The use of recombinant protein and RNA interference
approaches to study the reproductive functions of a
gonad-stimulating hormone from the shrimp
Metapenaeus ensis
Shirley Hiu-Kwan Tiu and Siu-Ming Chan
Department of Zoology, The University of Hong Kong, China
Neurosecretory structures in crustacean eyestalks are
known to produce the crustacean hyperglycemic hor-
mone (CHH), molt-inhibiting hormone (MIH) and
gonad-inhibiting hormone (GIH) of the CHH ⁄ MIH ⁄
GIH gene family. These neuropeptides can regulate a
variety of physiologic processes, including molting,
carbohydrate metabolism, and reproduction [1–3].
GIH is one of the most studied neuropeptides of this
group because of its potential importance in shrimp
aquaculture. In penaeid shrimp, GIH is produced in
the X-organs and stored in the sinus glands of eye-
stalks [4–7]. Although the precise mechanism is not
known, GIH is postulated to inhibit reproduction by
suppressing ovary growth or vitellogenesis [1,2]. Eye-
stalk ablation removes the source of GIH and results
in ovary growth. In contrast, when eyestalk-ablated
females were injected with eyestalk extract, the gonad
stimulatory effect of eyestalk ablation was abolished
[1,2]. In addition to GIH, a factor found in the brain
and thoracic ganglion of decapod has been implicated
Keywords
eyestalk neuropeptide hormone; RNA
interference; shrimp; vitellogenin gene
Correspondence
S M. Chan, Department of Zoology, The

Abbreviations
CHH, crustacean hyperglycemic hormone; GIH, gonad-inhibiting hormone; GSI, gonadosomatic index; MeVg1, Metapenaeus ensis
vitellogenin gene 1; MIH, molt-inhibiting hormone; RNAi, RNA interference; si, small interfering.
FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS 4385
in the stimulation of gonad maturation. Injection of
protein extract from thoracic ganglion or the brain can
stimulate gonad maturation [8].
In sand shrimp Metapenaeus ensis, two forms of
MIH-like cDNA (i.e. MeMIH-A and MeMIH-B) have
been cloned and characterized [4,5]. MeMIH-B shows
only 68% amino acid s imil arity to M eMI H-A, and
amino acid sequence alignment ind icates that M eMIH-B
is more closely related to GIH of the lobster Homarus
americanus [9] than to the mandibular organ-inhibiting
hormones of the crab Cancer pagarus [10]. MeMIH-A
and MeMIH-B are non-sex-specific and are expressed
in the eyestalks of males and females. The expression
of MeMIH-A is molt-stage-related, whereas the expres-
sion of MeMIH-B is correlated with the reproductive
cycle. In addition to the eyestalk, MeMIH-B is also
expressed in the brain [4]. MeMIH-B transcript level is
low in the initial phase of gonad maturation and
increases towards the end of maturation [4]. These
findings suggest that the two neuropeptides should
have different functions. As they share relatively high
sequence similarity, cross-bioactivity also occurs for
these two neuropeptides [4]. For example, injection of
recombinant MeMIH-B also delays the process of
molting [4,11]. At the time when we had characterized
MeMIH-B, only a few CHH type II neuropeptides

in vitro explant culture system and an RNAi technique
to demonstrate the reproductive function of MeMIH-B
in M. ensis.
Results
Expression of MeMIH-B in shrimp
Although we have previously studied the tissue distri-
bution of MeMIH-B in the female shrimp, the expres-
sion pattern of MeMIH-B in the central nervous
system of different reproductive stages has not
been fully investigated. Moreover, to ascertain that
MeMIH-B expression pattern is correlated with repro-
ductive developmental stages in females, we have rein-
vestigated the expression pattern of MeMIH-B in the
eyestalks and other nervous tissues of the adult females
by northern blot analysis. MeMIH-B transcripts could
be detected in the eyestalk, nerve cord, thoracic gan-
glion and brain of shrimp at early to middle stages of
gonad maturation (Fig. 1A). In female eyestalks,
MeMIH-B transcript level was low in immature
shrimp with low gonadosomatic index (i.e. GSI < 2).
As gonad development was in progress, a steady
increase in MeMIH-B transcript level was observed.
Similarly, the expression pattern of MeMIH-B in the
thoracic ganglia also followed that of the eyestalk
(Fig. 1B). For example, in both eyestalk and thoracic
ganglion, the highest MeMIH-B transcript level was
recorded at the late maturation stage in shrimp with
GSI ¼ 10. Similar to the previous results, expression
of MeMIH-B is sex-nonspecific, as the males also
expressed MeMIH-B (Fig. 1B).

