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Hemolymph ecdysteroids during the last three molt cycles of the blue crab Callinectes sapidusquantitative and qualitative analyses and regulation.

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A r t i c l e
Callinectes sapidus:
J. Sook Chung
Center of Marine Biotechnology, University of Maryland Biotechnology
Institute, Baltimore, MD, USA
The profiles of circulating ecdysteroids during the three molt cycles prior
to adulthood were monitored from the juvenile blue crab, Callinectes
sapidus. Ecdysteroid patterns are remarkably similar in terms of peak
concentrations ranging between 210–330 ng/ml hemolymph. Analysis of
hemolymph at late premolt stage revealed six different types of
ecdysteroids with ponasterone A (PoA) and 20-OH ecdysone (20-OH E)
as the major forms. This ecdysteroid profile was consistent in all three
molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes
inhibitory neurohormones including crustacean hyperglycemic hormone
(CHH) and molt-inhibiting hormone (MIH) and often results in
precocious molting in crustaceans. However, the inhibitory roles of these
neuropeptides in vivo have not yet been tested in C. sapidus. We
determined the regulatory roles of CHH and MIH in the circulating
ecdysteroid from ablated animals through daily injection. A daily
administration of purified native CHH and MIH at physiological
concentration maintained intermolt levels of ecdysteroids in the EA
animals. This suggests that Y organs (YO) require a brief exposure to
CHH and MIH in order to maintain the low level of ecdysteroids.
Compared to intact animals, the EA crabs did not exhibit the level of peak
ecdysteroids, and the major ecdysteroid turned out to be 20-OH E, not
Grant sponsor: NOAA Chesapeake Bay Office (to the Blue Crab Advanced Research Consortium); Grant number:
NA17FU2841; Grant sponsor: Binational Agricultural Research and Development (BARD) UMBI-Israel
program; Grant number: MB-8714-08.
Correspondence to: J. Sook Chung, Center of Marine Biotechnology, University of Maryland Biotechnology
Institute, 701 E. Pratt Street, Suite 236, Baltimore, MD 21202. E-mail:
Published online in Wiley InterScience (
& 2009 Wiley Periodicals, Inc. DOI: 10.1002/arch.20327
Archives of Insect Biochemistry and Physiology, January 2010
PoA. These results further underscore the important actions of MIH and
CHH in ecdysteroidogenesis, as they not only inhibit, but also control the
C 2009 Wiley Periodicals, Inc.
composition of output of the YO activity. Keywords: ecdysteroids; in vivo administration; molt-inhibiting hormone;
crustacean hyperglycemic hormone; eyestalk ablation
Animals belonging to the phylum Arthropoda require periodic shedding of the
exoskeleton for somatic growth. Each species experiences a fixed number of molt
processes during its life history. In the case of Callinectes sapidus, animals undergo
27–29 molts from hatching to reaching adulthood (Churchill, 1919). Within a molt
cycle, animals spend the longest duration in intermolt, premolt, postmolt, and ecdysis
in order, the last of which only lasts several hours. Molting, growth, and development
in arthropods are generally regulated by ecdysteroids (molting hormones). In
crustaceans, paired Y-organs (YO), the equivalent of prothoracic glands in insects
that are located in the anterior cephalothorax, are responsible for the synthesis of
ecdysteroids (Skinner, 1985).
The activity of YO in crustaceans is known to be negatively regulated by inhibitory
neurohormones such as molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). These hormones are produced in the neurosecretory cells
located in the medulla terminalis X-organ (MTXO), transported, and released from
the sinus gland (SG) into the hemolymph. Due to the MTXO-SG being located within
the eyestalk, the removal of eyestalks usually results in the synthesis and release of
ecdysteroids from YO, resulting in induction of molting or precocious molting.
