Báo cáo khoa học: Functional studies of crustacean hyperglycemic hormones (CHHs) of the blue crab, Callinectes sapidus – the expression and release of CHH in eyestalk and pericardial organ in response to environmental stress pot

Functional studies of crustacean hyperglycemic hormones
(CHHs) of the blue crab, Callinectes sapidus – the
expression and release of CHH in eyestalk and pericardial
organ in response to environmental stress
J. Sook Chung and N. Zmora
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, USA

Keywords
hypoxia; pericardial organ and sinus gland
crustacean hyperglycemic hormones;
temperature stress
Correspondence
J. S. Chung, Center of Marine
Biotechnology, University of Maryland
Biotechnology Institute, 701 East Pratt
Street, Columbus Center, Suite 236,
Baltimore, MD 21202, USA
Fax: +1 410 234 8896
Tel: +1 410 234 8841
E-mail:
(Received 9 October 2007, revised 5
December 2007, accepted 11 December
2007)
doi:10.1111/j.1742-4658.2007.06231.x

The rapid increase in the number of putative cDNA sequences encoding
crustacean hyperglycemic hormone (CHH) family in various tissues [either
from the eyestalk (ES) or elsewhere] underscores a need to identify the
corresponding neuropeptides in relevant tissues. Moreover, the presence of
provided structural CHH implies the level of the complexity of physiological regulation in crustaceans. Much less is known of the functions of nonES CHH than of those of its counterpart present in ESs. In the blue crab,
Callinectes sapidus, we know little of CHH involvement in response to the

stressful conditions that naturally occur in Chesapeake Bay. We have identified two isoforms of CHH neuropeptide in the sinus gland of the ES and
isolated a full-length cDNA encoding CHH from the pericardial organ
(PO). The functions of ES-CHH and PO-CHH in this species were studied
with regard to expression and release in response to stressful episodes:
hypoxia, emersion, and temperatures. Animals exposed to hypoxic conditions responded with concomitant release of both CHHs. In contrast, the
mRNA transcripts encoding two CHHs were differentially regulated:
PO-CHH increased, whereas ES-CHH decreased. This result suggests a
possible differential regulation of transcription of these CHHs.

In recent years, crustacean hyperglycemic hormones
(CHHs), traditionally identified in the medulla terminalis X-organ and sinus gland (SG) in the eyestalk
(ES), have been found in non-ES tissues. Reported
sites for the synthesis of CHH-like neuropeptides in
non-ES tissues include the gut [1,2], subesophageal
ganglion (SOG) [3], pericardial organs (POs) [4,5], and
cells in the abdominal segments of embryos [6]. In Carcinus maenas, the expression and translation of gut
CHH occurs exclusively during premolt [1,2], whereas
ES-CHH is molt stage independent [7]. The amino acid
sequence of gut CHH is identical to that in the ES

[1,8], but only 66% homologous to PO-CHH [4,5,8].
Thus, it seems that C. maenas [1,4,8], Homarus americanus [3,9,10], Pachygrapsus marmoratus [5] and Machrobrachium rosenbergii [11] exhibit multiple isoforms
and synthesis sites for CHH, suggesting that this may
be a common feature among crustaceans.
Many putative CHH sequences have been identified
with the aid of cDNA cloning, but there is little information about the localization or the physiological
function of active CHH neuropeptides in corresponding tissues. Prototypical actions attributed to ES-CHH
include induction of hyperglycemia, suppression of

Abbreviations

AK, arginine kinase; CHH, crustacean hyperglycemic hormone; CPRP, crustacean hyperglycemic hormone precursor-related peptide; eIF4A,
eukaryotic translation initiation factor 4A; ES, eyestalk; PO, pericardial organ; SALDI, surface-assisted laser desorption ⁄ ionization; SG, sinus
gland; SOG, subesophageal ganglion; TD-PCR, touchdown PCR; TG, thoracic ganglia.

FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS

693


Expression and release of CHH in the blue crab

J. S. Chung and N. Zmora

694

Results
Identification and bioactivity of CHHs from ES
sinus glands and PO
Two RP-HPLC peaks (1 and 2) as presented in Fig. 1
showed strong cross-reactivity with anti-ES-CHH
(Fig. 1). The result of a glucose bioassay with these
peaks confirmed that peak 2 is the major type of
Cal. sapidus ES-CHH-II, as it is for Car. maneas and
Cancer pagurus [29,30]. The molecular masses of
peaks 1 and 2, determined by ESI MS, were
8494.20 Da and 8478.01 Da, respectively.
The typical separation of PO extract (50 PO equivalents) by RP-HPLC is presented in Fig. 2. Fractions

Absorbance at 405 nm

Absorbance at 210 nm

0.2
2

1
0.0
0

10

20
30
Retention time, min

40

Fig. 1. CHH neuropeptide profile of single SG extract of Cal. sapidus on an RP-HPLC C18 column. The gradient condition was 30–
80% solution B over 45 min (solution A, 0.11% trifluoroacetic acid
in water; solution B, 0.1% trifluoroacetic acid in 40% water and
60% acetonitrile). Absorbance and flow rate were monitored at
210 nm at a flow rate of 1 mLỈmin)1. Peak 1, CHH-I; peak 2, CHHII. The mass of each neuropeptide determined by ESI MS was as
follows; CHH-I, 8494.2 Da; CHH-II, 8478.1 Da. Fractions that positively cross-reacted with Car. maenas CHH antiserum are shown
as bars.