the overall expression of vitellogenin in the hepatopan-
creas and ovary (Fig. 3A,B). In contrast, injection
of 6.6 nmol of rMeMIH-B stimulated an increase
(2–3-fold) in MeVg1 expression by the hepatopancreas
and ovary (Figs 3A,B) at 72 h, but only weakly for the
24 h time point (data not shown).
It is well accepted that the vitellogenin produced in
the hepatopancreas serves as an extraovarian source
for the final synthesis of vitellin. The newly made vitel-
logenin is expected to be secreted rapidly into the
hemolymph and transported to the ovary for oocyte
uptake. To demonstrate that the increase in expression
of the MeVg1 gene could also result in the appearance
of vitellogenin in the hemolymph for transport, we
also collected hemolymph samples of these injected
shrimp and analyzed the increase in vitellogenin-spe-
cific protein. As shown in Fig. 3C,D, when females
were injected with rMeMIH-B (i.e. 6.6 nmol), the
hemolymph and ovaries of most animals contained a
much higher level of vitellogenin (i.e. 148 kDa)
(Fig. 3C, left panel). These vitellogenin-specific pro-
teins are presumably derived from the translation of
the MeVg1 gene from the hepatopancreas after
rMeMIH-B stimulation. The results from SDS ⁄ PAGE
and western blot analysis of the hemolymph and
ovarian proteins from shrimp injected with 6.6 nmol of
rMeMIH-B demonstrated an increase in the overall
Es Br Tg Vn Hp Mu Ov
noisserpxe evitaleR
1.0

racic ganglia during the gonad maturation cycle. Each lane repre-
sents an RNA sample from the eyestalk or the thoracic ganglion of
one shrimp. The last lane shows the RNA samples from a male.
The bar indicates the SE.
Relative expressionRelative expression
1.4
B
A
1.2
1.0
0.8
0.6
0.4
0.2
0
Concentrations of rMeMIH-B
Concentrations of rMeMIH-B
*
*
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Fig. 2. Histogram showing the relative expression levels of MeVg1
in (A) hepatopancreas and (B) ovary explants after exposure to dif-
ferent concentrations (i.e. from 0.3 p

0
2
4
6
8
12
10
0
0.5
1
1.5
2
2.5
3
Ctrl rMeMIH-A rMeMIH-B
Ctrl rMeMIH-A rMeMIH-B
148 kDa
Hycn
148 kDa
NSP
MeVg1
rRNA
MeVg1
rRNA
Me MIH-Actrl Me MIH-B
treatment
Me MIH-Actrl
Me MIH-B
treatment
Me MIH-Actrl

nin in ovary of shrimp at 48 h after injection of NaCl ⁄ P
i
, MIH-A, and rMIH-B. Right: western blot detection (upper) of vitellogenin (148 kDa)
in ovary of shrimp injected with rMIH-B. NSP is the nonspecific protein unrelated to vitellogenin of the ovary samples. In the northern blot
(or western blot) analysis, each lane represents RNA (or protein) samples collected from individual shrimps.
Functional study of crustacean neuropeptide S. H K. Tiu and S M. Chan
4388 FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS
vitellogenin-specific protein (Fig. 3C,D). Unlike the
rMeMIH-B-injected group, shrimp injected with
rMeMIH-A (6.6 nmol) did not show any changes in
the overall MeVg1 transcript level in the hepatopan-
creas or a significant increase in MeVg1 protein level
in the hemolymph and ovary (Fig. 3A–D).
Inhibition of vitellogenin expression after RNAi
We have performed preliminary experiments using a
nonspecific dsRNA (from Tiger frog virus), and the
results show no effect on MeMIH-B gene silencing
(data not shown). In the following study, individual
shrimp (N ¼ 40; average GSI < 3) were injected with
3 lg of dsRNA for MeMIH-B, and RNA samples were
collected after 24, 48, 72, 96 and 120 h. Northern blot
results from the eyestalk (Fig. 4A) indicated no signifi-
cant reduction in MeMIH-B transcript level at all time
points. However, when we used RT-PCR to analyze
the same samples, a significant reduction of MeMIH-B
transcript was observed (Fig. 4B). In fact, by RT-PCR,
the MeMIH-B dsRNA appeared to knock down most
of the transcripts after 72 h of treatment (Fig. 4B). In
addition, hybridization signals representing small-size
RNAs were strong and persisted from 24 to 120 h after

