General and Comparative Endocrinology 172 (2011) 323–330

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General and Comparative Endocrinology

journal homepage: www.elsevier.com/locate/ygcen

Review

Regulation of crustacean molting: A review and our perspectives

Ernest S. Chang a,⇑, Donald L. Mykles b
a
Bodega Marine Laboratory, University of California-Davis, Bodega Bay, CA 94923, USA
b
Department of Biology, Colorado State University, Fort Collins, CO 80523, USA

a r t i c l e i n f o a b s t r a c t

Article history: Molting is a highly complex process that requires precise coordination to be successful. We describe the
Received 2 February 2011 early classical endocrinological experiments that elucidated the hormones and glands responsible for this
Revised 30 March 2011 process. We then describe the more recent experiments that have provided information on the cellular
Accepted 4 April 2011
and molecular aspects of molting. In addition to providing a review of the scientific literature, we have
Available online 8 April 2011
also included our perspectives.
Ó 2011 Elsevier Inc. All rights reserved.
Keywords:
20-Hydroxyecdysone
Crustacean
Decapod
Ecdysone
Ecdysteroid
Molting

1. Introduction
To achieve growth, arthropods must initially loosen the connec-
tives between their living tissues and their extracellular cuticles,
escape from the confines of these cuticles relatively rapidly, take
up water or air to expand the new, flexible exoskeletons, and then
quickly harden them for defense and locomotion. Each of the major
arthropodan taxa, the crustaceans and insects, has provided valu-
able models for the study of the endocrine regulation of molting.
In this review, we focus on the Crustacea. It is not comprehensive
and contains some of our personal accounts of the journeys that
led to our current state of knowledge of the regulation of crusta-
cean molting.
2. Classical endocrinological experiments
The molting process is a cyclical event, the culmination of
which is the actual ecdysis, or shedding of the exoskeleton
(Fig. 1). The prohormone of the steroid molting hormone, ecdy-
sone, was isolated from hundreds of kilograms of moth pupae
⇑ Corresponding author. Fax: +1 707 875 2091.
E-mail address: eschang@ucdavis.edu (E.S. Chang).
1
Abbreviations used: 20E, 20-hydroxyecdysone; CaM, calmodulin; CHH, crustacean
hyperglycemic hormone; GC, guanylyl cyclase; GC-I, nitric oxide-sensitive guanylyl
cyclase; ILP, insulin-like peptide; LAFan, Limb Autotomy Factor–Anecdysis; LAFpro,
Limb Autotomy Factor–Proecdysis; LB, limb bud; LBA, limb bud autotomy; mTOR,
metazoan target of rapamycin; MIH, molt-inhibiting hormone; NO, nitric oxide; NOS,
nitric oxide synthase; PDE, phosphodiesterase; TGFb, transforming growth factor-b;
YO, Y-organ.
[13]. Several years later, the structure of ecdysone was determined
and its source traced to the prothoracic gland [53,55]. From many
subsequent studies, 20-hydroxyecdysone (20E)1 has been charac-
terized as the physiologically active form of the ecdysteroids (a
class of steroid molting hormones related in structure to 20E) in
most species. Parallel studies on the ecdysteroids of crustaceans
led to observations that the primary ecdysteroid of crustaceans
was also 20E [46,52].
Classical morphological observations and endocrinological
experiments involving organ ablation [36,40] indicated that the
molting gland in the crab Carcinus maenas was the thoracic Y-organ
(YO). In brachyurans, the YOs are spheroidal glands located ven-
trally and anteriorly in the gill chambers. The macruan YO is more
difficult to locate since it is a monolayer sheet of cells. While in J.
Dennis O’Connor’s laboratory, Chang (EC) began organ culture
experiments using YOs from the crabs Cancer antennarius and
Pachygrapsus crassipes. These experiments involved the character-
ization of ecdysone as the primary secretory product of the YO
[18]. Those results indicated that ecdysone was the prohormone
for 20E. Experimental animals were collected by hand at night un-
der rocks at Palos Verdes, California. It was due partially to those
collection trips that convinced EC that future scientific endeavors
should be more laboratory-intensive as opposed to field-based.
Those YO experiments involved the determination of appropri-
ate organ culture experiments. Defined media, such as M199
provided prolonged secretory activity of the YO compared to a
simple saline solution. However, since identification of the secre-
tory product required collection and concentration of hundreds
of milliliters of culture media, the saline solution provided a less

