GENE-40122; No.

of pages: 10; 4C:

Gene xxx (2014) xxxxxx

Contents lists available at ScienceDirect

Gene
journal homepage: www.elsevier.com/locate/gene

Hongkun Bai a, Hui Qiao b, Fajun Li a,c, Hongtuo Fu a,b,, Shengming Sun b, Wenyi Zhang b, Shubo Jin a,b,
Yongsheng Gong a, Sufei Jiang b, Yiwei Xiong b

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a r t i c l e

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Article history:
Received 2 August 2014
Received in revised form 17 November 2014
Accepted 5 December 2014
Available online xxxx

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Keywords:
Macrobrachium nipponense
Vitellogenin
Eyestalk ablation
RNA interference

Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, PR China

Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081,
PR China
c
Weifang University of Science and Technology, Shouguang 262700, PR China
b

i n f o

a b s t r a c t

Vitellogenin (Vg) is the precursor of yolk protein, which functions as a nutritive resource that is important for
embryonic growth and gonad development. In this study, the cDNA encoding the Vg gene from the oriental
river prawn Macrobrachium nipponense was cloned using expressed sequence tag (EST) analysis and the rapid
amplication of cDNA ends (RACE) approach. The transcript encoded 2536 amino acids with an estimated molecular mass of 286.810 kDa. Quantitative real-time PCR indicated high expression of Mn-Vg in the female
ovary, hemocytes, and hepatopancreas. As ovaries developed, the expression level of Mn-Vg increased in both
the hepatopancreas and ovary. In the hepatopancreas, the expression level rose more slowly at the early stage
of vitellogenesis and reached the peak more rapidly compared to the expression pattern in ovary. The observed
changes in Mn-Vg expression level at different development stages suggest the role of nutrient source in embryonic and larval development. Eyestalk ablation caused the Mn-Vg expression level to increase signicantly compared to eyestalk-intact groups during the ovary development stages. Ablation accelerated ovary maturation by
removing hormone inhibition of Mn-Vg in the hepatopancreas and ovary. In adult females, Mn-Vg dsRNA injection resulted in decreased expression of Mn-Vg in both the hepatopancreas and ovary, and two injection treatment dramatically delayed ovary maturation. Vg RNA interference down-regulated the vitellogenin receptor
(VgR) expression level in the ovary, which illustrates the close relationship between Vg and VgR in the process
of vitellogenesis.
2014 Published by Elsevier B.V.

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China (Bureau of Fishery, 2011), with an annual production value of

more than 100 million RMB. As the scale of production expanded, sexual precocity began to appear. This term refers to early male and female
gonad development, which leads to excessive propagation and overpopulation; in the M. nipponense population this occurs especially in autumn. Precocity results in the coexistence of multiple generations,
intensive breeding density, and lack of oxygen, which can lead to short
life span and low market value of the product. Thus, this phenomenon
is restricting the sustainable development of M. nipponense. Understanding the reproductive process and the mechanisms that regulate ovarian
maturation in M. nipponense is crucial to improving production of this
commercially important species.
Vitellogenin (Vg), which is the precursor of vitellin (Vn), is synthesized by female shrimp during gonad maturation. In the mature female
prawn, gonad maturity depends on the rapid synthesis and accumulation of Vg in the oocytes during the breeding season (Wilder et al.,
2010). In many oviparous vertebrate and invertebrate animals, Vn provides the substrate and energy for embryonic and ovarian development

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Molecular characterization and developmental expression of vitellogenin

in the oriental river prawn Macrobrachium nipponense and the effects of
RNA interference and eyestalk ablation on ovarian maturation

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1. Introduction

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The oriental river prawn Macrobrachium nipponense (Crustacea;

Decapoda; Palaemonidae) is an important commercial prawn species
that is widely distributed in freshwater areas of China and other Asian
countries. The total shing production reaches 230,248 t per year in

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Abbreviations: M. nipponense, Macrobrachium nipponense; Vg, vitellogenin; Vn, vitellin;