nin remained relatively constant. However, only minute
quantities of vitellogenin subunits (i.e. 148, 97 and
78 kDa) were detected in the ovaries of the dsRNA-
injected females (Fig. 7B). These proteins were immuno-
reactive to the antibody to vitellogenin of M. ensis [27].
B-HIMeM
β nitca-
0 24 48 72 96 120
0 24487296120
+-+-+-+-+-Ctr
120967248240
Relative change in
transcript level
100
80
60
40
20
Time after injection (h)
Time after injection (h)
AB
Fig. 4. Effects of MeMIH-B RNAi in eyestalk of female shrimp. (A) Northern blot detection of eyestalk MeMIH-B transcript level in control (–)
and dsRNA-injected (+) females from animals at different time intervals (i.e. 0, 24, 48, 72, 96 and 120 h); the arrow indicates the MIH-B
transcript, and the smear indicates the residual dsRNA. (B) Top panel: RT-PCR detection of MeMIH-B gene knockdown using MIH-B-specific
primers. Lower panel: relative change in MIH-B transcript level at different time intervals. The bar diagram indicates the relative transcript level
of MeMIH-B after normalization with b-actin gene.
S. H K. Tiu and S M. Chan Functional study of crustacean neuropeptide
FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS 4389
Discussion
Structure–function research on crustacean eyestalk

72h48h24h
72h48h24h
1g
V
eM
ANR
r
yravoninoisserpxe1gVeM
% decrease in MeVg I
transcript level
% decrease in MeVg
I
transcript level
30
20
10
724824 724824
100
80
60
40
20
+-+-+-
+-+-+-
MeVg1 expression in hepatopancreas
Time (h) after injection Time (h) after injection
AB
Fig. 6. Expression of vitellogenin in hepatopancreas and ovary of dsRNA-injected (+) and control (–) females. (A) Upper: northern blot detec-
tion of hepatopancreas MeVg1 transcript level in shrimp. Lower: bar indicates the relative decrease in expression level of MeVg1. (B) Upper:
RT-PCR detection of ovary MeVg1 transcript level in shrimp. Lower: bar indicates the relative decrease in expression level of MeVg1.

cal assays using these criteria are nonspecific and
provide little information on the mechanism of GIH
regulation of reproduction. Previously, we have pro-
duced rMeMIH-B (formerly MeeMIH-B), but little
progress was made in developing a biological assay for
the recombinant protein. This is mainly attributed to
the lack of a biomarker for the reproductive process.
With the recent cloning of the vitellogenin gene in dif-
ferent crustaceans [19–23], a more precise role for GIH
can be defined with the vitellogenin as a biomarker.
Recently, there was a report on the effect of sinus
gland extract and neuropeptide on vitellogenin gene
expression in M. japonicus. In that study, the effect of
a CHH peptide and two MIH-like peptides on ovary
vitellogenin gene expression was investigated; the
results indicated that CHH causes inhibition, whereas
the MIH-like neuropeptides have no effect on vitello-
genin gene epression [23]. Unlike the CHH of M. japo-
nicus, MeMIH-B (a type II neuropeptide) has a
stimulatory effect on vitellogenin synthesis. Taken
together, the results suggest that a CHH-like (or type I)
neuropeptide may be inhibitory for gonad maturation,
321M65
4
detcejni-ANRsddetcejni-SBP
321M654
detcejni-ANRsdde
tcejni-SBP
yravOh
p