0016-6480/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygcen.2011.04.003
324 E.S. Chang, D.L. Mykles / General and Comparative Endocrinology 172 (2011) 323–330

Fig. 1. Molting (or ecdysis) of a juvenile lobster (Homarus americanus). The entire sequence took about 30 min from start to finish. Molting is the culmination of a cyclic
process mediated by the steroid hormone 20-hydroxyecdysone. (A) The lobster has resorbed much of its old exoskeleton’s mineralization. The exoskeleton splits at the
junction of the thorax and abdomen. (B) The anterior of the animal retracts from its old exoskeleton by pulling posteriorly. (C) The posterior of the lobster pulls anteriorly. (D)
The lobster is free of its old exoskeleton and begins to take up water to expand its new, flexible, larger exoskeleton. (E) The postmolt lobster is above its shed exoskeleton. It
will take several days for the epidermis to deposit layers of chitin, protein, and calcium carbonate into the exoskeleton. The molt cycle will continue for several days to
months, depending upon the size and age of the animal. Photos by staff of the Bodega Marine Laboratory. Reproduced with permission [16].

contaminated secretory product for eventual mass spectrophoto-
metric analyses compared to the product secreted into M199.
Similar results were obtained from the crayfish Orconectes limo-
sus [57] and the blue crab Callinectes sapidus [99]. More recent data
have revealed that there are other ecdysteroids secreted by the YOs
in some species. These ecdysteroids include 3-dehydroecdysone
from C. antennarius [114] and 25-deoxyecdysone from C. maenas
[64] (see [83,121], for reviews). A productive area of future re-
search would be the determination of the types and ratios of the
different ecdysteroids secreted by the YOs from various other crus-
tacean species. The functional significance of these differences is
unclear.
Hemolymph ecdysteroid concentration fluctuates dramatically
during the molt cycle (e.g., from <10 pg/ll in postmolt lobster to
>350 pg/ll in premolt lobster) [17] and these changes mediate
the various biochemical and physiological processes that occur
during the cycle. The rate of synthesis and/or secretion of ecdy-
sone by the YO vary during the molt cycle and partially explain
these hemolymph fluctuations in ecdysteroid titer. Just prior to
the substage of premolt in which the highest concentration of
ecdysteroids was observed in the hemolymph, explanted YOs
were found to secrete the greatest amount of ecdysone [19].
Low hemolymph concentrations were correlated with low secre-
tory rates. This led to the question – what controls the secretory
rate of the YO?
Over a century ago, removal of both stalked eyes of the crab Uca
pugilator [130] resulted in a dramatic shortening of the molt inter-
val (length of the molt cycle). This was later confirmed in crayfish
[107] and many other species. These observations led to the postu-
lation of an endocrine factor present in the eyestalks that normally
inhibits molting – a molt-inhibiting hormone (MIH). Detailed
microscopical examinations resulted in the description of a neuro-
hemal organ in the eyestalk of several decapod crustaceans [9,92].
This neurohemal organ is called the sinus gland and serves as a
storage site for neurosecretory products. It consists of the enlarged
endings of a group of neurosecretory neurons collectively called
the X-organ [47]. The shortened molt interval observed in eye-
stalk-ablated decapods is likely due to a rapid elevation in the con-
centration of circulating ecdysteroids, which is a result of X-organ/
sinus gland removal [15,22]. Conversely, the demonstration of de-
creased ecdysteroid titers in eyestalk-ablated crabs that have been
injected with eyestalk extracts [50,56] lends additional support to
the paradigm of the MIH–YO molt-controlling axis. Further evi-
dence of this hypothesis was provided by Gersch et al. [41], who
demonstrated that if crayfish (O. limosus) were initially injected
with sinus gland extracts, the subsequent culture of the YOs re-
sulted in a decreased production of ecdysone compared to vehi-
cle-injected animals. In vitro secretion of ecdysteroid by crab YOs
could be inhibited when cultured with either conditioned medium
that had previously been incubated with explanted sinus glands or