VgR, vitellogenin receptor; GIH, gonad-inhibiting hormone; Mn-Vg, Macrobrachium
nipponense vitellogenin; qPCR, quantitative real-time PCR; GSI, gonadosomatic index;
dsRNA, double-stranded RNA; ELISA, enzyme-linked immunosorbent assay; RNAi, RNA interference; O, ovary; B, brain; L, hepatopancreas; Fx, hemolymph; H, heart; E, eyestalk; AG,
abdominal ganglion; CS, cleavage stage; BS, blastula stage; GS, gastrula stage; NS, nauplius
stage; PS, protozoea stage; ZS, zoea stage; L1, the rst day larva after hatching; L5, the fth
day larva after hatching; L10, the tenth day larva after hatching; L15, the fteen day larva
after hatching; P10, the tenth day post-larvae after metamorphosis; P20, the twenty day
post-larvae after metamorphosis; P30, the thirty day post-larvae after metamorphosis
Corresponding author at: Wuxi Fisheries College, Nanjing Agricultural University,
Wuxi 214081, Jiangsu Province, PR China.
E-mail address: fuht@ffrc.cn (H. Fu).

http://dx.doi.org/10.1016/j.gene.2014.12.008
0378-1119/ 2014 Published by Elsevier B.V.

Please cite this article as: Bai, H., et al., Molecular characterization and developmental expression of vitellogenin in the oriental river prawn
Macrobrachium nipponense and the effects of RNA ..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.008

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Adult healthy M. nipponense were obtained from Tai lake in Wuxi,

China (1201344E, 312822N). The body weight of the female/male
prawns ranged from 1.26 to 4.25 g. Individuals, feed with paludina

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2.2. The isolation of RNA and the synthesis of cDNA

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Total RNA was extracted from mixed tissues using RNAiso Plus Reagent (TaKaRa, Japan). RNA samples were treated with DNase I to
remove contaminated genomic DNA for further experiments. First
strand cDNA was synthesized from 3 g total RNA using Reverse Transcriptase M-MLV Kit (TaKaRa, Japan).

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2.3. Cloning and sequence analysis of Mn-Vg

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2.1. Experiment animal

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2. Materials and methods

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twice per day, were acclimatized in a recirculating water aquarium

system lled with aerated freshwater (2528 C) before tissues and
embryos were collected. A variety of tissues including: ovary, heart, hepatopancreas, muscle, hemocytes, gill, eyestalk, gut and brain were dissected out from mature female/male prawn. As to the collection of
hemocytes, we drawed hemolymph from the body cavity, centrifuged
it at 12000 rpm for 10 min, removed the supernatant, and then collected
the hemocyte precipitate to extract RNA. Ovarian maturity was classied into six stages according to gonadosomatic index (GSI), color, external morphology and histological feature: Proliferation (stage I, GSI =
0.85 0.46), fusion nucleolus (stage II, GSI = 2.54 1.28), oil globules
(stage III, GSI = 4.67 0.98), yolk granule (stage IV, GSI = 7.55 0.40),
maturation (stage V, GSI = 9.85 2.74), and paracmasis (stage VI,
GSI = 1.24 0.62) (Wu et al., 2009). Each developmental stage of larvae was assessed by the criteria of Chen et al. (2012) and Kang et al.
(1996). These samples were maintained in RNA protect liquid (Takara)
until total RNA extraction.

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Initially, the partial fragment of Mn-Vg cDNA was obtained from

normalized cDNA library of ovary and testis (article is in press) in our
lab. Vg complete sequence was cloned by the method of rapid amplication of the cDNA ends (RACE). The 3-RACE and 5-RACE terminal
fragments were extended using 3-full RACE Core Set Ver. 2.0 Kit and
5-full RACE Kit (TaKaRa, Japan). In addition, we examined the middle
sequence by dividing it into ve fragments. All the primers used in the
clone were listed in Table 1. The PCR products were puried using Gel
Extraction kit (Sangon, China), and sequenced by ABI3730 Biosystem,
USA analyzer after insertion into PMD-20T vector (Takara, Japan). To
conrm the validity of the sequence data that was obtained, each fragment was sequenced at least three times. According to the sequences
acquired above, 3-RACE and 5-RACE products and the fragment of
middle sequence, the full length of Mn-Vg cDNA was spliced. Sequences
were analyzed based on the nucleotide and protein databases using the

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Table 1
Primers used for cDNA clone.