Therefore, it is tacitly accepted in a simplistic manner that the low level of ecdysteroids
during intermolt is controlled by a high circulating concentration of MIH, or CHH, or
both. Compared to the physiological level of these neuropeptides at 10 12 M (Chung
and Webster, 2005; Nakatsuji and Sonobe, 2004), recombinant MIH injections at a
very high micromolar level delayed the molting process (Gu et al., 2001; Okumura
et al., 2005), implying its inhibitory role in crustacean molting. However, it has never
been described in crustaceans if the injection of native MIH or CHH at a
physiologically relevant concentration suppresses the hemolymph ecdysteroids. In
contrast, there are numerous examples of the in vitro effect of MIH or CHH on the
YO by the incubation of YO in the presence of these hormones (Chung and Webster,
1996, 2003; Chung et al., 1996, 1998; Lee et al., 2007; Webster, 1991; Yang et al.,
1996; Yasuda et al., 1994).
As stated earlier, blue crabs undergo 27–29 molts prior to adulthood. In insects,
ecdysteroid patterns differ between a molt leading from larva to larva and from larva to
pupa (Margam et al., 2006; Nijhout, 1998; Sehnal et al., 1981). This indicates that there
may be a life stage–dependent specific regulation in the ecdysteroid profiles, particularly
when a molt accompanies metamorphosis. As stated above, two of the molts occurring in
C. sapidus accompany one complete molt and the other partial metamorphic molt: the
former, megalopa to the first crab (crab 1), and the latter, prepubertal to adult molt in
female C. sapidus. In view of the reports that differential ecdysteroid profiles are
reported in many insect species (Nijhout, 1998), we proposed that the blue crab may
employ a similar regulatory role of ecdysteroids, particularly at metamorphic molts. In
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Quantitative and Qualitative Analyses of Hemolymph Ecdysteroids
addition, as reported in Carcinus maenas, Homarus americanus, Menippe mercenaria
(Lachaise and Lafont, 1984; Snyder and Chang, 1991; Wang et al., 2000), it appears
that some crustacean species produce a rather complex profile of ecdysteroids in the
hemolymph. In particular, an earlier finding stated the presence of ponasterone A (PoA)
in the developing embryos of C. sapidus (McCarthy, 1979).
In the current study, we examined the profiles of ecdysteroids in the hemolymphs
of the last three molt cycles leading to adulthood: antepenultimate, juvenile to the last
juvenile stage; penultimate, the last juvenile to prepubertal stage; prepubertal to adult
of C. sapidus using an ecdysone RIA. Using a HPLC-RIA, we analyzed the composition
of ecdysteroids in hemolymphs, which was obtained from animals at premolt stages for
three molt cycles. We also tested in vivo effects of native Callinectes CHH and MIH at
physiological relevant concentrations on the concentration of circulating ecdysteroids.
Our results obtained from the analyses of profiles of ecdysteroids of the intact and EA
animals imply that these inhibitory neurohormones regulate the ecdysteroid levels,
quantitatively and qualitatively.
Female juvenile C. sapidus (20–25 mm carapace width, CW) were obtained from the
blue crab hatchery at the Aquaculture Research Center, Center of Marine
Biotechnology (University of Maryland Biotechnology Institute, Baltimore, MD).
Animals were cultured in separate compartments (15 15 cm) and fed daily with
chopped squid.
Hemolymph Collection From Normal and Eyestalk Ablated Animals
Hemolymph was collected at the same time of day, in order to avoid a possible
fluctuation over a 24-h period as observed in H. americanus (Snyder and Chang, 1991).
Hemolymph (50–100 ml) was collected from individual animals (n 5 15–20) twice a
week for 5 months during a period that spanned the last three molts: juvenile to
juvenile ( J to J) molt, juvenile to prepubertal ( J to PP) molt, and prepubertal to adult
(PP to A) molt. In order to avoid coagulation, all samples were stored in an
anticoagulant at a ratio of 1:1.
Animals undergoing J to J molt were eyestalk-ablated and hemolymphs were
sampled as described above.
