Molecular mechanisms regulating molting in a crustacean
Halagowder Devaraj and Ayithan Natarajan
Unit of Biochemistry, Department of Zoology, University of Madras, Tamil Nadu, India
Periodic shedding of exoskeleton is associated with
growth in crustaceans. The mechanisms that control
this phenomenon may also control the growth and dif-
ferentiation processes. Eyestalk ablation has been tra-
ditionally carried out to shorten the duration of the
molt cycle and to influence the growth, reproduction
and other metabolic activities of crustaceans [1]. Lack
of adequate knowledge on the molecular physiology
of eyestalk ablation, excessive loss of hemolymph,
labor intensiveness, increased incidence of shrimp mor-
tality and reduction in life span restrict the applicabil-
ity of the eyestalk ablation technique to modern
aquaculture practices. Efforts therefore have been
made to understand the molecular physiology of mol-
ting and its ramifications in aquaculture practices.
As the growth in crustaceans is not continuous
because of the rigid exoskeleton, it is often shed to
allow periodic growth [2]. Molting is controlled by
a complex interplay of hormones, in particular, the
negative regulation of molt-inhibiting hormone (MIH)
from X-organ sinus gland (XO-SG) complex which
suppresses the synthesis or secretion of molting hor-
mones (ecdysteroids) from the Y-organ [3,4].
As crustacean growth consists of tandem prolifera-
tive and growth arrest phases, we investigated the
expression of the growth arrest-specific protein (Gas7)
in the molting process of crustaceans. Gas7 was pri-
marily characterized in NIH 3T3 cultured fibroblast
cells which enter a quiescent state following serum
deprivation [5]. It promotes G
0
arrest and is preferen-
tially expressed in differentiated neuronal cells and
peripheral nerves in mouse and other animals [6]
Individual Gas7 genes have been implicated in a vari-
ety of biological functions, including the control of
Keywords
crustaceans; ecdysteroid; growth arrest-
specific protein (Gas7); molt-inhibiting
hormone; X-organ sinus gland complex
Correspondence
H. Devaraj, Unit of Biochemistry,
Department of Zoology, University of
Madras, Life Sciences Building, Guindy
Campus, Chennai 600 025, Tamil Nadu,
India
Fax: +91 44 22301003
Tel: +91 44 22200574
E-mail: hdrajum@yahoo.com
(Received 20 September 2005, revised
15 December 2005, accepted 22 December
2005)
doi:10.1111/j.1742-4658.2006.05117.x
Crustacean growth and development is characterized by periodic shedding
(ecdysis) and replacement of the rigid exoskeleton. Secretions of the
X-organ sinus gland complex control the cellular events that lead to growth
and molting. Western blot and ELISA results showed a progressive
increase in growth arrest-specific protein (Gas7) from early postmolt stage
to a maximum at late postmolt stage. Phosphorylation of ERK2, a down-
stream signaling protein, was also identified in the subsequent stages.
ERK2 phosphorylation resulted in the expression of molt-inhibiting hor-
mone (MIH). Specific ERK inhibitors (PD98059 and UO126) exhibited the
ability to reduce the molting duration of Fenneropenaeus indicus from
12–14 days to 7–8 days, suggesting that the ERK1 ⁄2 signaling pathway is
responsible for the expression of MIH, which controls the molt cycle. We
have identified the stage-specific expression of Gas7 (48 kDa) in the
X-organ sinus gland complex of eyestalk which is involved in the down-
stream signaling of the ERK1 ⁄2 pathway regulating the expression of MIH
during the molt cycle of the white shrimp, F. indicus. These are the first
data showing an association between the Gas7 signal-transduction process
and regulation of the molt cycle and provides an alternative molecular
intervention mechanism to the traditional eyestalk ablation in crustaceans.
Abbreviations
ERK, extracellular signal-regulated kinase; Gas7, growth arrest-specific protein 7; MIH, molt-inhibiting hormone; XO-SG complex, X-organ
sinus gland complex.
FEBS Journal 273 (2006) 839–846 ª2006 The Authors Journal compilation ª2006 FEBS 839
microfilament organization [6], nerve cell growth or
differentiation [7], tyrosine kinase receptor activity [7],
and the negative [8] and positive [7] control of cell
cycling in human Schwann cells.