t1:1
Q9
t1:2

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(e.g., carbohydrates, amino acids, lipids, vitamins, phosphorus, sulfur,

and trace elements) (Matozzo et al., 2008). The complete cDNA sequence encoding Vg has been cloned for many species of decapod crustacean. In addition, the molecular characteristics and the regulatory
mechanism of Vg have been widely studied (Tsutsui et al., 2000; Yang
et al., 2000; Raviv et al., 2006; Okumura et al., 2007; Jia et al., 2013).
However, molecular studies of M. nipponense Vg (Mn-Vg) are needed
to better understand the mechanisms involved in the reproductive process of this species.
In crustaceans, the site and the process of Vg synthesis are still controversial. Mainly have two kinds: extra-ovarian sources, namely by the
organ beyond ovary synthesis precursor, Vg was considered to be taken
into the developing oocytes from the hemolymph by the vitellogenin
receptor (VgR) via receptor-mediated endocytosis. The mechanisms
for endocytotic internalization of Vg have been well studied in certain
oviparous vertebrates (Schneider, 1992) and insects (Sappington and
Raikhel, 1998), but such studies in crustaceans are limited. Vg synthesis
also may be endogenous (i.e., auto-synthesis), whereby the oocyte itself
produces Vg with participation from relevant organelles. For example, the
ovary was found to be the site of Vg synthesis in Penaeus semisulcatus
(Browdy et al., 1990) and Callinectes sapidus (Lee and Watson, 1995).
However, the Vg gene was uniquely expressed in the hepatopancreas of
Macrobrachium rosenbergii (Yang et al., 2000) and Pandalus hypsinotus
(Tsutsui et al., 2004). Further studies indicated that both the ovary and
hepatopancreas were the Vg synthesis sites in Marsupenaeus japonicus
(Okumura et al., 2007), Fenneropenaeus merguiensis (Phiriyangkul et al.,
2007), Litopenaeus vannamei (Raviv et al., 2006), Penaeus japonicus
(Tsutsui et al., 2000), Metapenaeus ensis and Penaeus monodon (Tiu
et al., 2006a, 2006b).
In decapods, vitellogenesis is hormonally regulated, and it can be
inhibited by the occurrence of hormones in the neurosecretory cells of
the X-organ/sinus gland. For example, gonad-inhibiting hormone
(GIH), which is synthesized in the X-organ/sinus complex, is thought
to play an inhibitory role for the initiation of vitellogenesis in the
ovary (De Kleijn et al., 1994; De Kleijn et al., 1998; Gu et al., 2002).
Adiyodi and Adiyodi (1970) reported that eyestalk ablation removed
the inhibition of neuropeptides and accelerated the accumulation of
Vg. In addition, Jayasankar et al. (2002) found that eyestalk ablation
increased Vg synthesis in the hepatopancreas of the giant freshwater
prawn M. rosenbergii. In P. japonicus, Vg mRNA transcripts were measured
both in the hepatopancreas and the ovary in normal and eyestalk-ablated
adult shrimp. An obvious increment of mRNA levels was revealed in the
ovary, whereas mRNA levels were negligible in the hepatopancreas
(Tsutsui et al., 2005). Overall, existing data indicate that the mechanisms
for hormonal regulation of Vg synthesis vary among crustaceans. Thus,
molecular characterization and functional studies of Vg are critical to understand the reproductive mechanisms in M. nipponense. Such information can be used to improve aquaculture production in practice.
In this study, we cloned the cDNA encoding the Vg gene from
M. nipponense (Mn-Vg) and conducted structural and phylogenetic
analyses. The expression proles of different tissues and development
stages (embryo and larvae) were determined using quantitative realtime PCR (qPCR). qPCR was also used to evaluate the effects of eyestalk
ablation to gain a better understanding of the hormonal regulation
mechanism involved in Vg synthesis. RNAi technology was rstly applied to investigate the expression pattern of Vg in ovary cycles. The results of this study should be helpful for developing methods to cope
with the problem of sexual precocity in the aquaculture setting.