Isolation, Purification, and Quantification of CHH and MIH
The methods for isolation, purification, and quantification of C. sapidus of CHH and
MIH were as described (Webster, 1993). Briefly, batches of sinus gland (50 or 100)
were extracted in ice-cold 2N acetic acid by sonication (Bronson) and centrifuged for
10 min at 14,000 rpm. The supernatant was first isolated on a RP-HPLC with a phenyl
column (4.6 250 mm, Waters) using a gradient of 30–80% B over 45 min at a flow
rate of 1.0 ml/min (A 5 0.11% trifluoroacetic acid [TFA] in H2O, B 5 0.1% TFA in 60%
acetonitrile). The peaks of CHH and MIH were collected manually and dried for
further purification. Dried neurohormones were re-run on a C18 column
(4.6 250 mm, Phenomenex) using a gradient 45–60% B over 30 min for CHH and
45 min for MIH at same flow rate. Peaks were manually collected. The quantification
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Archives of Insect Biochemistry and Physiology, January 2010
of neuropeptides was performed by amino acid analysis of hydrolysates obtained by
gas-phase hydrolysis in vacuo at 1501C for 1 h using azeotropic hydrochloric acid
containing a trace of phenol. Vacuum-dried hyrolysates were then quantified for
amino acid composition by o-phthalaldehyde pre-column derivertization.
Ecdysteroid Radioimmunoassay (Ecd-RIA)
Hemolymph samples (1–10 ml) were analyzed for total concentration of ecdysteroid
using an Ecd-RIA with ecdysone specific antiserum and [3H] PoA (Perkin Elmer) and
Ecd antiserum, as described (Chung and Webster, 1996). Ecdysone (E) served as the
standard at concentrations ranging from 2.5 ng to 30 pg/tube. The results were
analyzed using the AssayZap program (Biosoft) and EC50 value of Ecd-RIA was
100710 pg/tube (n 5 10).
Reverse Phase-High Performance Liquid Chromatography and Ecd RIA (RP-HPLC and
Ecd RIA)
Hemolymphs obtained from late premolt stages were extracted in 80% methanol.
After centrifugation at 14,000 rpm for 10 min at room temperature, the supernatants
were dried in a vacuum centrifuge ( Jouan). The dried samples were re-suspended in
water and separated on a C18 column (Gemini, 4.6 mm 150 mm, Phenomenex) that
was connected into a HPLC (Waters Alliance) under the following solvent system (A:
100% water; B: 100% methanol) at a flow rate of 1 ml/min. The ecdysteroid standards
(10–20 ng each) were run on the HPLC at the same condition as the samples: E,
20-OH E, 3-deoxyecdysone, 25-deoxyecdysone, and PoA. These fractions of samples
and standards were collected, dried, and assayed as described above. The ecdysteroids
in samples were identified according to the retention time, which was compared with
authentic standards.
Daily Administration of CHH, MIH, or Both
Both eyestalks were removed from juvenile animals (60–70 mm CW) at intermolt prior
to hormone injection. Animals received daily injections of native CHH (25 pmol) or of
native MIH (5 pmol) or both for 7 days, while controls received saline. Hemolymph
samples were withdrawn on days 2 and 7 for the quantification of ecdysteroids as
described above.
Statistical Analysis
The data obtained were analyzed using GraphPad Instat 3 program (GraphPad
Software) and were examined using the Student’s t-test or one-way ANOVA followed
by Tukey-Kramer multiple comparison test. Statistical significance was presented at
Po0.05: , and Po0.001: .
Ecdysteroid Profiles of the Last Three Molt Cycles of the Life History of C. sapidus
Total hemolymph ecdysteroids were measured in the last three molts of female
C. sapidus using an Ecd-RIA. The ecdysteroid concentrations ranged from 50–70 ng/ml
hemolymph (n 5 15, small peaks) to 209–330 ng/ml hemolymph (n 5 15, large peaks).
Archives of Insect Biochemistry and Physiology
Quantitative and Qualitative Analyses of Hemolymph Ecdysteroids
The large peak concentration of ecdysteroids was significantly different from all the
sampling points within the same molt cycle (Pr0.01). However, these peak
concentrations from three separate premolts did not differ significantly. Prior to
ecdysis (arrow marked in Fig. 1), the circulating ecdysteroids were cleared from the
hemolymph and the level dropped to the lowest (o10 ng/ml hemolymph) after molt.