by the addition of eyestalk extracts [74,99,111,122]. In Homarus
americanus, EC’s laboratory demonstrated that injection of sinus
gland extracts decreased circulating titers of ecdysteroids and in-
creased the molt interval [12].
MIH from C. maenas was among the first of the MIHs to be char-
acterized [123]. EC’s laboratory worked on MIH from H. americanus
[21]. At that time, Prof. Rainer Keller was visiting EC’s laboratory.
Keller had remembered much of the C. maenas MIH sequence
and was very excited to see the similarities in amino acids when
he was shown the lobster sequence.
MIH is a member of a novel neuropeptide family. Representa-
tives of this family have only been found in arthropods so far
[37]. This neuropeptide family regulates such diverse functions
as molting, reproduction, and metabolism [10,15,34,120,125].
 E.S. Chang, D.L. Mykles / General and Comparative Endocrinology 172 (2011) 323–330
MIH binds to hormone receptors on the membranes of YO cells
[124] and likely mediates its action via cyclic nucleotide second
messengers [97](see [31], for review) or through alterations in
calcium ion fluxes and activity of protein kinase C that
phosphorylates enzymes involved in steroidogenesis [113]. The
mode of action of MIH is discussed below.
EC’s group has also worked on the hypothesis that ecdysteroids
may feed back upon the X-organ, the YO, and/or other target tis-
sues to mediate normal ecdysis. Chang and O’Connor [19] showed
that the activity of various tissues to hydroxylate ecdysone to 20E
was inhibited with the addition of exogenous 20E. Cheng and
Chang [23] demonstrated that normal ecdysis in lobsters could
be delayed by the injection of exogenous 20E. Those results indi-
cated that the normal completion of processes, such as ecdysis, re-
quired not only increases in hormone concentrations, but also
required their subsequent decline. The mechanism of this ecdy-
steroid inhibition is unknown. El Haj et al. [38] published findings
that ecdysteroid receptors are located in eyestalk neural tissue.
Those data hinted at a possible positive or negative feedback regu-
lation upon MIH secretion. We recently obtained preliminary data
that a non-steroidal ecdysteroid analog (tebufenozide) was able to
inhibit ecdysone secretion from crab YOs in vitro [90]. This effect
was observed at 50 and 100 pg/ll of culture medium, but not at
lower concentrations. This ecdysteroid inhibition of ecdysone
secretion may account for the observation that hormone titers in
eyestalk-ablated lobsters continue to cycle up and down, even
though the eyestalk tissue does not regenerate [14].
3. Molecular regulation of molting
3.1. Limb factors
A variety of signals affect YO activity. External cues, such as
stress, temperature extremes, crowding, and short photoperiod
can inhibit molting [1,8,48,78,93]. In the blackback land crab,
Gecarcinus lateralis, loss of five or more walking legs, termed multi-
ple leg autotomy or MLA, induces molting as growth of limb buds
(LBs) is restricted to premolt. Upon ecdysis the LBs are extended
and become functional appendages [8,104,106]. However, limb
bud autotomy (LBA) during early premolt suspends molting 2–
3 weeks, allowing time for a secondary (2°) LB to grow, so that ani-
mals molt with a full complement of legs [49,82]. These studies led
Dorothy Skinner to propose that LBs produce stimulatory and
inhibitory factors that she designated Limb Autotomy Factor–
Anecdysis or LAFan and Limb Autotomy Factor–Proecdysis or LAF-
pro, respectively [104]. LAFan is produced by primary (1°) LBs and
stimulates molting in response to MLA. The identity of LAFan
remains unknown. LAFpro is a MIH-like factor produced by 2° LBs
that inhibits 1° LB growth by lowering hemolymph ecdysteroid
titers [128]. By mid premolt the animal becomes committed to
molt and, consequently, interventions such as LBA and MIH injec-
tions fail to suspend premolt and animals molt without delay
[49,128].
Gecarcinus lateralis is an excellent experimental organism for
the study of the endocrine control of molting. These air-breathing
animals occur throughout the Caribbean, living in burrows they dig
in dune areas above coral sand beaches [6,7]. They are easy to
maintain in the laboratory and molting is easily manipulated.
Dorothy Bliss pioneered the use of the G. lateralis in the 1950s
[9] (see [73] for a complete bibliography). In 1962, Skinner pub-