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Primer name

Sequence (5 3)

Purpose

Q10
t1:3

Mn-Vg-R1
Mn-Vg-R2
5-RACE outer
5-RACE inner

ATTACAGGTGTGCAGAGTTCCCTC
AAGTACCCTACCTGAACCACCT
CATGGCTACATGCTGACAGCCTA
CGCGGATCCACAGCCTACTGATGA
TCAGTCGATG
TACCGTCGTTCCACTAGTGATTT
CGCGGATCCTCCACTAGTGATTTCAC
TATAGG
TCTTGTTAACTGGATCGTCCACG
AAGCTCTCTTCGTACCTGTTCAG
TCTGGCGACAGCCTCAGCTGGT
TTGATGACAGTGAACGTTCCTGA
CTGGAGCAGTCAAGGTTATGGT
TACAGAGCACACGATTCCAGAC
GCCAGAGAAAATGGAGTTGGTG
TTGGTACTGAGAGCTTCCTTGG
GTCAGGCGAAACATCACAAGTC
CCTGTGACCTTCTGTTCCTCTC
CTGTTGCTTGATGTCACCCTCTC
ACCGTGCATTATGGTGGCTTGA

Primer for 5-RACE

primer for 5-RACE
Primer for 5-RACE
Primer for 5-RACE

t1:4
t1:5
t1:6
t1:7

Primer for 3-RACE

Primer for 3-RACE

t1:8
t1:9

Primer for 3-RACE

Primer for 3-RACE
Middle segment A
Middle segment A
Middle segment B
Middle segment B
Middle segment C
Middle segment C
Middle segment D
Middle segment D
Middle segment E
Middle segment E

t1:10
t1:11
t1:12
t1:13
t1:14
t1:15
t1:16
t1:17
t1:18
t1:19
t1:20
t1:21

3-RACE outer
3-RACE inner
Mn-Vg-F1
Mn-Vg-F2
VG-A-F1
VG-A-R1
VG-B-F1
VG-B-R1
VG-C-F1
VG-C-R2
VG-D-F1
VG-D-R1
VG-E-F1
VG-E-R1

Please cite this article as: Bai, H., et al., Molecular characterization and developmental expression of vitellogenin in the oriental river prawn
Macrobrachium nipponense and the effects of RNA ..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.008

H. Bai et al. / Gene xxx (2014) xxxxxx

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BLASTX and BLASTN search program (http://www.ncbi.nlm.nih.gov/

BLAST/) of GenBank. The homology search for the nucleotide and
protein sequences was carried out by the BLAST algorithm at NCBI
(http://www.ncbi.nlm.nih.gov/). Deduced amino acid sequences were
obtained using an ORF nder program (http://www.ncbi.nlm.nih.gov/
gorf/gorf.html). The signal sequence was predicted using program
SignalP (http://www.cbs.dtu.dk/services/SignalP/). The NetNGlyc 1.0
Server (http://www.cbs.dtu.dk/services/NetNGlyc/) (N-X-S/T) was
used to identify glycosylation site. The phosphorylation site was found
by the NetPhos 2.0 Server (http://www.cbs.dtu.dk/services/NetPhos/).
Phylogenetic analysis of M. nipponense Vg was constructed using the
neighbor-joining method of MEGA 5.0.

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2.4. Expression of Vg in different development stage and tissue

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Adult female M. nipponense prawns used in this study were selected

from Tai lake Wuxi, approximately 1.50 g3.0 g in wet weight. The
ovary development stage of each prawn was identied in the proliferation (stage I). For eyestalk ablation experiment, about 80 prawns were
divided into two groups, one group was served as intact (un-eyestalk
ablated) and another one was eyestalk ablated (experimental group).
The experiments were performed in duplicate and maintained for a period of 15 days at 28 C. Female prawn eyestalks were removed by cauterization with red hot tweezer experiment. Samples from different
tissues were collected from prawns at 1, 3, 5, 7, 9, 11 and 13 days after

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t2:1
Table 2
t2:2
Q11 Primers used for quantitative PCR and RNA inference.
t2:3
t2:4

Primer
name

Sequence (5 3)

Purpose

t2:5
Q12
t2:6
t2:7

VG-Q-F
VG-Q-R
VG-I-F

Primer for MnVG expression

Primer for MnVG expression
Primer for MnVG dsRNA preparation

t2:8

VG-I-R

t2:9
t2:10
t2:11
Q13
t2:12
t2:13
t2:14

VG-QI-F
VG-QI-R
VGR-QI-F
VGR-QI-R
-Actin F
-Actin R

GAAGTTAGCGGAGATCTGAGGT
CCTCGTTGACCAATCTTGAGAG
TAATACGACTCACTATAGGTGC
CAAGAAAAAGCTCCTGT
TAATACGACTCACTATAGGGCC
AAAGGTTGGTGCATAGT
TGCTCTTGCTCTACTCAAGTCC
CTGATGACAGTGAACGTTCCTG
TACCACTTCGTCACAGATGCAG
CTTGTCGCACCAGTAGATCCTC
TATGCACTTCCTCATGCCATC
AGGAGGCGGCAGTGGTCAT