Qualitative Analysis of Hemolymph Ecdysteroids Using a High-Performance Liquid
Chromatography Combined Radioimmunoassay (HPLC-RIA)
Ecdysteroids extracted in 80% methanol were separated by RP-HPLC. The elution
profile of ecdysteroid standards was as follows: 20-OH E, E, 3-dehydroecdysone, PoA,
and 25-deoxyecdysone. Hemolymphs of C. sapidus contained at least six different types
of ecdysteroids, except for the hemolymph of crabs undergoing a J to J molt that
exhibited five. In all three separate molt cycles, PoA was the major ecdysteroid in the
hemolymphs of C. sapidus, comprising 45–65% of the total ecdysteroids. However, the
highest level of PoA (6873.4%, n 5 5) was determined at the J to J molt versus those of
J to PP and PP to A molts: 52.371.9 % (n 5 5) and 48.874.3% (n 5 5), respectively.
20-OH E was the second major ecdysteroid in all crab molt cycles, comprising 12–25%
of the total ecdysteroids. As opposed to PoA, the levels of 20-OH E were significantly
higher in the J to PP and PP to A molts: 24.670.4% (n 5 8) and 21.571.5 (n 5 5),
respectively versus that of J to J molt: 12.870.5% (n 5 8).
Ecdysone and 25-deoxyecdysone, precursors of 20-OH E and PoA, respectively,
were detected at similar levels, 5%, in all three molt cycles. Interestingly,
3-dehydroecdysone was detected during J to PP and PP to A molts, but not during
J to J molt (Fig. 2). A small amount of an unknown ecdysone present in all three molt
cycles eluted between 3-dehydroecdysone and PoA.
Effects of Daily Administration of Native CHH, MIH, or Both on Ecdysteroid Levels in
To determine the in vivo effect of neurohormones on circulating ecdysteroids,
eyestalks were ablated and crabs were injected with native CHH (25 pmol), MIH
(5 pmol), or both. Control animals received saline alone. A hemolymph sample was
removed prior to and 3 and 7 days post EA. Hemolymph ecdysteroid levels of control
crabs increased significantly (1872.7 (n 5 6) to 45.876.2 (n 5 6) ng/ml, Po0.05) on
sampling day 3, and continued to increase to 83.579.9 (n 5 6) ng/ml hemolymph on
day 7 (Fig. 3).
The initial level of ecdysteroids in groups injected with hormones was higher than
that of control (1872.7, n 5 6) as shown above, ranging from 37.5715.2 (CHH
injected, n 5 6) to 48.0712.0 ng/ml hemolymph (n 5 6) for both hormones injected.
However, the continual daily injections of CHH, MIH, or both maintained ecdysteroid
concentration at low intermolt levels with no significant change during the duration of
the experiment.
Effect of Eyestalk Ablation on the Levels of Hemolymph Ecdysteroids
Hemolymph samples were collected to determine the effect of EA on ecdysteroid levels
in the animals that would undergo a J to J molt. The patterns of ecdysteroid
concentration were compared to those of normal animals as shown in Figure 1. The
results presented in Figures 1 and 4 contained two significant differences between
normal and EA marked as ‘‘1’’: the absence of an ecdysteroid peak and the lack of a
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Archives of Insect Biochemistry and Physiology, January 2010
Figure 1. Ecdysteroid profiles in hemolymphs during last three molt cycles of the life history of C. sapidus.
The levels of ecdysteroids (ng/ml hemolymph) were presented as mean71SE (n 5 15–20 for J to J, 15–20 for
J to PP, and 15–17 for PP to A). Arrow indicates the time of ecdysis. Closed circle: juvenile to juvenile molt;
closed square: juvenile to prepubertal molt; closed triangle: prepubertal to adult molt. Arrow: time of
ecdysis. Peak concentration of ecdysteroids did not differ in all three molts, noted as ‘‘a’’; Pr0.001.
Figure 2. Analyses of total hemolymph ecdysteroids using a RP-HPLC and Ecd RIA. Six different
ecdysteroids were determined in the hemolymph obtained from animals at late premolt stage. Results were
presented as mean71SE (n 5 5). Black bar: juvenile to juvenile molt, light grey bar: juvenile to prepubertal
molt; dark grey bar: prepubertal to adult molt. Letters show statistical significance at Pr0.05.
post peak of ecdysteroid clearance in hemolymph of the ablated crabs. Circulating
ecdysteroids increased after the eyestalk removal (marked ), but only reached a
concentration of 70 ng/ml hemolymph. Surprisingly, the ecdysteroids in hemolymph
of the ablated animals remained significantly high at 50–60 ng/ml hemolymph at
ecdysis and postmolt, compared to those in control animals.