Receptor tyrosine kinases play a central role in
transducing the external signals across cell membranes
into the intracellular signaling systems, which in turn
lead to cell proliferation, differentiation, and other
responses in human Schwann cells [7]. Receptor tyro-
sine kinases transduce the signals via extracellular
signal-regulated kinases (ERKs), the serine ⁄threonine
protein kinases belonging to the family of mitogen-
activated protein kinases in cardiac myocytes [9].
ERKs play an important role in the downstream regu-
lation of several cellular processes, such as prolifer-
ation and differentiation, and directly modulate
cellular functions that influence gene transcription and
translation in cancer cells [10].
The role of Gas7 has not been studied so far in crus-
taceans. As the XO-SG complex is a structural and
functional homologue of vertebrate hypothalamus,
which is involved in the secretion of neurohormones
that control growth and differentiation processes such
as molting [11], we focused on the expression of Gas7
in the XO-SG complex and its role in the regulation of
MIH expression.
Therefore this study aimed to illustrate the signal-
transduction pathway in the regulation of the molt
cycle in Fenneropenaeus indicus and to devise a mole-
cular intervention technology as an alternative to the
traditional eyestalk ablation process.
Results
Western blot analysis
Western blot analysis of extracts prepared from the
eyestalk of F. indicus during different molting stages
showed a single dominant band of Gas7 only in the
early postmolt (A) and late postmolt (B) stages
(Fig. 1A). The immunoreactive band of Gas7 had a
molecular mass of 48 kDa. It was prominently detec-
ted in the XO-SG complex using rabbit polyclonal
anti-Gas7 serum, but was not observed in other stages
of molting (Fig. 1A).
Immunohistochemical localization
Immunohistochemical analysis of eyestalk neural gan-
glia sections showed a highly positive immunoreactive
expression of Gas7 in the XO-SG complex of the eye-
stalk of F. indicus only in the early postmolt (A) and
late postmolt (B) stages but not in the intermolt (C),
early premolt (D
0
and D
1
) and late premolt (D
2-3
) sta-
ges using antibody to Gas7 (Fig. 2). The immunoreac-
tivity of Gas7 was observed prominently as a cluster
of neurosecretory cell bodies or group of cell mass
exclusively over the neurosecretory centers of the
XO-SG complex. Most of the Gas7-expressing neuro-
secretory cell bodies were found in the peripheral
regions and the neuropile regions of eyestalk neural
ganglia and more adjacent to the medulla terminalis
X-organ and sinus gland.
A
B
Fig. 1. Western blot analysis of proteins from the eyestalk of
F. indicus. (A) The supernatant from the XO-SG complex of the
eyestalk of F. indicus was subjected to SDS ⁄PAGE (10% gel) and
electroblotted on to nitrocellulose membrane. The 48-kDa Gas7
was detected only in the early postmolt and late postmolt stages
by western blot analysis using rabbit Gas7 polyclonal antibody. (B)
Protein samples were extracted from the eyestalk of different
molting stages of F. indicus and subjected to SDS ⁄PAGE (10%
gel); proteins were transferred on to nitrocellulose membrane.
Phosphorylation of ERK2 protein was found in the intermolt and
early premolt stages by a shift in electrophoretic mobility on west-
ern blot analysis using rabbit ERK1 ⁄2 antibody, goat anti-rabbit
IgG–horseradish peroxidase conjugate and developed with 3,3¢-di-
aminobenzidine tetrahydrochloride substrate. KD, kDa; M, broad
range of standard protein markers run on SDS ⁄10% polyacryl-
amide gel, electroblotted on to nitrocellulose membrane, and
stained with Ponceau S red; A, early postmolt; B, late postmolt;
C, intermolt; D
0
, early premolt; D
1
, early premolt; D
2-3
, late
premolt.
Gas7 and the ERK1 ⁄2 signaling pathway in MIH expression H. Devaraj and A. Natarajan
840 FEBS Journal 273 (2006) 839–846 ª2006 The Authors Journal compilation ª2006 FEBS
ELISA
Gas7 concentrations were determined by direct ELISA
using antibody to Gas7. A progressive increase from
the early postmolt stage to a maximum at the late
postmolt stage followed by a sudden fall in the sub-
sequent stages (Fig. 3) was observed. The results are
consistent with the results of western blot and immu-
nohistochemical analysis.