Primer for MnVG dsRNA preparation

Primer for MnVG dsRNA detection
Primer for MnVG dsRNA detection
Primer for MnVGR dsRNA detection
Primer for MnVGR dsRNA detection
Primer for -actin expression
primer for -actin expression

2.6. RNA interference (RNAi)

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The specic primers containing T7 promoter site of RNA interference

were designed using Snap Dragon tools (http://www.yrnai.org/cgibin/RNAind_primers.pl). Vg dsRNA synthesis primers were shown in
Table 2. The PCR products were puried with a gel extraction kit
(Sangon, Shanghai, China). According to the Transcript Aid T7 High
Yield Transcription kit (Fermentas, Inc, USA) manufacturer's instructions, gene-specic dsRNA was synthesized in vitro. Purity and integrity
of the dsRNA were examined by standard agarose gel electrophoresis. The
concentration of dsRNA was measured at 260 nm by using a BioPhotometer (Eppendorf, Hamburg, Germany), and then save at 20 C. All
primers used were listed in Table 2. VgR detection primers were designed
according to the sequence of NCBI under accession (GenBank KJ768658).
For the short-term in vivo dsRNA injection experiment, 150 health
mature female M. nipponense (each weighing 1.62.3 g) were selected
to inject into pericardial cavity. The female prawns, selected in the proliferation stage (stage I), were assigned to the three treatment groups:
Vg-dsRNA injection (N = 50), two Vg-dsRNA injection (N = 50) and
vehicle injection (N = 50). Each prawn was injected with 4 g/g VgdsRNA or 4 g vehicles. For two injection group, supplemented another
4 g/g Vg-dsRNA in the seventh day after the rst injection. The Vg
mRNA expression of the ovary and hepatopancreas were investigated
to detect the interference efciency by qPCR after injection for 1, 3, 5,
7, 9, 11, 13, 15 and 17 days (N 3). The body weight and ovary weight
of each prawn was recorded to measure the GSI.
For two injection group, samples were captured respectively in the
1, 5, 9, 13, 17, 21, and 25 days after the injection (N 3). All tissues
were saved in the RNA protect liquid.

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2.7. Statistical analysis

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The expression level of Mn-Vg in different development stages of

embryo, post-larvae, juvenile prawns and different tissues was demon182
strated by qPCR. Total RNA treated with RNase-free DNase I (Sangon,
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China) to eliminate possible genomic DNA contamination. The concen184
tration of RNA was quantied by BioPhotometer (Eppendorf). 1 g of
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total RNA was reverse-transcribed by iScript cDNA Synthesis Kit per186
fect Real Time (BIO-RAD, USA) following the manufacturer's instruction.
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The qPCR amplications were carried out in a total volume of 25 L, con188
taining 1 L cDNA (50 ng), 10 l SsoFast EvaGreen Supermix (Bio-Rad,
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USA), 0.5 L 10 M of genes specic forward and reverse primer
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(Table 2), and 13 L of DEPC-water. The reaction mixture was initially
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incubated at 95 C for 30 s to activate the Hot Start Taq DNA polymerase,
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followed by 40 cycles of 95 C for 10 s and 60 C for 10 s, melting cure
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was performed at the end of qPCR reaction at 6595 C (in 0.5 C inc)
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for 10 s. At least three replicate qPCRs were performed per sample
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and three prawns were analyzed for each sample, and amplication of
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-actin was used as the internal control (Zhang et al., 2013). The signif197
icant differences of expressions were showed at p b 0.05. The relative
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copy number of Vg mRNA was calculated according to the 2CT com199
Q17 parative CT method (Livak and Schmittgen, 2001).

eyestalk ablation (N 3). All collect samples were stored at RNA sample
protect liquid. qPCR was used to detect the expression pattern after eyestalk ablation. The specic method of transcription and qPCR process is
the same with different development stages and tissues.