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Quantitative and Qualitative Analyses of Hemolymph Ecdysteroids
Figure 3. Effects of daily administrations of 25 pmol of native CHH, 5 pmol of native MIH, or both on the
concentration of circulating ecdysteroids of the EA animals. Controls received saline alone and hormones
were injected daily for 7 days. Bar graph is shown as mean71SE (n 5 6). Black bar: day 0; light grey bar:
day 2; dark grey bar: day 7. Statistical significance was analyzed using ANOVA followed by Tukey-Kramer
multiple comparison. No statistical significance was found in any hormone-injected groups (CHH, MIH, or
Figure 4. Profiles of circulating ecdysteroids during the molt cycle of intact (n 5 15) and EA animals
(n 5 8). Each point represents as mean71 SE. Total ecdysteroids in hemolymph of animals undergoing
juvenile to juvenile molt. Closed circle: intact animals; open circle: EA animals. : time of ablation. Arrows:
time of molt. Statistical significance was marked as ‘‘1’’ at Pr0.05.
Qualitative Secretion of Ecdysteroids in the EA Animals
The differential secretion of ecdysteroids in EA animals was determined during late
premolt and is shown in Figure 5. The proportion of ecdysteroids was significantly
different between EA and intact animals. In contrast to results shown in Figure 2 that
were obtained from intact animals, 20-OH E was the major ecdysteroid in EA animals
and was 4-fold higher than the amount measured in intact animals, thus comprising
over 55.079.5% (n 5 5) of total ecdysteroids. This was followed by PoA, of 34.378.3%
(n 5 5), which was only 50% in the intact animals. E and 25 deoxyecdysone in the EA
animals were also lower than intact animals. It was also noted that the EA animals had
3-dehydroecdysone in the total ecdysteroids.
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Archives of Insect Biochemistry and Physiology, January 2010
Figure 5. Quantitative analyses of the circulating ecdysteroid of the intact and EA animals using a RPHPLC and Ecd RIA. The data are presented as mean71 SE (n 5 6). Black bar: intact animals; white bar: EA
animals. Pr0.001; Pr0.05.
The current study describes the quantitative and qualitative profiles of ecdysteroids in
hemolymph obtained from female C. sapidus undergoing antepenultimate, penultimate, and final molt. In all molts, our results show a similar concentration of
ecdysteroids ranging between 209–330 ng/ml hemolymph consisting of at least six
different ecdysteroids. PoA as the major ecdysteroid is followed by 20-OH E. For the
first time, we demonstrate that injections of native CHH, MIH, or both inhibit levels of
circulating ecdysteroids from the EA animals, thereby probably suppressing molt
induction. Bilateral EA causes changes in the composition and quantity of the total
ecdysteroids, which implies the selective specific inhibition of CHH and MIH on the
activity of YO.
The concentrations of the total ecdysteroids in hemolymph at premolt stage vary
widely in different crustacean species, while these concentrations may differ
during the life stages of an animal. In C. sapidus, animals were monitored for
hemolymph ecdysteroid through the three successive molts, leading to adulthood.
The highest level of ecdysteroids is comparable to that reported earlier
(Lee et al., 1998) and was maintained at a similar level in all three molts. This
consistency over three different molts in C. sapidus contrasts with a result reported in
Chionoecetes opilio. In C. opilio, known to undergo a terminal molt, the circulating
ecdysteroid was over 20 times higher in juveniles than in adult males and females at
2–4 ng/ml hemolymph (Tamone et al., 2005). The adult molt in female C. sapidus is
considered to be a terminal molt, as they cease molting upon maturity for
reproduction. The level of ecdysteroids in these adult females at intermolt was
maintained at 45 ng/ml hemolymph lower than those of juveniles and prepubertal
crabs at intermolt. The result that adult females at intermolt had lower ecdysteroids
than juveniles at intermolt is congruent with previously reported results (Soumoff and
Skinner, 1983).