Absorbance at 405 nm

0.5
Absorbance at 210 nm

ecdysteroid and methyl farnesoate synthesis, inhibition
of ovarian protein synthesis and osmoregulation [12–
19]. Hyperglycemia, the commonly observed adaptive
response, is caused by the release of CHH from the ES
in response to changes in environmental conditions
such as oxygen, temperature, and salinity [19–24].
CHHs from the gut and the cells in embryonic abdominal segments seem to be particularly involved in the
process of water uptake during ecdysis and hatching,
respectively [1,6]. The physiological roles of SOGCHH and PO-CHH have not been defined [3–5,11].
Although CHH secretion from the ES in response to
stress is well documented [19–24], the effect of stresses
on CHH transcription in the ES remains unanswered.
Thus, despite > 90 putative cDNA structures for
CHH being deposited in GenBank, it is apparent that
much work is still required to address the physiological
roles and the localization of the corresponding neuropeptides in the tissues from which many CHH cDNAs
were derived.
The blue crab, Callinectes sapidus, an economically
valuable euryhaline species in Chesapeake Bay, experiences migration and seasonal changes in environmental conditions, including temperature, salinity
and dissolved oxygen ( />monitoring/water/index.html). In particular, it is noted
that in the Bay, low temperatures during winter and
anoxia during summer, in combination with the
changes in salinity, are associated with high mortality
in this species [25–27]. In view of CHH involvement in
response to stress in other crustacean species, it is reasonable to think that Cal. sapidus CHH may also play
an important regulatory role in adaptation to naturally occurring stressful conditions. Thus, we were
interested in isolating the cDNA of PO-CHH and
identifying the native CHH neuropeptide in the ES,
after the recent report of CHH cDNA from the ES
[28]. Also, we examined the physiological responses of

the release and expression of these two CHHs under
stressful conditions, especially severe hypoxia, hypothermia and hyperthermia, in an attempt to define
their functions.
To address these questions, we cloned the fulllength cDNA of PO-CHH and identified the presence
of the neuropeptide forms in the ES and PO. For the
first time in crustaceans, the expression profiles of
these two CHHs in response to oxygen and temperature changes were documented using quantitative
real-time RT-PCR. To further define the physiological
role of PO-CHH, the levels of this CHH were measured from the same animals, along with ES-CHH,
using RIAs under control normoxic and hypoxic conditions.

0.0

0

10

20
30
Retention time, min

40

Fig. 2. Separation of the extract of PO (50 equivalents) on an RPHPLC C18 column. Elution conditions were the same as described
for Fig. 1. The cross-reacted fractions initially tested with Car. maenas CHH antiserum are shown as bars.

FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS


Glucose (ug/1 mL hemolymph)

J. S. Chung and N. Zmora

Expression and release of CHH in the blue crab

1200

C
***

900

D

600

A

300
0
Saline

E

ES-CHH PO-CHH

Fig. 3. In vivo bioassay of PO-CHH and ES-CHH. (A) Glucose
assay: open bars, controls at time = 0; closed bar, t = 2 h. Results
are presented as mean ± 1 SE (n = 6–8). ES-CHH showed a significant increase in hemolymph glucose (***P < 0.001, paired t-test).

analyzed by ELISA are marked on a bar graph that
shows positive cross-reactivity with PO-CHH antiserum. Immunopositive fractions (numbers 30–32)
were further analyzed by surface-assisted laser desorption ⁄ ionization (SALDI) to localize fraction 31 containing PO-CHH. PO-CHH retained in fraction 31
showed two masses with a 16 Da difference: 8373 Da
and 8356 Da. This fraction was subjected to further
purification, and the final peak was collected for bioassay.
Native PO-CHH (20 pmol) or ES-CHH (10 pmol)
of the blue crab was injected into intact blue crabs.
ES-CHH increased hemolymph glucose five-fold, as
compared to controls, whereas the injection of
PO-CHH did not elevate hemolymph glucose (Fig. 3).
Animals injected with 20 pmol of oxidized ES-CHH,
in which one of the Met residues was oxidized to give
a 16 Da higher molecular mass than that of non-oxidized ES-CHH (peak 2, CHH-II), showed only a modest two-fold increase in glucose level from 77.79 ± 7.3
to 151.58 ± 13.4 lgỈmL)1 hemolymph (n = 10,
P < 0.01, paired t-test). Interestingly, the injection of
20 pmol of Car. maenas ES-CHH also induced a significant increase (P < 0.05, paired t-test) in hemolymph glucose from the blue crab (257 ± 34.6 to
633 ± 229 lgỈmL)1 hemolymph, n = 10).
PO immunohistochemistry
Figure 4A shows the intrinsic multipolar cells in the
posterior bar of the PO;  50 cells, 30–40 lm in
length, were positive with anti-PO-CHH serum. Most
cells (35–40) were located in the anterior (Fig. 4A) and
posterior (Fig. 4B) bars, whereas the rest were located
in trunks. Two types of cells were observed: the majority of cells showed homogeneous CHH staining in the
cytoplasm (Fig. 4C) with a visibly large nucleus,
whereas others had less intensive but punctuated and