Concentrations of the immunoreactively expressed
MIH protein were also quantified by direct ELISA
from the extracts prepared from the XO-SG complex
of the eyestalk from control groups as well as groups
treated with specific inhibitors (PD98059 and UO126).
In control stages, MIH concentrations were increased
significantly from early postmolt to a maximum at
intermolt, but were dramatically reduced to a mini-
mum at early premolt and late premolt stages. In the
case of the PD98059-treated and UO126-treated stages
(intermolt and early premolt), MIH concentrations
were reduced to a basal level (Fig. 4A,B).
Phosphorylation of ERK2
Phosphorylation or activation of ERK2 protein was
found only in intermolt and early premolt by a shift
in mobility on western blot analysis (Fig. 1B). No
shift in electrophoretic mobility was observed in early
postmolt, late postmolt, early premolt and late
premolt stages.
Fig. 2. Immunohistochemical localization of Gas7 expression was
observed only in (A) early postmolt and (B) late postmolt stages in
the eyestalk tissue of F. indicus using rabbit polyclonal antibody to
Gas7. (N), Negative control treated with normal goat serum not
showing expression of Gas7; ME, medulla externa; MI, medulla
interna; MT, medulla terminalis; SG, sinus gland; XO, X-organ; NSC,
neurosecretory cell bodies. Scale bar represents 100 lm.
Fig. 3. Quantitative determination of Gas7 concentrations in
extracts prepared from the XO-SG complex of F. indicus by direct
ELISA using rabbit polyclonal antibody to Gas7. The line diagram
represents high concentrations of Gas7 immunoreactivity only in
early postmolt, maximum at late postmolt, and basal level in subse-
quent stages. Values are expressed as mean ± SD (n¼5).
H. Devaraj and A. Natarajan Gas7 and the ERK1 ⁄2 signaling pathway in MIH expression
FEBS Journal 273 (2006) 839–846 ª2006 The Authors Journal compilation ª2006 FEBS 841
Treatment with inhibitors and blocking
of ERK2 activation
Phosphorylation of ERK2 and expression of MIH were
studied in both control and inhibitor-treated (PD98059
and UO126) stages. Phosphorylation of ERK2 was
detected as a shift in mobility during the intermolt
and early premolt stages of control shrimps, whereas
PD98059-treated stages (intermolt and early premolt)
showed no shift in ERK2 protein mobility using rabbit
anti-ERK1 ⁄2 IgG (Fig. 5A). Mouse anti-phospho-
ERK1 ⁄2 IgG specifically detected only phosphorylated
ERK2 protein in control groups of intermolt and early
premolt stages, but the antibody could not detect the
ERK2 protein in UO126-treated stages (Fig. 5B). Like-
wise, the immunoreactive band of MIH was also detec-
ted only in the control stages, i.e. early postmolt, late
postmolt, intermolt and early premolt, but not in the
PD98059-treated and UO126-treated stages (intermolt
and early premolt) (Fig. 6A,B). The results are consis-
tent with the results of direct ELISA.
Fig. 4. Effect of (A) PD98059 and (B) UO126 on MIH concentra-
tions of F. indicus. MIH concentrations were determined by direct
ELISA in extracts prepared from control (Untreated) and PD98059
or UO126 treated stages of F. indicus using anti-r-Pej-MIH IgG.
The bar diagram shows the highly significant concentrations
(P<0.0001; analysis of variance) of MIH in the intermolt stage of
the control samples, but PD98059 and UO126 treated stages (inter-
molt and early premolt) show very low concentrations of MIH.
Values are expressed as mean ± SD (n¼5).
A
B
Fig. 5. Effect of (A) PD98059 and (B) UO126 on the phosphoryla-
tion of the ERK2 protein from the XO-SG complex. (A) Protein sam-
ples from the XO-SG complex of different molting stages were run
on SDS ⁄10% polyacrylamide gel and electroblotted on to nitrocellu-
lose membrane. Phosphorylation of the ERK2 protein was detected
by a shift in electrophoretic mobility on western blot analysis only
in control stages (intermolt and early premolt), but PD98059-treated
stages did not show ERK2 phosphorylation using rabbit antibody to
ERK1 ⁄2. (B) Mouse anti-phosphoERK1 ⁄2 IgG specifically detected
only phosphorylated ERK2 protein in control groups of intermolt
and early premolt stages, but the antibody could not detect the
ERK2 protein in UO126-treated stages. KD, kDa; M, broad range
of standard protein markers run on SDS ⁄10% polyacrylamide gel,
electroblotted on to nitrocellulose membrane, and stained with
Ponceau S red; A, early postmolt; B, late postmolt; C, intermolt;
D
0
, early premolt; D
1
, early premolt; D
2-3
, late premolt.