Molting results in an incremental increase in carapace width (20–50%) with a
proportional increase in hemolymph volume. Animals at the last molt (PP to adult) are
at least twice as large as animals undergoing the first molt ( J to J), assuming a 40% molt
Archives of Insect Biochemistry and Physiology
Quantitative and Qualitative Analyses of Hemolymph Ecdysteroids
increment at each molt. Therefore, it is pertinent to suggest that the constant peak
concentration of ecdysteroids shown in Figure 1 resulted from the activity of YO
proportionally increasing in order to maintain a similar level of circulating
The activity of YO is cyclic: low at intermolt stage but high during premolt.
However, the mechanism (s) regulating such activity is not clearly defined, compared
to the current information in insects. The increase in YO activity is generally believed
to be due to the absence or low level of circulating MIH through a negative feedback of
ecdysteroids on the eyestalk (Mattson and Spaziani, 1986). On the contrary, the
injection of ecdysteroids reduced the activity of YO (Dell et al., 1999), perhaps through
induction of ecdysone metabolic enzyme (Williams et al., 1997). This result suggests a
possible feedback mechanism that activates the clearance of ecdysteroids from
hemolymph prior to ecdysis.
Profiles of ecdysteroid compositions appear to be complex. Most crustacean
species and some insects carry several different ecdysteroids in hemolymph, some as
active forms, while others are inactive and metabolites (Cuzin-Roudy et al., 1989;
Lachaise and Lafont, 1984; Snyder and Chang, 1991; Wang et al., 2000). In C. sapidus,
six different ecdysteroids are consistently present in the three molts. This suggests that
C. sapidus YO, as in other crustacean species, are capable of producing three different
ecdysteroids: E, 3-dehydroecdysone, and 25-deoxyecdysone (Subramoniam, 2000).
Similarly, the peripheral tissues of C. sapidus may convert into 20-OH E and PoA.
It appears in some decapod crustaceans that PoA is the major form of ecdysteroid,
while in H. americanus it is present as a minor form, compared to 20-OH E (Snyder and
Chang, 1991).
As stated above, native CHH and native MIH had been tested for their inhibitory
action on the YO in vitro (Chung and Webster, 1996, 2003; Chung et al., 1996, 1998).
In contrast, only one study of rMIH showed the delayed molting in vivo after the
injection of a high concentration of rMIH at 10 5–10 4 M (Okumura et al., 2005). Our
results demonstrated that daily administration of CHH, MIH, or both at physiologically relevant concentrations of 10 9–10 10 M and at the same time of day suppressed
circulating ecdysteroids from the EA animals, whereas those animals receiving saline
had significantly higher levels of circulating ecdysteroids. As found in C. maenas
(Chung and Webster, 2003), C. sapidus CHH also retains the role of MIH (unpublished
observation). Overall, it appears that one brief exposure to these hormones is enough
for the inhibition of YO activity for a 24-h period, despite the rapid clearance and short
half-lives of these hormones (Chung and Webster, 2003, 2008). However, it remains to
be determined if there is a specific regulatory role of MIH and CHH on
ecdysteroidogenesis in YO, which may result in a difference in the profile of
Our results showed significant differences in the peak concentration and
composition of ecdysteroids in the EA animals. The different effects of EA on
ecdysteroid levels are reported as either no effect (Keller and Schmid, 1979) or
suppression of ecdysteroid peak (Cuzin-Roudy et al., 1989). Our results obtained from
the ablated C. sapidus are congruent with the latter case in which the ecdysteroid peak
was absent, but ecdysteroids were present in hemolymph. It is generally suggested that
EA animals show elevated levels of ecdysteroids immediately after bilateral ablation;
hence bilateral EA induces molting. However, it was observed that the effect of EA of
molt interval is less at the molt immediately following ablation than at subsequent
molts (Snyder and Chang, 1986).
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Archives of Insect Biochemistry and Physiology, January 2010
As reported in H. americanus, C. sapidus exhibited a similar pattern of molt after EA
in that successive molts after ablation shorten the molt interval more dramatically than
the immediate molt after ablation. This phenomenon may be explained by the
persistent presence of relatively high levels of ecdysteroids, which may also contribute
to the larger molt increment of ablated animals, but at a poor success rate of ecdysis.