k67
Fig. 7. Western blot analysis of the hemolymph and ovary total protein of dsRNA-injected females. (A) Hemolymph sample of NaCl ⁄ P
i
-
injected and dsRNA-injected females. Lanes 1–3: NaCl ⁄ P
i
-injected and 4–6 dsRNA-injected animals. Individual lanes represent protein sam-
ples collected from injected shrimps, and the arrows indicate the vitellogenin-specific protein (148, 97 and 76 kDa) using antibody to vitellog-
enin [19,27]. (B) Ovary sample of NaCl ⁄ P
i
-injected and dsRNA-injected females. Lanes 1–3: Ovary from the corresponding NaCl ⁄ P
i
-injected
individual and 4–6 dsRNA-injected animals. Each lane represents protein samples collected from individuals, and the arrow indicates the vitel-
logenin-specific protein determined using antibody to vitellogenin [19].
S. H K. Tiu and S M. Chan Functional study of crustacean neuropeptide
FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS 4391
whereas the MIH-like neuropeptide (i.e MeMIH-B,
type II) may be a vitellogenin-stimulatory factor of
shrimp. Because of the existence of multiple forms of
the CHH family neuropeptides, there may be a dis-
crepancy in defining the function of many cDNAs
cloned using a molecular biology approach. For
example, although the MIH-like gene of Litopenaeus
vannamei was reported [24], detailed analysis revealed
that the MIH-like deduced protein was more closely
related to the CHH as described in other crustaceans.
Since our report of a second form of MIH subtype
cDNA in M. ensis, the naming of this MeMIH-B as a
GIH was based on its similarity to the lobster GIH, as

that MeMIH-B may act directly on the hepatopan-
creas and ovary to increase the rate of vitellogenin
gene expression, indicating that both the hepatopan-
creas and ovary are the targets of MeMIH-B. This
result will provide the basis for identifying and
characterizing the receptor for the neuropeptide.
Furthermore, rMeMIH-B acted on the hepatopan-
creas and ovary in a dose-dependent manner. As the
optimal concentration (i.e. 30 nm in vitro and
6.6 nmol in vivo) for the stimulatation of MeVg1
expression is low, the result also suggests that
rMeMIH-B is highly potent in stimulating vitellogenin
gene expression. As subadult (i.e. < 15 g) and adult
females also responded to rMeMIH-B in a similar
dose-dependent manner, the results would be useful
for us to develop a strategy to induce gonad matura-
tion in shrimp aquaculture.
RNAi is defined as the gene-silencing effect medi-
ated by dsRNA. RNAi technology was developed in
the mid-1990s, based on the antisense RNA techno-
logy developed in the 1980s. RNAi can silence or
knock down the expression of a gene, and the phe-
nomenon appears to be universal, as it has been
reported in both plants, animals, and even cultured
cells. There are two major types of RNAi, with slight
differences in the mechanism. They are mediated by
either: (a) dsRNA; or (b) small interference (si)RNA.
The longer dsRNA may generate a large population
of siRNA (with 21–23 nucleotides), and the use of
longer dsRNA may be advantageous over siRNA. In

animal was about 3–5 lg for each shrimp (23–28 g).
At present, the mechanism of RNAi in shrimp is not
known, but we expected that these dsRNA molecules
Functional study of crustacean neuropeptide S. H K. Tiu and S M. Chan
4392 FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS
could circulate by way of hemolymph and would be
taken up by a variety of tissues. In the target tissues
that express MeMIH-B (i.e. neuronal cells of the eye-
stalk and ⁄ or central nervous system), the dsRNA
enters the cell to initiate gene knockdown. The
dsRNA appears to be stable in the target tissues. For
example, a strong hybridization signal representing the
residual MeMIH-B dsRNA still remains in the eye-
stalk at 120 h after injection (Figs 4A and 5A).
In conclusion, the use of recombinant protein or
RNAi alone may not be sufficient to confirm the func-
tion of a neuropeptide. The combined use of recombi-
nant protein and RNAi described in this study has
provided unequivocal evidence for a stimulatory func-
tion of MeMIH-B in vitellogenesis. We can also apply
similar approaches to study the structure–function
relationships of other CHH ⁄ MIH ⁄ GIH neuropeptide
members.
Experimental procedures
Animals
Shrimp were purchased from a local seafood market. They
were acclimated in the laboratory at 25–28 °C in an indoor
aquarium for 2 days before rMeMIH-B or dsRNA injec-
tion. The GSI was calculated as the percentage of ovary
weight per total body weight.

