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.. Hemolymph t
Trang 1(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
In recent years, crustacean hyperglycemic hormones
(CHHs), traditionally identified in the medulla
termi-nalis 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
Car-cinus 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 americ-anus [3,9,10], Pachygrapsus marmoratus [5] and Mach-robrachium 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 infor-mation about the localization or the physiological function of active CHH neuropeptides in correspond-ing tissues Prototypical actions attributed to ES-CHH include induction of hyperglycemia, suppression of
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: chung@comb.umbi.umd.edu
(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 physiologi-cal regulation in crustaceans Much less is known of the functions of
non-ES CHH than of those of its counterpart present in non-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 iden-tified 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 condi-tions 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
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.
Trang 2ecdysteroid 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
abdom-inal segments seem to be particularly involved in the
process of water uptake during ecdysis and hatching,
respectively [1,6] The physiological roles of
SOG-CHH and PO-SOG-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
neuro-peptides in the tissues from which many CHH cDNAs
were derived
The blue crab, Callinectes sapidus, an economically
valuable euryhaline species in Chesapeake Bay,
experi-ences migration and seasonal changes in
environ-mental conditions, including temperature, salinity
and dissolved oxygen (http://www.dnr.state.md.us/bay/
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
rea-sonable to think that Cal sapidus CHH may also play
an important regulatory role in adaptation to
natu-rally 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,
hypo-thermia and hyperhypo-thermia, in an attempt to define
their functions
To address these questions, we cloned the
full-length 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
tempera-ture changes were documented using quantitative
real-time RT-PCR To further define the physiological
role of PO-CHH, the levels of this CHH were
mea-sured from the same animals, along with ES-CHH,
using RIAs under control normoxic and hypoxic
con-ditions
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 equiva-lents) by RP-HPLC is presented in Fig 2 Fractions
Retention time, min
0 10 20 30 40
0.0
0.2
1 2
Fig 1 CHH neuropeptide profile of single SG extract of Cal sapi-dus 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 mLÆmin)1 Peak 1, I; peak 2,
CHH-II The mass of each neuropeptide determined by ESI MS was as follows; CHH-I, 8494.2 Da; CHH-II, 8478.1 Da Fractions that posi-tively cross-reacted with Car maenas CHH antiserum are shown
as bars.
0.0
0.5
Retention time, min
0 10 20 30 40
Fig 2 Separation of the extract of PO (50 equivalents) on an RP-HPLC C18 column Elution conditions were the same as described for Fig 1 The cross-reacted fractions initially tested with Car mae-nas CHH antiserum are shown as bars.
Trang 3analyzed by ELISA are marked on a bar graph that
shows positive cross-reactivity with PO-CHH
anti-serum Immunopositive fractions (numbers 30–32)
were further analyzed by surface-assisted laser
desorp-tion⁄ ionization (SALDI) to localize fraction 31
con-taining 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
bio-assay
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-oxi-dized ES-CHH (peak 2, CHH-II), showed only a
mod-est two-fold increase in glucose level from 77.79 ± 7.3
to 151.58 ± 13.4 lgÆmL)1 hemolymph (n = 10,
P< 0.01, paired t-test) Interestingly, the injection of
20 pmol of Car maenas ES-CHH also induced a
sig-nificant increase (P < 0.05, paired t-test) in
hemo-lymph glucose from the blue crab (257 ± 34.6 to
633 ± 229 lgÆmL)1hemolymph, 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
major-ity of cells showed homogeneous CHH staining in the
cytoplasm (Fig 4C) with a visibly large nucleus,
whereas others had less intensive but punctuated and
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 ampli-con of 500 bp On the basis of this sequence, gene-specific 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 touch-down 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 walk-ing 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
Saline ES-CHH PO-CHH
Glucose (ug/1 mL hemolymph) 0
300
600
900
1200
***
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
signifi-cant increase in hemolymph glucose (***P < 0.001, paired t-test).
A
B
C
D
E
Fig 4 Immunohistochemistry of PO staining with PO-CHH anti-serum (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.
Trang 4Hemolymph 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 fmolÆmL)1) in hemolymph was 9-fold
higher than that of PO-CHH (45.6 ± 8.6 fmolÆmL)1)
One hour of exposure of hypoxia induced CHH
secre-tion both from the ES and PO ES-CHH doubled in
level from 376 ± 67 to 762 ± 143 fmolÆmL)1
hemo-lymph (n = 9, P < 0.05) The level of PO-CHH
increased from 45.6 ± 8.6 to 74.5 ± 19.8 fmolÆmL)1
hemolymph (n = 9) but there was no statistical
differ-ence Interestingly, ES-CHH levels in the hemolymph
of animals 10 min after they were returned to
con-trol normoxic seawater, after previous exposure to
hypoxia for 1 h, significantly decreased to 156 ± 50
and 44.5 ± 12.8 fmolÆmL)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
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 initia-tion 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 experi-enced 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 con-trols (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) Lev-els 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 (29C) conditions caused a 2.5-fold increase in glucose levels in hemo-lymph, as shown in Fig 8A, whereas in controls and under hypothermic conditions (4C) 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 com-pared 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 22C
The effect of different temperatures on gene expres-sion in the ES and PO is shown in Fig 8C The expression of ES-CHH was modestly increased at
29C (P = 0.08, one-way anova) as compared with
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
quantita-tive 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¢)
Trang 5that in controls at 22C; whereas the level of
PO-CHHexpression was only slightly higher, at 29C 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 22C
and 29C, except that animals exposed to 4 C
showed higher AK expression in the PO, as compared
with those exposed to 22C 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,
molecu-lar 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 environ-mental conditions: hypoxia, emersion, and tempera-ture 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 sug-gests 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 mae-nas [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
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.