B

Fig. 4. Immunohistochemistry of PO staining with PO-CHH antiserum. (A, B) Intrinsic multipolar cells, located in anterior (A) and
posterior (B) bars. (C, D) Cells at · 1200 magnification. (E) PO-CHH
staining shown in nerve fibers located in trunk. Scale
bars = 50 lm.

granulated cytoplasm (Fig. 4D). Figure 4E shows the
possible release sites on the surface of the PO and
many nerve fiber tracts and varicosities in trunks of
the PO.
Cloning and sequencing of PO-CHH cDNA
The first PCR of PO cDNA with a combination of a set
of primers LF1 and LR1 (Table 1) produced an amplicon of 500 bp. On the basis of this sequence, genespecific primers for 5¢-RACE (LR and LR2) and
3¢-RACE (LF2) were generated. A nested PCR with a
combination of LF2 and 3¢ nested primer (Invitrogen,
Carlsbad, CA, USA), using the template from a touchdown product, generated an amplicon of  1.7 kbp.
The sequencing of the cloned vector of this amplicon as
an insert was completed using M13 F and R and a walking primer (PWF1, ATGGGATATGTTCTCAGT),
revealing the presence of a long 3¢-UTR ( 1.5 kbp).
The amplicon (132 bp) produced from the nested
PCR of 5¢-RACE cDNA contained a 5¢-UTR and the
remaining sequence of the 5¢-end of PO-CHH. The
complete cDNA sequence of PO-CHH, shown in
Fig. 5, contained a 5¢-UTR, a signal peptide (MQS
IKTVCQITLLVTCMMATLSYTHA), a crustacean
hyperglycemic precursor-related peptide (RSAEG
LGRMGRLLASLKSDTVTPLRGFEGETGHPLE), a
CHH, a non-amidated C-terminus (QIYDSSCK
GVYDRAIFNELEHVCDDCYNLYRNSRVASGCR
ENCFDNMMFETCVQELFYPEDMLLVRDAIRG)
and a 3¢-UTR of  1.5 kbp.

FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS

695


Expression and release of CHH in the blue crab

J. S. Chung and N. Zmora

Table 1. List of primers used for cloning of 5¢-3¢-RACE of PO-CHH.
LF and LR primers were used for standard cRNA production for
quantitative RT-PCR, whereas SF and SR primers were for quantitative RT-PCR. A combination of CHH-SF and ES-SR was used for
ES-CHH, and for PO-CHH, PO-SR primer was used. Cal. sapidus
AK, Q9NH49; Cal. sapidus CHH, AY536012; Cal. sapidus eIF4A,
DQ667140; Cal. sapidus PO-CHH, DQ667141.
Primers

Sequence (5¢- to 3¢)

LF1
LF2
LR1
LR2
LR3
PWF1
CHH-SF
ES-SR
PO-SR
AK LF

AK SF
AK LR
AK SR
eIF4A LF
eIF4A SF
eIF4A LR
eIF4A SR

CAATCCATCAAAACCGTGTG
TGCTACAGCAACTGGTGATCAGAAGGG
CCCTTCCTGATCACCATGTTGCTGT
GGCCATCATACAGGTGACTAGGAGGGT
GGGTGATTTGACACACGGTTTTGATGGA
ATGGGATATGTTCTCAGT
ACAGATTTACGACTCCTCCTG
CATGTTGCTGTAGCAGTTTGAT
ATATAAGCTTATCCTCTGATAGC
GACCCATCATCGAGGACTA
ACCACAAGGGTTTCAAGCAG
CCACACCAGGAAGGTCTTGT
GGTGGAGGAAACCTTGGACT
ACGTCAACATGTCCGACAAA
CGGTGGAGACAACAAGGACT
TGCGTTTCGTTTGACTTCAC
GGCTGATGGCTTCTCAAAAC

Hemolymph titers of PO-CHH and ES-CHH in
response to changes in dissolved oxygen
Hypoxia induced the release of CHHs from the PO
and ES of the juvenile crabs (Fig. 6). At the initial

control normoxic condition, the amount of ES-CHH
(376 ± 67 fmolỈmL)1) in hemolymph was  9-fold
higher than that of PO-CHH (45.6 ± 8.6 fmolỈmL)1).
One hour of exposure of hypoxia induced CHH secretion both from the ES and PO. ES-CHH doubled in
level from 376 ± 67 to 762 ± 143 fmolỈmL)1 hemolymph (n = 9, P < 0.05). The level of PO-CHH
increased from 45.6 ± 8.6 to 74.5 ± 19.8 fmolỈmL)1
hemolymph (n = 9) but there was no statistical difference. Interestingly, ES-CHH levels in the hemolymph
of animals 10 min after they were returned to control normoxic seawater, after previous exposure to
hypoxia for 1 h, significantly decreased to 156 ± 50
and 44.5 ± 12.8 fmolỈmL)1 hemolymph (n = 9,
P < 0.05), respectively.
Effects of stresses on the levels of glucose and
lactate in hemolymph and gene expression in the
ES and PO
Hypoxia and emersion
Hypoxia and emersion caused hyperglycemia and
hyperlactemia (Fig. 7A,B). Crabs exposed to hypoxia
696

and emersion showed a  3-fold increase in glucose
and a more than 30-fold increase in lactate, whereas
the levels in controls remained constant. The arginine
kinase gene (AK) and the eukaryotic translation initiation factor 4A gene (eIF4A, DQ667140) were initially
selected as control genes, but the expression levels in
both tissues were significantly increased in response to
hypoxia and emersion. Thus, all the expression levels
were presented as total copy number ⁄ tissue, as in
Fig. 7. The ES and PO from the animals that experienced 1 h of hypoxia and emersion showed significant
changes in CHH gene expression. As shown in
Fig. 7C, hypoxia and emersion greatly reduced

