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
University of Hong Kong, Pokfulam Road,
Hong Kong
Fax: +852 2857 4672
Tel: +852 2299 0864
E-mail: chansm@hkucc.hku.hk
(Received 25 January 2007, revised 15 June
2007, accepted 2 July 2007)
doi:10.1111/j.1742-4658.2007.05968.x
Although the crustacean crustacean hyperglycemic hormone ⁄molt-inhibi-
ting hormone ⁄gonad-inhibiting hormone neuropeptides have been studied
extensively in the last two decades and several neuropeptides from the
shrimp Metapenaeus ensis have been cloned, the functions of most of these
neuropeptides remained putative. In this article, we describe the use of
recombinant protein and an RNA interference approach to study the
reproductive function of the previously reported molt-inhibiting hormone
(MeMIH-B) in M. ensis. When hepatopancreas and ovary explants were
cultured in medium containing recombinant MeMIH-B, the vitellogenin
gene (MeVg1) expression level was upregulated in a dose-dependent man-
ner, reaching a maximum in explants treated with 0.3 nmrecombinant
MeMIH-B. Shrimp injected with recombinant MeMIH-B showed an
increase in vitellogenin gene expression in the hepatopancreas. Moreover, a
corresponding increase in the vitellogenin-like immunoreactive protein was
detected in the hemolymph and ovary of these females. Injection of
MeMIH-B dsRNA into the female shrimp caused a decrease in MeMIH-B
transcript level in thoracic ganglion and eyestalk. These shrimp also
showed reduction of vitellogenin gene expression in the hepatopancreas
and ovary. Furthermore, the hemolymph vitellogenin level was also
reduced in these animals. In summary, the results from recombinant
protein and RNA interference experiments have demonstrated the gonad-
stimulatory function of MeMIH-B in shrimp.
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 ª2007 The Authors Journal compilation ª2007 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 similarity to MeMIH-A, and
amino acid sequence alignment indicates that MeMIH-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
were reported [2,3]. Despite its potential involvement
in reproduction, no further research on the reproduc-
tive function of MeMIH-B has been attempted, as
there is a lack of a good bioassay system for the neu-
ropeptide. The recent cloning and characterization of
the gene encoding the major yolk protein, vitellogenin,
may provide a potential biomarker for analysis of
genes that regulate ⁄control reproduction [12].
Concurrently, the recently developed RNA interfer-
ence (RNAi) technique has been used to define the bio-
logical function of many genes. This technique is based
on the gene-silencing effect of dsRNA [13]. The tech-
nique has revolutionized ‘reverse genetic’ research by
introducing dsRNA to organisms or cells. dsRNA can
knock down a gene and will produce a phenotypic loss
of function of that gene [14–16]. Although the com-
plete mechanism has yet to be revealed, successful
RNAi has been reported for many animal models. For
example, Caenorhabditis elegans can be soaked in
dsRNA or can be fed plasmids that make dsRNA and
consequently exhibit RNAi effects. In many studies,
dsRNA can move across cell boundaries freely. Thus,
it is not necessary to inject dsRNA directly into the
gonad to get progeny that exhibit RNAi effects [13].
As RNAi works in many organisms, it might also
work in shrimp. Gene function analysis by RNAi may
be advantageous as compared to other conventional
approaches. This article describes the production of
recombinant protein and dsRNA for reproduction-
related eyestalk neuropeptide gene, and use of an
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 ¼10. Similar to the previous results, expression
of MeMIH-B is sex-nonspecific, as the males also
expressed MeMIH-B (Fig. 1B).
Functional study of recombinant MeMIH-B
in vitro and in vivo
The rMeMIH-B produced by pRSET expression was
purified on an Ni
2+
-charged column. To study the
function of rMeMIH-B in reproduction, hepatopan-
creas explants from females at early stage of gonad
maturation (GSI < 2) were used. A dose-dependent
increase of MeVg1 expression was recorded when the
concentration of rMeMIH-B was increased (i.e.
0.3 pm,3pmand 30 pm). The maximum increase of
MeVg1 transcript level was observed in the hepatopan-
creas explants treated with 0.3 nmrMeMIH-B; further
increase of rMeMIH-B (i.e. 3 nm,30nmand 300 nm)
resulted in a decrease in the overall MeVg1 expression
level (Fig. 2A). When the ovary explants were treated
Functional study of crustacean neuropeptide S. H.-K. Tiu and S.-M. Chan
4386 FEBS Journal 274 (2007) 4385–4395 ª2007 The Authors Journal compilation ª2007 FEBS
with 0.3 nmrMeMIH-B, an increase of about 25% of
MeVg1 expression was recorded (Fig. 2B).