Gas7 and the ERK1 ⁄2 signaling pathway in MIH expression H. Devaraj and A. Natarajan
842 FEBS Journal 273 (2006) 839–846 ª2006 The Authors Journal compilation ª2006 FEBS
Molting duration of F. indicus
The inhibitor-treated (PD98059 and UO126) stages
showed an absence of MIH, which reduces the molting
duration of F. indicus. The shrimps in the control
groups required 12–14 days to complete the molt
cycle, whereas in those injected with either PD98059 or
UO126 the duration of the molt cycle was reduced to
7–8 days (Fig. 7). The available data also show a
correlative and significant change in the shortening of
the molt cycle which leads to frequent molting and
growth of shrimps.
Discussion
Gas7 is a multifunctional protein involved in the mat-
uration of neurons and release of neurotransmitters
[5]. The stage-specific expression of the Gas7 gene
using RT-PCR of Gas7 mRNA and coimmunoprecipi-
tation of Sky receptor has been identified in the early
and late postmolt stages of Penaeus monodon, suggest-
ing that Gas7 may act as a ligand for tyrosine kinase
Sky receptor (unpublished work). ERK2 is part of the
Ras ⁄Raf ⁄MEK downstream signaling pathway for
many growth factors that act by binding to tyrosine
kinase receptors in cardiac myocytes [19]. The mito-
gen-activated protein kinase pathway is involved in a
number of cellular changes inducing growth and differ-
entiation in human Schwann cells [7]. In cardiac myo-
cytes, protein kinase C generates a positive feedback
loop to ERK2 phosphorylation [19], and this effect
has also been demonstrated in P. monodon (unpub-
lished work).
In decapod crustaceans, the growth and differenti-
ation processes are controlled by a complex interplay
of neuropeptides that are stage specific and inducible
[3,4]. To elucidate the functional necessity of Gas7
in MIH expression, highly selective ERK inhibitors
(PD98059 and UO126) were used, which completely
inhibited the Gas7-dependent downstream signaling
during ERK2 phosphorylation. ERK2 is active only
when it is phosphorylated [7]. Inhibition of ERK2 with
ERK ⁄MEK inhibitors (PD98059 and UO126) appears
A
B
Fig. 6. Effect of (A) PD98059 and (B) UO126 on 7–8-kDa MIH
from the XO-SG complex. Protein samples prepared from the
XO-SG complex of the eyestalk of F. indicus were run on
SDS ⁄15% polyacrylamide gel. Immunoblotting was performed
using anti-r-Pej-MIH IgG in different molting stages. The 7–8-kDa
MIH protein was detected only in the control stages (early post-
molt, late postmolt, intermolt and early premolt), whereas shrimps
treated with PD98059 and UO126 (intermolt and early premolt sta-
ges) show an absence of MIH immunoreactivity. KD, kDa; M, broad
range of standard protein markers run on SDS ⁄15% polyacrylamide
gel, electroblotted on to nitrocellulose membrane, and stained with
Ponceau S red; A, early postmolt; B, late postmolt; C, intermolt;
D
0
, early premolt; D
1
, early premolt; D
2-3
, late premolt.
Fig. 7. Molt cycle duration of F. indicus in controls and shrimps
injected with specific inhibitors (PD98059 and UO126) on day 3
after the first molt was recorded as hours between the first molt
and the second molt. Values are mean ± SD (n¼5). The highly
significant (P< 0.0001, analysis of variance) reduction in molt cycle
duration was observed only in the D
1
stage.
H. Devaraj and A. Natarajan Gas7 and the ERK1 ⁄2 signaling pathway in MIH expression
FEBS Journal 273 (2006) 839–846 ª2006 The Authors Journal compilation ª2006 FEBS 843