lished her first paper on G. lateralis, reporting the first microscopic
analysis of the changes in integumentary structure during the molt
cycle [102], and it became her primary experimental animal for
nearly four decades (see [86], for a complete bibliography). Mykles
(DM) was introduced to G. lateralis when he joined the Skinner
325
laboratory at Oak Ridge National Laboratory in 1979 to study a
molt-induced claw muscle atrophy that Skinner reported in 1966
[80,103]. DM continued studies on G. lateralis when he moved to
Colorado State University in 1985. While in residence at the
Bodega Marine Laboratory 1998–1999, DM began a collaboration
with EC to study the endocrine regulation of molting in G. lateralis,
resulting in the characterization of LAFpro [128]. Subsequent work
has been directed toward neuropeptide signaling pathways in the
YO and ecdysteroid control of muscle atrophy [29,30,33,58–60,66–
70,76,129]. Thus, G. lateralis has been an important experimental
animal for crustacean molting physiology for more than 60 years.
3.2. Regulation of the Y-organ by eyestalk neuropeptides
Both MIH and a related peptide, crustacean hyperglycemic hor-
mone (CHH), inhibit YO ecdysteroidogenesis through separate sig-
naling pathways that converge at cGMP [24,39]. YO membranes
have distinct receptors for CHH and MIH [2,24,27,124]. The CHH
receptor appears to be a membrane guanylyl cyclase (GC)
[26,43], whereas the identity of the MIH receptor is not firmly
established. Thus, binding of CHH leads directly to an increase in
cGMP [26]. By contrast, MIH signaling usually involves a transient
increase in cAMP, followed by a larger, sustained increase in cGMP
[4,11,31,87,89,97,100,119]. The delayed increase in cGMP suggests
that MIH activates a soluble GC, as activation of a membrane GC
would result in an immediate increase in cGMP. Both cAMP and
cGMP inhibit YO ecdysteroidogenesis in vitro (see [31], for review).
Our recent work indicates that nitric oxide (NO)-sensitive GC (GC-
I) is involved. GC-I agonists (NO donors and YC-1) can inhibit YO
ecdysteroidogenesis in vitro [29,84]. Moreover, YOs express a
Ca2+/CaM-dependent NO synthase (NOS) and the catalytic (b) sub-
unit of GC-I [58,68,70,76].
Much of the data support the organization of the MIH signaling
pathway into a cAMP/Ca2+-dependent ‘‘triggering’’ phase and a NO/
cGMP-dependent ‘‘summation’’ phase linked by calmodulin (CaM)
[32,70,84]. A transient increase in cAMP triggers the influx of Ca2+,
which activates CaM (Fig. 2). A sustained activation of NOS results
from the combined effects of direct binding of Ca2+/CaM to NOS
and the dephosphorylation of NOS by calcineurin, which is a
Ca2+/CaM-dependent protein phosphatase. Phosphorylation of
NOS reduces its activity; dephosphorylation by calcineurin en-
hances NOS activity [63]. NOS is phosphorylated in the activated
YO, which is consistent with inactivation of NOS by protein kinases
in the absence of MIH [70]. This is similar to the signaling mecha-
nism that stimulates fluid secretion in Drosophila Malpighian tu-
bules by the decapeptide cardioacceleratory peptide 2b (see [5],
for review). MIH is released in pulses, with each pulse having a
half-life of 5–10 min; this results in low MIH titers (<5 fmol/ml)
in the hemolymph of most intermolt animals [25]. The sustained
activation of GC-I in the model provides a mechanism for the inhi-
bition of the YO between pulses, thus explaining how the YO is
suppressed for extended periods when average MIH titers are
low in intermolt animals.
3.3. Changes in the Y-organ over the molt cycle
The YO is a dynamic organ that transitions through four physi-
ological states during the molt cycle (Fig. 3). During postmolt
(stages A, B, and C1–3) and intermolt (stage C4), the YO is in the ba-
sal state, resulting in low (<20 pg/ll) ecdysteroid titers in the
hemolymph. A reduction in MIH triggers the transition from the
basal state to the activated state; the increasing hemolymph ecdy-
steroid titers initiate premolt processes in the integument (e.g.,
separation of the epithelium from the exoskeleton or apolysis),
gastric epithelium (e.g., gastrolith formation in certain species),
skeletal muscle (e.g., claw muscle atrophy), and limb regenerates
326 E.S. Chang, D.L. Mykles / General and Comparative Endocrinology 172 (2011) 323–330