Trang 6these 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
neurotransmit-ter, 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
open-ings 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
pro-duced 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
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
C o n t
r o l n
o r
o x i a
H y p o
x i a
C o n t
r o l n
o r
o x i a
0
20
40
60
80
100
ES-CHH (fmol/mL hemolymph) 0
200
400
600
800
1000
a
b
c
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.
Control Emersion
Glcuose (ug/mL hemolymph) 0
100
200
300
400
**
***
ES-
C
H ES-AK
E S-
eI F 4A
PO
CH
H
P O -A K PO-e
IF 4A
10 10
10 9
108
107
106
a
b
b a
b
b
b
b
b
a a
a
a a b
Hypoxia
Lactate (ug/mL hemolymph) 0
150
300
450
600
** *
** *
Control Emersion Hypoxia
A
B
C
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 signifi-cance 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 ).
Trang 7anti-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 bio-assays of Cal sapidus ES-CHHs reflect high sequence identity (> 75%) of ES-CHHs between two crab spe-cies 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 ES-CHH and PO-ES-CHH shows that the greatest homology
is in the first 40 amino acids, with much more differ-ence in the latter half of the sequdiffer-ence Such differdiffer-ences are common in all CHH sequences currently available
in GenBank, indicating that functionality inducing hyperglycemia may lie in the first 40 amino acid resi-dues [36–40] Therefore, it is suggested that these two CHHs may have separate receptors in their target tis-sues, where PO-CHH may be mobilizing glucose but not be directly involved in hyperglycemia in hemo-lymph PO-CHHs among Cal sapidus, Car maenas and P marmoratus share overall 67% sequence iden-tity, 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)11m, 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 pmolÆES)1) It is noteworthy that the basal level
of ES-CHH is an order of magnitude greater in Cal sapidus (10)10m) than in Car maenas (10)11m) [24] or Can pagurus (< 10)11m) [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
60
120
180
*
**
***
22 to 22 22 to 29 22 to 4
100
200
300
Temperature (°C)
22 to 22 22 to 29 22 to 4 Temperature (°C)
**
S
-H C E A -S K -S I e
A 4
F -CH O P
K -O P O P
-eIF
A 4
C 1e+6
1e+7
1e+8
1e+9
a a b
A
B
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 ).
Trang 8sternal 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
expres-sion 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
hemo-cyanin gene in the hepatopancreas in the same species
[46] Moreover, the degree of decrease in CHH
expres-sion 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
tran-scription of PO-CHH are in proportion to the demand
for its release, whereas the inhibition of ES-CHH
tran-scription may be caused by high glucose levels in
he-molymph 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
We have shown the presence of ES-CHH neuropep-tides 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 sensi-tive 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 partic-ular, in response to oxygen level Defining the phy-siological 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 mar-moratus, the function of this neuropeptide may be simi-lar 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], Aqua-culture 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 22C] under ambient daylight conditions Experimen-tal animals were fed daily with chopped frozen squid and pelleted sea bream (EWOS, Surrey, Canada) All experi-mental 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 Dir-cksen et al [4], respectively CHH peaks were initially iden-tified 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 RP-HPLC fraction containing PO-CHH and its mass determi-nation were performed using SALDI
Trang 9RACE 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¢-5¢-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 94C for 3 min;
three cycles at 94C for 30 s, 54 C for 30 s, and 72 C for
2 min; three cycles at 94C for 30 s, 52 C for 30 s, and
72C for 2 min; three cycles at 94 C for 30 s, 50 C for
30 s, and 72C for 2 min; 24 cycles at 94 C for 30 s,
55C for 30 s, and 72 C for 2 min, and a final extension
at 72C 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 94C for 3 min, 30 cycles at
94C for 30 s, 60 C for 30 s, and 72 C for 2 min, and a
final extension at 72C 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
prim-ers (Table 1) Further cRNA synthesis and RNA
quantifi-cation were performed as described in Chung & Webster
[6,7], with a modification for purification of in vitro
tran-scribed 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
equiva-lent 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
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,
KIQVV-NH2; Invitrogen) were synthesized with the addi-tion 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 (Pro-teintech 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 CHH, and Car maenas ES-CHH, 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 hemo-lymph 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 ED50values 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 sea-water (15 p.p.t., 22 C) was continuously purged with nitro-gen to reduce the level of dissolved oxynitro-gen to < 0.5%
Trang 10(YSI 58 Dissolved Oxygen Meter) Test juvenile animals
(60–80 mm carapace width) were exposed for 1 h to
hyp-oxic 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
hypobran-chial 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
col-lected 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
sam-ples 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
(Univer-sity 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
main-taining the water quality of the recirculation system
This article is contribution no 07-177 of the Center of
Marine Biotechnology (University of Maryland
Bio-technology Institute, Baltimore, MD) The work is
supported by a program grant (NA17FU2841) from
NOAA Chesapeake Bay Office to the Blue Crab
Advanced Research Consortium
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