ES-CHH gene expression ( 10-fold and five-fold)
from 1.36 ± 0.42 · 108 to 1.06 ± 0.16 · 107 and
2.49 ± 0.35 · 107 (copy number ⁄ tissue, one-way
anova, P < 0.05), respectively. In contrast, the
expression level of the PO-CHH gene was dramatically
increased from 1.95 ± 0.5 · 107 to 1.45 ± 0.45 · 109
and 2.21 ± 0.79 · 109 (copy number ⁄ tissue, one-way
anova, P < 0.05).
Emersion caused significantly higher expression
of eIF4A (5.85 ± 2.38 · 107) than in controls
(2.86 ± 0.73 · 106) in the ES, at P < 0.05 (one-way
anova), whereas hypoxia induced slightly higher
expression of eIF4A (3.81 ± 2.49 · 106) than in controls (2.86 ± 0.73 · 106), which was not statistically
significant. Hypoxia and emersion, on the other hand,
greatly increased eIF4A expression in the PO
(1.34 ± 0.637 · 108, 9.99 ± 2.68 · 107, respectively),
as compared with controls (1.31 ± 0.163 · 107). Levels of increase in AK expression in the ES and PO were
approximately 3–5-fold, as compared with controls.
Temperature
A 2 h exposure to hyperthermic (29 °C) conditions
caused a 2.5-fold increase in glucose levels in hemolymph, as shown in Fig. 8A, whereas in controls and
under hypothermic conditions (4 °C) there was a
slightly higher glucose level. In contrast to the modest
increase in glucose, the levels of lactate from all three
groups were significantly elevated after 2 h, as compared with those at the beginning of the experiment.
The increase in lactate levels was pronounced, in that
both thermal stresses caused increases from seven-fold
(hypothermal) to 13-fold (hyperthermal), whereas a
3.5-fold increase was observed in animals maintained
at 22 °C.

The effect of different temperatures on gene expression in the ES and PO is shown in Fig. 8C. The
expression of ES-CHH was modestly increased at
29 °C (P = 0.08, one-way anova) as compared with

FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS


J. S. Chung and N. Zmora

Expression and release of CHH in the blue crab

Fig. 5. Sequence alignments of nucleotide and deduced amino acids of full-length PO-CHH (DQ667141) and ES-CHH (AY536012). PO-CPRP
and ES-CPRP are in bold italic, and both CHHs are in bold. The 5¢-UTR and a signal peptide are italicized. A dibasic cleavage site (KR) is
underlined. The stop codon (TAA) is marked as *, and a putative polyadenylation site (AATAAA) is underlined.

that in controls at 22 °C; whereas the level of POCHH expression was only slightly higher, at 29 °C. In
contrast to the results obtained with hypoxia and
emersion, as shown in Fig. 7, there was little change in
AK and eIF4A expression in the ES and PO at 22 °C
and 29 °C, except that animals exposed to 4 °C
showed higher AK expression in the PO, as compared
with those exposed to 22 °C and 29 °C.

Discussion
In this article, we describe studies on the identification,
localization and bioactivity of CHH neuropeptides of
the blue crab, Cal. sapidus, using biochemical, molecular and immunological methods. Blue crabs produce a
CHH neuropeptide in the PO that shows a 66%
deduced amino acid sequence identity with ES-CHH
[28]. We have also demonstrated, for the first time in

crustaceans, the differential expression of these two

CHHs in response to changes in the following environmental conditions: hypoxia, emersion, and temperature. More important with respect to physiology, we
measured the hemolymph titers of these two CHHs
under different dissolved oxygen levels in seawater.
Our results indicate that the regulatory mechanisms
governing the expression of ES-CHH and PO-CHH
are different. Yet, the release of both CHHs seems to
be sensitive to dissolved oxygen in seawater, suggesting
an adaptive role.
A single SG in the ES of Cal. sapidus contains two
isoforms of CHH. The molecular mass difference suggests that the major CHH may have pyroglutamate at
the N-terminus via post-translational cyclization of
Glu of peak 1. This feature appears to be common in
CHHs of brachyuran crab species, including Car. maenas [29] and Cancer pagurus [30], and differs from
those in astacuran species [10,31–33]. The relative
abundance of these CHH isoforms in the SG among