Next, we performed an in vivo injection of
rMeMIH-B into females to further confirm its gonad-
stimulatory effect. To demonstrate the specificity of
rMeMIH-B in gonad maturation, a control group
injected with rMeMIH-A was included. As compared
to the NaCl ⁄P
i
control, injection of an equal amount
(6.6 nmol) of rMeMIH-A did not cause any change in
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
A
B
0.75
0.5
0.25
%2
%4
%
6
%7
%9
M
1.0
0.75
0.5
0.25
noisserpxe evitaleR
Es MeMIH-B
Tg MeMIH-B
Fig. 1. Expression of MeMIH-B in different tissues of early
(GSI < 2) mature females (N¼5). (A) The relative expression levels
of MeMIH-B in nervous tissues (ES, eyestalk; Br, brain; Tg, thoracic
ganglia; Vn, ventral nerve) and non-nervous tissues (Hp, hepatopan-
creas; Mu, muscle; Ov, ovary); the bar indicates the SE. (B) The
expression pattern of MeMIH-B (N> 20) at different gonad matura-
tion stages of the eyestalk (open bar) and thoracic ganglia (diago-
nally shaded bars) of females. The percentage indicates the GSI of
the females. M (B) indicates the expression pattern of MIH-B in
the same tissues in males (N¼5). The lower panel is the northern
blot analysis of MeMIH-B expression in the eyestalk (Es) and tho-
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 pMto 0.3 lM) of rMeMIH-B.
The sample size (or numbers of shrimp) is 10 for the in vitro assay.
Relative MeVg1 mRNA levels (A) are shown as means + SEM of
10 prawns. The shrimp that show significant differences (P<0.05)
in the relative MeVg1 mRNA levels are indicated by an asterisk.
S. H.-K. Tiu and S.-M. Chan Functional study of crustacean neuropeptide
FEBS Journal 274 (2007) 4385–4395 ª2007 The Authors Journal compilation ª2007 FEBS 4387
0
0.5
1
1.5
2
2.5
3
3.5
4
A
B
C
D
treatment
0
0.5
1
1.5
2
2.5
3
rMeMIH-BCtrl rMeMIH-A
Ctrl rMeMIH-A rMeMIH-B
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 Me MIH-B
treatment
Me MIH-Actrl Me MIH-B
Fold changes of Vg transcript
level in hepatopancreas
Fold changes of Vg transcript
level in ovary
Fold changes of Vg
level in hemolymph
Fold changes of Vg
content in ovary
Fig. 3. Effect of recombinant MeMIH-B and MeMIH-A on vitellogenin expression in shrimp. (A) Left: relative expression levels of MeVg1 in
hepatopancreas of females (N¼10) at 48 h after injecting NaCl ⁄P
i
, rMIH-A and rMIH-B. Right: a typical northern blot analysis of the shrimp
MeVg1 transcript level after injection of NaCl ⁄P
i
, rMIH-A, and rMIH-B. (B) Left: relative expression levels of MeVg1 in ovary of females
(N¼10) at 48 h after injection of NaCl ⁄P
i
, rMIH-A, and rMIH-B. Right: a typical northern blot analysis of the shrimp MeVg1 transcript level
after injection of NaCl ⁄P
i
, rMIH-A, and rMIH-B. (C) Left: relative levels of vitellogenin in hemolymph of females (N¼10) at 48 h after injec-
tion of NaCl ⁄P
i
, rMIH-A, and rMIH-B. Right: western blot analysis (upper) of the hemolymph level of vitellogenin for shrimp injected with
rMIH-B. The 148 kDa protein is one of the vitellogenin subunits recognized by the shrimp antibody to vitellogenin [27]. The lower panel
shows the Coomassie blue staining of the hemocyanin (Hcy) corresponding to the same protein samples. (D) Left: relative levels of vitelloge-
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 ª2007 The Authors Journal compilation ª2007 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 (N¼40; average GSI < 3) were injected with
3lg 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
injection (Fig. 4A). This suggests that dsRNAs are very
stable, as residual MeMIH-B dsRNA remained. Unlike
in the eyestalk, there was a significant decrease in the
MeMIH-B transcript level in the nerve cord as early as
24 h after MeMIH-B dsRNA injection. The knock-
down also persisted 120 h after dsMIH-B injection
(Fig. 5A). MeMIH-B transcript level was lowest in
nerve cord at 72 h after injection, but started to
increase afterwards (Fig. 5B). With regard to the effect
of MeMIH-B dsRNA on hepatopancreas MeVg1
expression, it was observed that there was a significant
drop in MeVg1 transcript level in the hepatopancreas.
For example, at 24, 48 and 72 h after dsRNA treat-
ment, drops of 20%, 71% and 23% of the overall
MeVg1 transcript level were recorded. (Fig. 6A).
Unlike in the hepatopancreas, the reduction of MeVg1
expression in ovaries of these female was small after
the injection of MeMIH-B dsRNA. For example, the
reduction in MeVg1 transcript level in the ovary repre-
sented only 6%, 7% and 22% decreases at 24, 48 and
72 h post-dsRNA treatment (Fig. 6B).
Similar SDS ⁄PAGE analysis and western blot analy-
sis were performed for these females. In the hemolymph
sample of the NaCl ⁄P
i
-injected control, vitellogenin-spe-
cific protein could be detected using antibody to vitel-
logenin. In contrast, no vitellogenin-specific protein was
detected in the hemolymph of the dsRNA-injected
females (Fig. 7A). In the ovary, the amount of vitelloge-
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
βnitca-
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 ª2007 The Authors Journal compilation ª2007 FEBS 4389