MIH Ca2+ channel

MIH-R Cell membrane

G AC
PKA

Triggering phase
ATP
cAMP

Ca2+
PDE1

PKA
AMP
CaM

NOS
Pi
CaN

Arginine NOS
dephosphorylation

Summation phase
Pi

Citrulline
NO

GC-I

PDE5
GTP cGMP GMP

PKG

Ecdysteroid synthesis
(Transcriptional/Translational Regulation)

Fig. 2. Proposed MIH signaling pathway regulating ecdysteroidogenesis in decapod crustacean molting gland. The ‘‘triggering’’ phase is initiated by binding of MIH to a G
protein-coupled receptor (MIH-R) and activation of adenylyl cyclase (AC); cAMP increases intracellular Ca2+ via cAMP-dependent protein kinase (PKA) phosphorylation of
Ca2+ channels. Sensitivity to MIH is determined by phosphodiesterase 1 (PDE1) activity, which varies during the molting cycle. The ‘‘summation’’ phase is mediated by NO and
cGMP. Calmodulin (CaM) links the two phases by activating NO synthase (NOS) directly and indirectly via calcineurin (CaN). Dephosphorylation of NOS by CaN can potentially
prolong the response to MIH. CaM can also activate PDE1 to inhibit the triggering phase (PDE1 can also hydrolyze cGMP, thus inhibiting the summation phase). cGMP-
dependent protein kinase (PKG) inhibits ecdysteroidogenesis. Chronic activation of PKA may directly inhibit ecdysteroidogenesis. Chronic elevated intracellular cAMP can
inhibit ecdysteroidogenesis directly, perhaps by inhibiting protein synthesis. Other abbreviations: G, G protein; GC-I, NO-sensitive guanylyl cyclase; PDE5, cGMP PDE. From
[32].

(e.g., LB growth) in early premolt (stage D0) (see [51,80–
82,96,105,126], for reviews). At mid premolt (stage D1), the YO
transitions from the activated to committed state; the YO increases
ecdysteroid biosynthesis and hemolymph ecdysteroid titers reach
a peak (>300 pg/ll) at stage D2. LB growth, claw muscle atrophy,
and gastrolith formation are completed by stage D2 [105]. The high
ecdysteroids trigger the transition of the YO from the committed
state to the repressed state in late premolt (D3–4). At ecdysis, the
YO transitions back to the basal state in postmolt when ecdysteroid
titers are low.
YO activation at stage D0 is triggered by a decrease in the re-
lease of MIH from the sinus gland. This de-represses the YO, pre-
sumably through inactivation of MIH signaling components,
resulting in hypertrophy and increased ecdysteroid biosynthetic
capacity (Fig. 3). NOS is phosphorylated in YOs from 1 day eye-
stalk-ablated animals, suggesting the NOS is inactivated in the ab-
sence of MIH [70]. The downstream targets are largely unknown,
but activation probably involves transcriptional, translational,
and posttranslational regulation. YO activation results in signifi-
cant (>3-fold) changes in the levels of hundreds of proteins [70].
 E.S. Chang, D.L. Mykles / General and Comparative Endocrinology 172 (2011) 323–330 327

Early Mid Late

Intermolt Premolt Premolt Premolt E Postmolt
(C4) (D 0) (D 1,2) (D3,4) (A, B, C1-3)

Hemolymph
ecdysteroid
synthetic capacity
YO ecdysteroid

YO State: Basal Activated Committed Repressed Basal

Sensitivity to MIH: High High Low Low High
Limb regeneration (R Index): 0 - 10 11 - 15 16 - 23 23

Fig. 3. Hormonal regulation of molting in the blackback land crab, Gecarcinus lateralis. Diagram shows the relationship between molt stage, YO state, YO sensitivity to MIH,
limb regeneration (R index), YO ecdysteroid synthetic capacity, and hemolymph ecdysteroid titer. During postmolt (A, B, C1–3), intermolt (C4), early premolt (D0), and mid
premolt (D1,2), hemolymph ecdysteroid titers are correlated with YO synthetic capacity; during late premolt (D3,4), high ecdysteroid represses YO ecdysteroidogenesis and
ecdysteroid titer falls. The YO transitions through four physiological states during the molt cycle: basal (B), activated (A), committed (C), and repressed (R). The B to A
transition is triggered by a reduction in MIH; the YOs hypertrophy, but remain sensitive to MIH, as premolt is suspended by MIH injection or by limb bud autotomy (LBA). At
the A to C transition, the animal becomes committed to molt, as the YO is less sensitive to MIH and premolt is not suspended by LBA; this transition may be triggered by an
increase in MIH or an unidentified tropic factor. At the C to R transition, YO ecdysteroid synthetic capacity remains high, but high hemolymph ecdysteroid titer inhibits
ecdysteroid secretion. Molting, or ecdysis (E), marks the R to B transition, during which the YO atrophies and becomes sensitive to MIH.