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697


J. S. Chung and N. Zmora

Control

Emersion

Hypoxia

B

***
450

***

300
150
0

Control

Hypoxia

Emersion

C
1010
b

b

9

10

b
a

8

10

bb

b
b

a

b
b

a

7

10

a

b

a

b

a

a

6

10

H
H
PO
PO AK
-e
IF
4A

these species varies as a ratio of two isoforms (peaks 1
and 2), ranging from 1 : 5 for Cal. sapidus, to 1 : 8 in
Car. maenas, to 1 : 10 in Can. pagurus [29,30], yielding
peak 2 as the major CHH.
The immunohistochemistry studies show that the
intrinsically staining cells are responsible for PO-CHH
synthesis. The staining patterns are similar to those of
Car. maenas, in that most cells (approximately 35–40)
were localized in the anterior and posterior bars [4].
However, PO-CHH staining appears to be somewhat
different from that of crustacean cardioactive peptide
(CCAP) in the PO, where it is known mainly at its
release site [34]. Overall, the immunohistochemisty of
PO-CHH in the PO indicates its role as a neurotransmitter, as it may be directly targeted into the specific sites in
which the nerve fibers of the PO innervate the anterior

ramifications. On the other hand, as a neurohormone,
PO-CHH may be released from the surface of the PO
and into branchiocardiac veins through the direct openings of anterior and posterior bars [35]. Furthermore,
considering the localization of PO-CHH-producing
cells in the pericardial chamber, it may be pertinent to
suggest that these intrinsic multipolar PO-CHH cells
may be sensitive to homeostasis of hemolymph.
Cloning of cDNA of PO-CHH of Cal. sapidus produced only one size (2004 bp) that is translated into
PO-CHH neuropeptide. The cDNA sequence of
PO-CHH encodes a preprohormone containing a
signal peptide (26 amino acid residue), one crustacean
hyperglycemic hormone precursor-related peptide
(CPRP, 36 amino acid residue), and PO-CHH, not

600

4A

Fig. 6. Changes in hemolymph titers of PO-CHH and ES-CHH in
response to dissolved oxygen. CHH control values were obtained
from animals in control normoxic water. The closed bar shows a
significantly increased level of ES-CHH (P < 0.05, one-way ANOVA,
Krustal–Wallis test, INSTAT); the open bar is PO-CHH. Bars represent
mean ± 1 SE (n = 9). Note the difference in the y-axis scales
between ES-CHH and PO-CHH.

698

0

PO
C

ia
xia
ox
po
orm
Hy
ln
tro
on
C

K

or
l n
tro
n
Co

0
xia
mo

100

-A

0

20

-e
IF

c

200

200

ES

40

H

a
400

**

ES

60

***
300

-C
H

600

400

ES

80

Glcuose (ug/mL hemolymph)

800

Lactate (ug/mL hemolymph)

b

A

Copy number of genes (tissue)

100

1000

PO-CHH (fmol/mL hemolymph)

ES-CHH (fmol/mL hemolymph)

Expression and release of CHH in the blue crab

Fig. 7. Effect of hypoxia and emersion on glucose (A), lactate (B)
and gene expression (C). (A, B) Open bars: controls at t = 0. Closed
bars: 2 h after exposure. Paired t-test showed the statistical significance at **P < 0.01 and ***P < 0.001. (C) The profiles of CHH,
AK and eIF4A expression in the ES and PO in response to hypoxia
and emersion. Closed bar: controls. Hatched bars: hypoxia. Crossed
bars: emersion. Bars represent mean ± 1 SE (n = 6). Statistical
analysis was performed using one-way ANOVA (P < 0.05, Krustal–
Wallis test, INSTAT).

amidated at the C-terminus (71 amino acids). This
cDNA of Cal. sapidus PO-CHH contains a much
longer long 3¢-UTR ( 1.5 kbp) than that of Car.
maenas (518–759 bp) [4]. Interestingly, a putative
cDNA encoding a PO-CHH type has been cloned in
thoracic ganglia (TG) of Cal. sapidus and other tissues
[28]. However, the immunohistochemisty study using

FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS


J. S. Chung and N. Zmora

Expression and release of CHH in the blue crab

Glucose (ug/mL hemolymph)

A
300

**
200

100

0
22 to 22 22 to 29 22 to 4
Temperature (°C)

Lactate (ug/ mL hemolymph)

B
180

***
120

**

60

*
0
22 to 22 22 to 29 22 to 4
Temperature (°C)
1e+9

1e+8
b

1e+7

aa

H

PO AK
-e
IF
4A

PO

-C
H

PO

-e
IF

ES

ES

4A

1e+6
-C
H
H
ES
-A
K

Copy no. of genes (ES/PO)

C

Fig. 8. Effect of hypothermia and hyperthermia on glucose (A),
lactate (B), and gene expression (C). (A, B) Open bars: controls at
t = 0. Closed bars: 2 h after exposure. The results were analyzed
for statistical significance using a paired t-test (*P < 0.05,
**P < 0.01, ****P < 0.001). (C) The profiles of CHH, AK and eIF4A
expression in the ES and PO in response to hypothermia and
hyperthermia. Closed bar: controls. Hatched bar: hypothermia.
Crossed bar: hyperthermia. Bars represent mean ± 1 SE (n = 6).
Statistical analysis was performed using one-way ANOVA (P < 0.05,
Krustal–Wallis test, INSTAT).