The clearance of ecdysteroids in hemolymph is required for normal and successful
ecdysis as it was notable in three different molts as shown in Figure 1 and a similar
finding was reported in H. americanus that prolonged presence of ecdysteroids by
injection caused delayed molting in these animals (Cheng and Chang, 1991).
Moreover, the major composition of total ecdysteroids in the ablated animals is
distinguishable from that of normal animals, as 20-OH E presents as the major
ecdysone in the EA animals.
Eyestalk ablated animals often exhibited a larger molt increment than
demonstrated in intact animals but at a poor rate of completion of ecdysis
(Burnette et al., 2003; Gross and Knowlton, 1999, 2002; Terwilliger et al., 2005).
Ablation caused incomplete metamorphosis, especially in metamorphic molts (Gross
and Knowlton, 2002; Snyder and Chang, 1986). The mechanism of ecdysis of
crustaceans is closely associated with the expression and release of gut CHH
during premolt and ecdysis (Chung et al., 1999; Webster et al., 2000), as the
presence of CHH in the hemolymph accelerates drinking for the water uptake
required in the swelling process. In vivo injection studies provide additional
evidence that the amount of circulating CHH during ecdysis is molt increment
through increase in water uptake (Chung et al., 1999). Particularly, the pattern
of gut CHH expression during premolt implies that CHH may be induced by the high
level of circulating ecdysteroids at premolt (unpublished observation). Thus, it is
reasonable to suggest that the differences in the ecdysteroid profiles of the ablated
animals may affect the expression level of CHH in the gut endocrine cells during
premolt, resulting in a larger molt increment at a poorer completion rate than normal
Although the pathway of ecdysteroidogenesis is not completely understood, the
last three steps of the process are a series of sequential hydroxylations at C-25, C-22,
and C2 of 2, 22, 25-trideoxyecdysone, and the very first step of cholesterol conversion
to 7-dehyrocholesterol is well established (Grieneisen, 1994; Kappler et al., 1988). In
locusts, incubation of 3-dehyroecdysone in hemolymph samples resulted in its
conversion into E (Roussel, 1992). The conversion of E to 20-OH E is carried out
through a gene product of Shade in Drosophila (Petryk et al., 2003), while that of 25
deoxyecdysone to PoA is carried out by another gene product, Phantom (Warren et al.,
2004). PoA exhibited a higher affinity for the ecdysteroid receptor than 20-OH E in
insects (Sonobe and Yamada, 2004), implying it may be a bioactive molting hormone.
However, it remains to be determined if PoA is an active molting hormone in many
crustacean species including C. sapidus.
Nevertheless, the differences in the major ecdysone between the intact and EA
animals are notable. The inhibitory neurohormones regulate the circulating
ecdysteroids both quantitatively and qualitatively perhaps at a molt stage–specific
manner, as suggested in C. maenas (Lachaise et al., 1988; Saidi et al., 1994).
Furthermore, these results indicate that CHH and MIH may have regulatory roles in
biosynthesis and the metabolism of ecdysteroids, resulting in the changes in the
composition of ecdysteroids as well as their clearance from hemolymph of the ablated
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Quantitative and Qualitative Analyses of Hemolymph Ecdysteroids
The author is indebted to O. Zmora for the juvenile C. sapidus, Professor E. Chang
(Bodega Bay Marine Laboratory, University of California, Davis) for ecdysone
antiserum, and Dr. S. G. Webster (Bangor University) for the standard ecdysone.
This article is contribution no. 08-198 of the Center of Marine Biotechnology
(University of Maryland Biotechnology Institute, Baltimore, MD) and the work is
supported by a program grant (NA17FU2841) from NOAA Chesapeake Bay Office to
the Blue Crab Advanced Research Consortium and by a Binational Agricultural
Research and Development (BARD) UMBI-Israel program MB-8714-08
Burnette J, Broders S, Quackenbush S. 2003. Growth in eyestalk ablated juvenile Callinectes
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