hormones and the
gonad-inhibiting hormone during the reproductive cycle
of the female American lobster Homarus americanus.
J Endocrinol 156, 291–298.
11 Khayat M, Yang W, Aida K, Nagasawa H, Tietz A,
Funkenstein B & Lubzens E (1998) Hyperglycaemic
hormones inhibit protein and mRNA synthesis in
in vitro-incubated ovarian fragments of the marine
shrimp Penaeus semisulcatus. Gen Comp Endocrinol 110,
307–318.
12 Fanjul-Moles ML (2006) Biochemical and functional
aspects of crustacean hyperglycemic hormone in deca-
pod crustaceans: review and update. Comp Biochem
Physiol C Toxicol Pharmacol 142, 390–400.
13 Chung JS & Webster SG (2003) Moult cycle-related

changes in biological activity of moult-inhibiting hor-
mone (MIH) and crustacean hyperglycaemic hormone
(CHH) in the crab, Carcinus maenas. From target to
transcript. Eur J Biochem 270, 3280–3288.
14 Webster S (1998) Neuropeptides inhibiting growth and
reproduction in crustaceans. In Recent Advances in
Arthropod Endocrinology (Coast GM & Webster SG,
eds), Seminar Series 65, pp. 33–52. Cambridge Univer-
sity Press, Cambridge.
15 Chang ES, Bruce MJ & Newcomb RW (1987) Purifica-
tion and amino acid composition of a peptide with
molt-inhibiting activity from the lobster, Homarus
americanus. Gen Comp Endocrinol 65, 56–64.
16 Wainwright G, Webster SG, Wilkinson MC, Chung JS
& Rees HH (1996) Structure and significance of man-
dibular organ-inhibiting hormone in the crab, Cancer
pagurus. Involvement in multihormonal regulation of
growth and reproduction. J Biol Chem 271, 12749–
12754.
17 Lu W, Wainwright G, Webster SG, Rees HH & Turner
PC (2000) Clustering of mandibular organ-inhibiting
hormone and moult-inhibiting hormone genes in the
crab, Cancer pagurus, and implications for regulation of
expression. Gene 253, 197–207.
18 Kegel G, Reichwein B, Weese S, Gaus G, Peter-Katali-
nic J & Keller R (1989) Amino acid sequence of the
crustacean hyperglycemic hormone (CHH) from the
shore crab, Carcinus maenas. FEBS Lett 255, 10–14.
19 Keller R (1969) Untersuchungen zur Artspezifita
¨

t eines
Crustaceenhormons. Z verg Physiol 63, 137–145.
20 Leuven RS, Jaros PP, Van Herp F & Keller R (1982)
Species or group specificity in biological and immuno-
logical studies of crustacean hyperglycemic hormone.
Gen Comp Endocrinol 46, 288–296.
21 Lorenzon S, Giulianini PG & Ferrero EA (1997) Lipo-
polysaccharide-induced hyperglycemia is mediated by
CHH release in crustaceans. Gen Comp Endocrinol 108,
395–405.
22 Marco HG, Brandt W, Stoeva S, Voelter W & Ga
¨
de G
(2000) Primary structures of a second hyperglycemic
peptide and of two truncated forms in the spiny lobster,
Jasus lalandii. Peptides 21, 19–27.
23 Soyez D, Laverdure AM, Kallen J & Van Herp F
(1998) Demonstration of a cell-specific isomerization of
invertebrate neuropeptides. Neuroscience 82, 935–942.
24 Ollivaux C, Vinh J, Soyez D & Toullec JY (2006) Crus-
tacean hyperglycemic and vitellogenesis-inhibiting hor-
mones in the lobster Homarus gammarus. FEBS J 273,
2151–2160.
25 Toullec JY, Serrano L, Lopez P, Soyez D & Spanings-
Pierrot C (2006) The crustacean hyperglycemic
hormones from an euryhaline crab Pachygrapsus
marmoratus and a fresh water crab Potamon ibericum:
N. Montagne
´
et al. CHH characterization in Anomura

FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works 1051
eyestalk and pericardial isoforms. Peptides 27, 1269–
1280.
26 Mettulio R, Edomi P, Ferrero EA, Lorenzon S & Giu-
lianini PG (2004) The crustacean hyperglycemic hor-
mone precursors a and b of the Norway lobster differ
in the preprohormone but not in the mature peptide.
Peptides 25, 1899–1907.
27 De Kleijn DP, Janssen KP, Martens GJ & Van Herp F
(1994) Cloning and expression of two crustacean hyper-
glycemic-hormone mRNAs in the eyestalk of the cray-
fish Orconectes limosus. Eur J Biochem 224, 623–629.
28 Dircksen H, Webster SG & Keller R (1988) Immunocy-
tochemical demonstration of the neurosecretory systems
containing putative moult-inhibiting hormone and
hyperglycemic hormone in the eyestalk of brachyuran
crustaceans. Cell Tissue Res 251, 3–12.
29 Ahyong ST & O’Meally D (2004) Phylogeny of the
Decapoda Reptantia: resolution using three molecular
loci and morphology. Raffles Bull Zool 52, 673–693.
30 Chen SH, Lin CY & Kuo CM (2005) In silico analysis
of crustacean hyperglycemic hormone family. Mar
Biotechnol 7, 193–206.
31 Lago-Lesto
´
n A, Ponce E & Mun
˜
oz ME (2007) Cloning
and expression of hyperglycemic (CHH) and molt-
inhibiting (MIH) hormones mRNAs from the eyestalk

of shrimps of Litopeaneus vannamei grown in different
temperature and salinity conditions. Aquaculture 270,
343–357.
32 Tiu SH, He JG & Chan SM (2007) The LvCHH-ITP
gene of the shrimp (Litopenaeus vannamei) produces a
widely expressed putative ion transport peptide (LvITP)
for osmo-regulation. Gene 396, 226–235.
33 Davey ML, Hall MR, Willis RH, Oliver RW, Thurn
MJ & Wilson KJ (2000) Five crustacean hyperglycemic
family hormones of Penaeus monodon: complementary
DNA sequence and identification in single sinus glands
by electrospray ionization-Fourier transform mass spec-
trometry. Marine Biotechnol 2, 80–91.
34 Gu PL & Chan SM (1998) The shrimp hyperglycemic
hormone-like neuropeptide is encoded by multiple
copies of genes arranged in a cluster. FEBS Lett 441,
397–403.
35 Gu PL, Yu KL & Chan SM (2000) Molecular charac-
terization of an additional shrimp hyperglycemic hor-
mone: cDNA cloning, gene organization, expression
and biological assay of recombinant proteins. FEBS
Lett 472, 122–128.
36 Bendtsen JD, Nielsen H, von Heijne G & Brunak S
(2004) Improved prediction of signal peptides: Sig-
nalP 3.0. J Mol Biol 340, 783–795.
37 Fu Q, Christie AE & Li L (2005) Mass spectrometric
characterization of crustacean hyperglycemic hormone
precursor-related peptides (CPRPs) from the sinus gland
of the crab, Cancer productus. Peptides 26, 2137–2150.
38 Marco HG, Hansen IA, Scheller K & Ga

¨
de G (2003)
Molecular cloning and localization of a cDNA encoding
a crustacean hyperglycemic hormone from the South
African spiny lobster, Jasus lalandii. Peptides 24, 845–
851.
39 Abascal F, Zardoya R & Posada D (2005) ProtTest:
selection of best-fit models of protein evolution.
Bio-
informatics 21, 2104–2105.
40 Jones DT, Taylor WR & Thornton JM (1992) The
rapid generation of mutation data matrices from pro-
tein sequences. Comput Appl Biosci 8, 275–282.
41 Guindon S & Gascuel O (2003) A simple, fast, and
accurate algorithm to estimate large phylogenies by
maximum likelihood. Syst Biol 52, 696–704.
CHH characterization in Anomura N. Montagne
´
et al.
1052 FEBS Journal 275 (2008) 1039–1052 ª 2008 FEBS. No claim to original French government works