anti-PO-CHH revealed exclusive positive staining in
the PO but not in TG (our unpublished observation),
suggesting that the putative cDNA encoding the
PO-CHH type cloned in TG is not translated into a
protein. Similarly, for Car. maenas, nine putative

cDNA sequences of PO-CHH are listed in GenBank,

despite the fact that only one of these encodes the
conceptual neuropeptide sequence of PO-CHH [4].
The results of homologous and heterologous bioassays of Cal. sapidus ES-CHHs reflect high sequence
identity (> 75%) of ES-CHHs between two crab species. Our finding is in agreement with a previous report
that the injection of Cal. maenas ES-CHH triggered
hyperglycemia in Can. pagurus [30]. Overall, such
results of heterologous bioassays indicate that CHH
receptors among these crab species may share some
degree of similarity.
Cal. sapidus PO-CHH injection (20 pmol) did not
cause hyperglycemia in the blue crab. This finding is
not unexpected, as a similar result was observed in
Car. maenas [4]. The close sequence analysis of ESCHH and PO-CHH shows that the greatest homology
is in the first 40 amino acids, with much more difference in the latter half of the sequence. Such differences
are common in all CHH sequences currently available
in GenBank, indicating that functionality inducing
hyperglycemia may lie in the first 40 amino acid residues [36–40]. Therefore, it is suggested that these two
CHHs may have separate receptors in their target tissues, where PO-CHH may be mobilizing glucose but
not be directly involved in hyperglycemia in hemolymph. PO-CHHs among Cal. sapidus, Car. maenas
and P. marmoratus share overall 67% sequence identity, of which 85% and 68% are contributed by the
first 40 residues and by the C-terminus, respectively
[4,5]. Thus, on the basis of the sequence identity
among PO-CHHs, it is proposed that the physiological
function of this CHH may be conserved in at least
these crab species, although it has not yet been fully
defined and understood.
To define the physiological function of PO-CHH,
the release pattern was evaluated along with that of
ES-CHH in response to hypoxia. The basal level in the
PO was surprisingly high at 10)11 m, although it was

10-fold less than that of ES-CHH. This difference in
the concentrations of two CHHs seems to reflect the
amount present in these tissues, approximately 2–
5 pmol, at least 20–50-fold less than that of ES-CHH
(100 pmolỈES)1). It is noteworthy that the basal level
of ES-CHH is an order of magnitude greater in
Cal. sapidus (10)10 m) than in Car. maenas (10)11 m)
[24] or Can. pagurus (< 10)11 m) [20,23]. Such a high
CHH concentration in hemolymph of Cal. sapidus may
explain the high basal level of hemolymph glucose,
which may reflect the behavioral differences among
these species. Moreover, crabs under hypoxia and
emersion have shown differential redistribution of
hemolymph to increase the flow, especially to the

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Expression and release of CHH in the blue crab

J. S. Chung and N. Zmora

sternal artery, and overall cardiac output [41–43]. Our
finding of the elevated ES-CHH level in hemolymph is
congruent with previous reports on CHH secretion
under hypoxia or emersion [20–24]. Thus, our finding
supports the suggestion that PO-CHH and ES-CHH
have an adaptive role in the physiological response to

hypoxia.
In crustaceans, it seems to be rather difficult to find
an ideal control gene for quantitative RT-PCR, the
level of which does not change during molting or
the reproductive cycle [7,44]. As eIF4A, a member of
the DEAD box and ATP-dependent RNA helicase
family, may be considered as a temporal translation
indicator, because of its involvement in the assembly
of active polysome by unwinding secondary structure
in the 5¢-UTR of mRNAs [45], we reasoned that eIF4A
would be a good control gene as an indicator of the
level of translation. Therefore, Cal. sapidus cDNA of
the eIF4A gene was initially isolated, but the expression level of this gene was unexpectedly changed,
suggesting that it is sensitive to oxygen level.
One hour of acute hypoxia and emersion induced
different expression levels of PO and ES-CHH.
Chronic hypoxia caused downregulation of the hemocyanin gene in the hepatopancreas in the same species
[46]. Moreover, the degree of decrease in CHH expression in the ES is in proportion to that of CCAP
expression in the same tissue, as described in Chung
et al. [47], but there is no change in CCAP of TG,
indicating the tissue-specific regulation of CCAP
expression. Likewise, the present results concerning
PO-CHH and ES-CHH expression in response to
oxygen level suggest that the regulation of CHH
expression is different and tissue specific, perhaps via
tissue-specific alternative splicing or tissue-specific gene
expression through different regulatory arrangements
in the upstream promoter regions [37,38].
It is reasonable to suggest that the levels of transcription of PO-CHH are in proportion to the demand
for its release, whereas the inhibition of ES-CHH transcription may be caused by high glucose levels in hemolymph. On the other hand, the elevated hemolymph

glucose might have inhibited ES-CHH expression, as
a previous report indicated that CHH neurons are
hyperpolarized in response to 25 mm glucose in the
media [48]. Hyperglycemia may also inhibit the further
release of CHH, whereas the low glucose level in
hemolymph may have a positive influence on the
release of CHH from SG in the ES, as described in
Chung & Webster [24]. With these observations, it
would be interesting to see if in vitro incubation of the
ES in high-glucose media causes the inhibition of
CHH gene expression.
700