At the transcriptional level, Phantom (Phm), a key enzyme in the
ecdysteroid biosynthetic pathway, is up-regulated during premolt;
it encodes a cytochrome P-450 mono-oxygenase that hydroxylates
ecdysteroid precursors at the #25 carbon (see [83], for review). In
shrimp, Marsupenaeus japonicus, Phm expression in the YO in-
creases as much as 7-fold during premolt and is decreased about
2.5-fold by sinus gland extract and recombinant MIH [3].
Translational regulation is also involved in YO activation. In the
C. antennarius YO, the translation inhibitor cycloheximide inhibits
both protein synthesis and ecdysteroidogenesis [75]. By contrast,
the transcriptional inhibitor actinomycin D inhibits RNA synthesis
without having any effect on ecdysteroidogenesis [75]. A potential
target of MIH signaling is metazoan target of rapamycin (mTOR),
which functions as a nutrient sensor for cellular growth in animals
(see [94], for review). In insects, mTOR stimulates ecdysteroido-
genesis of the prothoracic gland. Nutrients and insulin-like pep-
tides activate mTOR, which phosphorylates components of the
protein synthetic machinery, such as p70-S6 kinase and eIF4E-
binding protein, to increase translation of mRNA (see [116], for re-
view). Ecdysteroidogenesis in the prothoracic gland of Bombyx mori
is stimulated by insulin [45]. In Drosophila melanogaster, the mTOR
pathway controls prothoracic gland size and ecdysteroidogenic
capacity [28,65,77]. mTOR activity may be required for full ecdys-
teroidogenesis in the crustacean YO, as rapamycin, an mTOR inhib-
itor, is a potent inhibitor of ecdysteroid secretion by G. lateralis and
C. maenas YOs in vitro [95].
A hallmark feature of the ‘‘committed’’ YO is that it becomes
refractory to MIH and CHH. In C. maenas and crayfish (Procambarus
clarkii), the sensitivity to MIH declines during premolt and is least
sensitive to MIH by the end of mid premolt and late premolt
[24,88,89]. As neither MIH nor CHH binding to high-affinity recep-
tors is reduced during premolt [124], the reduction in sensitivity
likely results from a down-regulation of signaling components
‘‘downstream’’ from the receptors. Increased phosphodiesterase
(PDE) activity contributes to the reduced response to MIH by keep-
ing intracellular cyclic nucleotides low [89]. However, the magni-
tude of the increase in PDE activity (2.5-fold) is less than that
of the decrease in MIH sensitivity (10-fold), suggesting that other
components of the MIH signaling pathway, such as NOS and GC-I,
are down-regulated. In G. lateralis and C. maenas YOs, NOS and GC-
Ib mRNA levels are increased by eyestalk ablation, indicating that
the expression of these genes is responsive to an acute withdrawal
of MIH and other neuropeptides [68,76].
The regulation of the transition from the activated to the com-
mitted state is largely unknown. It may be triggered by an increase
in MIH (see [32], for review). However, other autocrine/paracrine
factors may be involved. Members of the transforming growth fac-
tor-b (TGFb) superfamily of cytokines are potential candidates.
They are grouped into the TGFb/Activin/Nodal and BMP/GDF/MIS
families (see [71], for review). TGFb activates Smad transcription
factors, which regulate gene expression through transcriptional
activation or repression (see [79,118], for reviews). The only
TGFb/Activin/Nodal members that have been characterized in
decapods are myostatin (Mstn)-like factors; cDNAs have been
cloned from G. lateralis, C. maenas, and other decapods
[33,61,62,72] and may play a role in muscle protein turnover
[30]. Sequences encoding a TGFb factor distinct from Mstn are re-
ported for lobster and shrimp in the GenBank database (Accession
Nos. CN854252 and CAA72411). We hypothesize that a factor like
TGFb is synthesized as an autocrine factor during early premolt and
328 E.S. Chang, D.L. Mykles / General and Comparative Endocrinology 172 (2011) 323–330
alters gene expression in the YO by down-regulating MIH signaling
components and up-regulating mTOR signaling components and
ecdysteroid synthetic enzymes, such as Phm. This transitions the
YO to the committed state. The animal is now committed to molt,
as interventions (e.g., LBA or MIH injections) fail to delay molting
[44,49,88,91].
Repression of the YO in late premolt involves a negative feed-