–nitrilotriacetic acid–agarose
(Qiagen, Hilden, Germany) affinity column that was pre-
equilibrated with denaturing binding buffer. The column
was then washed three times with washing buffer (8 m urea,
20 mm Tris ⁄ HCl, 0.5 m NaCl, and 25 mm imidazole,
pH 7.9). The fusion protein was eluted with elution buffer
(6 mm Tris ⁄ HCl, 0.5 m NaCl, 8 m urea, and 300 mm imid-
azole, pH 7.9). The denatured recombinant protein was
refolded by both dilution and dialysis. The concentration of
urea present in the solubilized protein was decreased step-
wise by addition of an equal volume of renaturing buffer
(6 mm Tris ⁄ HCl, 0.5 m NaCl, and 300 mm imidazole,
pH 7.9) for every 3 h until the concentration of urea was
decreased to 1 m. The diluted recombinant protein was then
dialyzed in a dialysis bag (Sigma; cut-off 6–7 kDa) in a
large volume of renaturing buffer for 16 h at 4 °C, with
three changes of buffer. The recombinant protein was
further dialyzed in a large volume of 0.1 · NaCl ⁄ Tris over-
night, and the final dialysis was against 0.1 · NaCl ⁄ P
i
over-
night. A Bradford protein assay (Bio-Rad, Hercules, CA,
USA) was performed to determine the concentration of the
refolded protein. The recombinant MeMIH-A was
expressed using the same strategy and was used as negative
control in the following in vitro and in vivo bioassay.
Functional study of rMeMIH-B by explant assay
and in vivo injection
The functional study of rMIH-B involving a shrimp in vitro
explant culture system was based on a previously developed

3 min at 95 °C, followed by 35 cycles of denaturation at
95 °C for 1 min, annealing at 58 °C for 1 min, and exten-
sion at 72 ° C for 1 min. In the last cycle, the PCR product
was incubated at 72 °C for 10 min to allow the completion
of DNA synthesis. PCR products were analyzed on a 1.5%
agarose gel, and Southern Blot was performed to determine
the specific amplification of the cDNA.
For in vivo injection of rMIH-B, adult females in the
nonreproductive stage were injected with 20 lL of either
6.6 nmol or 0.66 nmol of rMIH-B at the arthropodial
membrane of the periopod and returned to the culture
tanks. At 24, 48 and 72 h after injection, the hepatopan-
creas and ovary of the shrimp were dissected for total
RNA preparation, and the hemolymph samples were col-
lected for SDS ⁄ PAGE and western blot analysis.
Functional study of MeMIH-B by RNAi
To prepare a DNA template for the synthesis of dsRNA,
DNA corresponding to the mature peptide of MeMIH-B
was amplified by PCR using T7 promoter-linked primers
(forward, 5¢-TAATACGACTCACTATAGGTACTATG
TATCGCATGCCAAT-3¢; reverse, 5¢-TAATACGACTC
ACTATAGGTACTTTAAAGTCCCGGGTTGA-3¢). For
PCR, the reaction mix consisted of 1 · Taq buffer contain-
ing 1.5 mm MgCl
2
, 0.2 mm dNTP mix, 0.5 lm MIH
T
7
-linked primers, and 0.25 lLofTaq DNA polymerase
(Life Technologies, Carlsbad, CA, USA). PCR conditions

tanks for culture before being killed for total RNA prepara-
tion from different tissues. The relative level of MeMIH-B
expression in the nerve cord was used as an indication of the
RNAi effect between the treatment and control groups.
Statistical analysis of northern blots and western
blots
Northern blot or western blot signals from the films (or
blots) were scanned with the free software imagej (Image
processing and analysis in Java: />to obtain quantitative numbers representing expression lev-
els of the gene or protein. The expression level was normal-
ized with either the rRNA (for the northern blot) or
hemocyanin (for the western blot). Either simple t-test or
anova was used to perform statistical analysis.
Acknowledgements
This research was supported by the Research Grant
Council of the Hong Kong Special Administrative
Region, China (HKU 7214 ⁄ 05M) awarded to S M.
Chan.
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