We have shown the presence of ES-CHH neuropeptides and the putative cDNA sequence of PO-CHH that
is translated into a neuropeptide in the PO. The location
of intrinsic multipolar cells and structure of nerve
branches in the PO indicate that these cells may be sensitive to changes in hemolymph homeostasis. Changes in
dissolved oxygen levels in seawater immediately affect
the release of CHHs from the PO and SG, strongly
suggesting that PO-CHH has an adaptive role, in particular, in response to oxygen level. Defining the physiological function of PO-CHH may seem to be a
challenge, as the structural similarity of PO-CHH places
it as ‘a tacit CHH’, despite the fact that it does not
induce hyperglycemia in hemolymph. However, we have
taken a positive step towards identifying a physiological
function of PO-CHH, as its release is recorded under the
changes in dissolved oxygen levels in seawater. For the
future, binding studies are required to identify its target
tissues and second messenger, as the next step towards
defining the physiological function of PO-CHH.
Furthermore, given the high sequence similarity of

PO-CHH among Cal. sapidus, Car. maenas and P. marmoratus, the function of this neuropeptide may be similar in these crab species, as is the function of ES-CHH.

Experimental procedures
Animals
Juvenile Cal. sapidus (carapace width: 45–80 mm) were
obtained from the blue crab hatchery program [49], Aquaculture Research Center, Center of Marine Biotechnology,
University of Maryland Biotechnology Institute, Baltimore,
MD and were maintained in individual compartments in
artificial seawater [15 parts per thousand (p.p.t.) salinity
and 22 °C] under ambient daylight conditions. Experimental animals were fed daily with chopped frozen squid and
pelleted sea bream (EWOS, Surrey, Canada). All experimental animals were at intermolt stage [50].

Identification, isolation and quantification of
neuropeptides from SGs and POs
Neuropeptides of SGs in the ES and PO were purified using
RP-HPLC, as described in Chung & Webster [29] and Dircksen et al. [4], respectively. CHH peaks were initially identified using ELISA with a combination of Carcinus CHH
antisera, the method described in Wilcockson et al. [23].
Amino acid analysis was carried out for the quantification
of neuropeptides [29], and ESI MS of SG neuropeptides
was used for mass determination. Confirmation of the RPHPLC fraction containing PO-CHH and its mass determination were performed using SALDI.

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J. S. Chung and N. Zmora

RACE of PO-CHH
Total RNA from POs was extracted in TRIzol reagent and
quantified using RIBO green (Invitrogen). The GeneRacer
protocol (Invitrogen) was employed for the synthesis of 5¢RACE and 3¢-RACE cDNAs from 1 lg of total RNA. For

the first amplification of 3¢-RACE cDNA, a touchdown
PCR (TD-PCR) was used with a forward gene-specific
primer (LF1: 5¢-CAATCCATCAAAACCGTGTG-3¢) and
3¢ universal primer (Invitrogen). Conditions of TD-PCR
were as follows: initial denaturation at 94 °C for 3 min;
three cycles at 94 °C for 30 s, 54 °C for 30 s, and 72 °C for
2 min; three cycles at 94 °C for 30 s, 52 °C for 30 s, and
72 °C for 2 min; three cycles at 94 °C for 30 s, 50 °C for
30 s, and 72 °C for 2 min; 24 cycles at 94 °C for 30 s,
55 °C for 30 s, and 72 °C for 2 min, and a final extension
at 72 °C for 7 min. A nested PCR was carried out with
a forward gene-specific primer (LF2: 5¢-TGCTACAG
CAACTGGTGATCAGAAGGG-3¢)
and
3¢ universal
nested primer (Invitrogen), with the following conditions:
after initial denaturation at 94 °C for 3 min, 30 cycles at
94 °C for 30 s, 60 °C for 30 s, and 72 °C for 2 min, and a
final extension at 72 °C for 7 min. The PCR product was
electrophoresed on 1% agarose gel, and the band was
excised, extracted and cloned into a TOPO-TA vector for
sequencing. The sequence of the 3¢-UTR was obtained by
primer walking using the gene-specific forward primer
(5¢-ATATAAGCTTATCCTCTGATAGC-3¢).
For 5¢-RACE, the same PCR conditions were employed
as described above for 3¢-RACE, using a gene-specific
reverse primer (LR1: 5¢-TTCCTGATCACCATGTT
GCTGT-3¢) and a 5¢ universal primer for the first PCR.
Following this, the nested PCR was conducted using LR2
(5¢-GGGTGATTTGACACACGGTTTTGATGGA-3¢) and

5¢ nested universal primers (Invitrogen). The methods of
PCR analysis and cloning were as described above.