back inhibition by ecdysteroids. At the end of premolt there is a
sudden drop in hemolymph ecdysteroids within a few days of
ecdysis [105]. This drop appears to determine the timing of ecdysis,
as artificially elevated ecdysteroid during late premolt delays ecdy-
sis [23,42]. It results from an increase in ecdysteroid excretion and
a decrease in YO ecdysteroid production [20,83,108–110,112]. 20E
inhibits YO ecdysteroidogenesis when injected into crayfish [35].
RH-5849 (1,2-dibenzoyl-1-tert-butylhydrazine), a non-steroidal
ecdysteroid agonist that mimics the effects of 20E in insects
[127], inhibits ecdysteroid secretion by crayfish YOs in vitro [35].
Both treatments produce significant reductions in ecdysteroido-
genesis within 1 h [35], suggesting a non-genomic response
mediated by G protein-coupled and/or membrane-associated
ecdysteroid receptors [54,98,115,117]. This inhibition lasts at least
24 h after a single 20E injection [35], which suggests that ecdyster-
oid may also affect gene expression.
4. Future research
For many years, crustacean endocrinologists were guided by a
very simple model, in which molting is regulated solely by the le-
vel of MIH circulating in the hemolymph. If this were so, then MIH
levels should be high in intermolt and postmolt and low in pre-
molt. However, measurements of MIH in the hemolymph during
the molt cycle do not show this pattern. During intermolt MIH le-
vel is not always high, and during mid- to late-premolt MIH levels
can be higher than that during intermolt [25,88]. This indicates
that the hormonal control of molting is more complex. Any model
must accommodate the dynamic changes in the YO during the
molt cycle. The release of MIH from the X-organ/sinus gland com-
plex is episodic rather than continuous, resulting in relatively low
average MIH concentrations in the hemolymph [25]. As brief expo-
sures of YOs to MIH have lasting effects on ecdysteroidogenesis
[101,111], it is thought that periodic releases of MIH are sufficient
to keep the YO inactive during intermolt [25]. YOs from late pre-
molt (stage D2–D3) animals are refractory to MIH [24,88,89], which
may result, at least in part, from increased degradation of cyclic
nucleotides by PDEs [89]. Ecdysteroids inhibit YO ecdysteroid
secretion, thus contributing to the rapid decrease in hemolymph
ecdysteroids at the end of premolt [35].
Future research should be directed toward understanding the
signaling mechanisms underlying the transitions between physio-
logical states. A biosystems approach is needed to fully understand
the regulation of the YO at transcriptional, translational, and post-
translational levels, and how these interact with environmental
signals [70,85]. High throughput sequencing and mass spectrome-
try technologies now make it possible to obtain complete tran-
scriptome and proteomic databases of the YO and to quantify
mRNA levels and protein levels and posttranslational modifications
at critical transitions during the molt cycle (Fig. 3). A complete
transcriptome will facilitate identification of proteins and phos-
phorylation sites by mass spectrometry. We expect that this anal-
ysis will identify genes in the MIH, mTOR, and TGFb signaling
pathways, as well as identify other genes important in YO regula-
tion and function (e.g., Phm and other Halloween genes [83]). It
may also identify candidate genes encoding the MIH receptor.
Acknowledgments
The authors are grateful to Howard Bern for his mentorship,
beginning when EC was an undergraduate student and DM was a
graduate student at UC Berkeley. DM acknowledges the unfailing
guidance and generous support of Dorothy Skinner until her death
in 2005. Her discoveries in crustacean molting biology are a lasting
legacy. The authors are grateful to the many students, technicians,
postdoctoral fellows, and visiting scientists who contributed to this
work over the last 25 years. The authors thank the Editors for the
invitation to participate in this project and Sharon Chang for edito-
rial assistance. Finally, the authors acknowledge the support of
grants from the California and Connecticut Sea Grant College Pro-
grams, the National Institutes of Health, and the National Science
Foundation.
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