Quantitative real-time RT-PCR
The cRNA standards of quantitative RT-PCR, including
PO-CHH and ES-CHH, AK and eIF4A, were initially
PCR amplified using a combination of LF and LR primers (Table 1). Further cRNA synthesis and RNA quantification were performed as described in Chung & Webster
[6,7], with a modification for purification of in vitro transcribed cRNAs (Ambion, Austin, TX, USA), eluting on a
spin-column (BD Biosciences, Mountainview, CA, USA).
Total RNA extracted from the tissues as described above
was treated with DNase, and each of 1 lg or 0.5 equivalent of PO RNAs were primed with random hexamers
for cDNA synthesis using avian myeloblastosis virus or
Moloney murine leukemia virus reverse transcriptase.
Final cDNA samples were diluted to 40 lL, and 2 lL of
each sample was analyzed for the expression of genes
using SYBR gold (ABI, Foster City, CA, USA) on ABI

Expression and release of CHH in the blue crab

Prism with a pair of gene-specific primers (SFs and SRs;
Table 1).

PO-CHH and ES-CHH antisera production
C-terminal fragments of PO-(FDNMMFETCVQELFY
PEDMLLVRDAIRG; Proteintech Group Inc., Chicago,
IL,
USA)
and
ES-CHH
(EDLLIMDNFEEYAR

KIQVV-NH2; Invitrogen) were synthesized with the addition of Cys at the N-terminus. These modified synthetic
peptides, after being N-terminally conjugated with bovine
thyroglobulin using m-maleimidobenzoyl-N-succinimide
ester [47], were used for antisera production in rabbits (Proteintech Group Inc., Chicago).

Whole-mount immunohistochemistry of PO
Immediately after dissection, POs were fixed in a fixative
containing 7% picric acid and 4% paraformaldehyde
in 0.1 m phosphate buffer (pH 7.4) overnight. Procedures
for washing and application of primary (· 1000 dilution)
and secondary antiserum (Vector Laboratories, Burlingame,
CA, USA) were as described in Chung & Webster [5].
Z-stacked images of PO preparations were collected using a
Zeiss Confocal Microscope with a BioRad program
(COMB, UMBI).

Bioassays of CHH neuropeptides
Levels of glucose in hemolymph were estimated before and
2 h after injection of 10 or 20 pmol of each of ES-CHH
and PO-CHH, oxidized ES-CHH, and Car. maenas ESCHH, using the glucose assay described in Webster [20].

Iodination of neuropeptides and RIA
The detailed procedures for iodination of ES-CHH and
C-terminal synthetic peptide of PO-CHH, RIA and hemolymph sample preparation were as described in Chung &
Webster [24,29]. Standards of both RIAs ranged from
500 fmol per tube to 3.8 fmol per tube, and the detection
limit was < 3 fmol per tube for both CHHs. ED50 values
were 120 ± 5 and 134 ± 8 (fmol per tube) for SG-CHH
and PO-CHH, respectively. The results were analyzed using
assayzap (Biosoft, Cambridge, UK).

Effects of environmental factors on the levels
of hemolymph and gene expression of CHH –
dissolved oxygen, emersion and temperature
Thirty minutes before the experiment, 5 L of artificial seawater (15 p.p.t., 22° C) was continuously purged with nitrogen to reduce the level of dissolved oxygen to < 0.5%

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701


Expression and release of CHH in the blue crab

J. S. Chung and N. Zmora

(YSI 58 Dissolved Oxygen Meter). Test juvenile animals
(60–80 mm carapace width) were exposed for 1 h to hypoxic seawater under continuous nitrogen purging, whereas
controls remained in aerated normoxic seawater. At the
end of the 1 h exposure, either to anoxia or the control,
hemolymph samples were withdrawn from the hypobranchial sinus through the arthrodial membrane between the
chelae and the first walking leg, using a 1 mL syringe with
a 23-gauge needle. The ES and PO were dissected out,
immediately frozen on dry ice, and kept at )80° C until
further processing.
For emersion experiments, juvenile crabs were exposed to
air for 1 h at 22° C, whereas controls were maintained in
15 p.p.t. artificial seawater at 22° C. At the end of the
experiment, the hemolymph and tissue samples were collected as described above.
Hemolymph CHHs were estimated using RIAs after the
elution of hemolymphs on a Sep-Pak C18 column (Waters;

360 mg cartridges), as described in Chung & Webster
[24,29].

Temperatures
Juvenile crabs (45–60 mm carapace width) were initially
held at 22° C (15 p.p.t.). Once hemolymph samples were
drawn as described above at time 0, animals (n = 6) were
subjected for 2 h to the following temperatures: 29° C,
22° C, and 4° C. The second hemolymph and tissue samples were collected at the end of exposure. Total RNA was
extracted using the method described above.

Statistical analysis
The data were tested for statistical significance using
graphpad instat version 3.0 (GraphPad Software, San
Diego, CA, USA).

Acknowledgements
The authors wish to thank Drs S. G. Webster (University of Wales, Bangor, UK) for amino acid analysis of
neuropeptides, and M. M. Ford for comments on the
manuscript. We also wish to thank to Dr S. Moore for
SALDI (Ciphergen) and Mr M. Prescott for ESI MS
analyses (University of Liverpool). We are indebted to
Mr O. Zmora and hatchery personnel for the young
juvenile crabs, and S. Rogers and E. Evans for maintaining the water quality of the recirculation system.
This article is contribution no. 07-177 of the Center of
Marine Biotechnology (University of Maryland Biotechnology Institute, Baltimore, MD). The work is
supported by a program grant (NA17FU2841) from
NOAA Chesapeake Bay Office to the Blue Crab
Advanced Research Consortium.

702

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