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Q18
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The prawns were weighed and the ovaries were dissected out and 244
indexes were determined using the following formula:
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gonadsomatic index GSI

wet weight of the ovary

 100%:
wet weight of the prawn
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Quantitative data were expressed as means SD.

Statistical differences were estimated by one-way ANOVA followed 248
by Duncan's multiple range test.
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3. Results

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3.1. Molecular cloning and structural analysis of the Mn-Vg gene

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The full-length Mn-Vg gene is 7804 base pairs (bps) long and includes a 5-terminal untranslated region (UTR) of 34 bp, a 7611 bp
open reading frame (ORF) encoding 2536 amino acid (aa) residues,
and a 159 bp 3-terminal UTR (excluding the poly(A) + tail). The MnVg cDNA sequence was submitted to GenBank under the accession
number KJ768657. Fig. 1 shows the sketch map of the deduced amino
acid sequences, and the specic sequence information for Mn-Vg is provided in Fig. 1s. Mn-Vg has an estimated molecular mass of 286.810 kDa
and a theoretical isoelectric point of 9.08. SignalP software analysis revealed that the deduced peptides contained a putative 20 aa signal peptide and a cleavage site between Pro20 and Ser21. The deduced amino
acid sequences included six N-glycosylation sites (N-X-S/T) and two putative subtilisin cleavage sites (RQKR). In addition, 151 phosphorylation
sites (90 Ser, 40 Thr, and 21 Try) were identied by the NetPhos 2.0
Server. Alignment with other species revealed that the Mn-Vg protein

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Please cite this article as: Bai, H., et al., Molecular characterization and developmental expression of vitellogenin in the oriental river prawn
Macrobrachium nipponense and the effects of RNA ..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.008

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RERR

Signal peptide

GLLG

RERR

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3.3. Expression of the Mn-Vg gene in larvae and post-larvae

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The expression pattern of Mn-Vg at different developmental stages

(embryo, larvae, and post-larvae) was evaluated by qPCR. Mn-Vg was
highly expressed in the cleavage, gastrula, and zoea stages, but expression declined slightly in the blastula and nauplius stages (Fig. 4). Once
larvae ruptured the embryonic membrane, Vg expression decreased signicantly and was maintained at relatively low level. However, a sharp
increase in expression occurred beginning 20 days after metamorphosis,
and the highest expression level was measured 30 days after metamorphosis. The expression level of 30 days post-larval showed signicant difference with other development stages.

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3.4. Expression of the Mn-Vg gene in different stages of ovarian development

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Fig. 5 shows the expression pattern of Mn-Vg in the ovary and hepatopancreas during the reproductive cycle. The qPCR results show that
the Mn-Vg level increased in both organs as ovarian development
progressed. In the hepatopancreas, the expression level gradually increased from stages I to III and then abruptly increased and peaked at
stage IV. After reproductive molting, the expression of Mn-Vg in stage
VI decreased rapidly to reach the same level of stage I. In the ovary,
Mn-Vg gene expression increased gradually from stage I to stage V
and reached the peaked. In the ovary, the expression level rose more
rapidly at the early stage of vitellogenesis and reached the peak more

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The distribution and expression of the Mn-Vg gene were analyzed by

qPCR in selected female prawn tissues. Mn-Vg was highly expressed in
the hepatopancreas (Fig. 3) (P b 0.001), followed by the ovary and hemocytes. Expression was signicantly lower in the other tissues (i.e., brain,
gill, heart, and abdominal ganglion). In male prawn tissues, few mRNA
transcripts were detected, and they were present at extremely low levels
(i.e., testis, brain, gill, heart, and abdominal ganglion). The expression
level in male tissues is less than one per few million when compared
with the expression level of female hepatopancreas.

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The expression level of the Mn-Vg after eyestalk removal was monitored by qPCR in the ovary and hepatopancreas (Fig. 6). When compared with the control female prawn group, the reproductive molt
cycle in the eyestalk ablation group was dramatically shortened both
in two tissues. In the hepatopancreas, Mn-Vg expression was upregulated 3 days after eyestalk ablation and reached a level of 3000fold higher than that of the control group (P b 0.01). Overall, the magnitude of gene expression level detected in the experimental group was
much higher than that in the control group. As the ovary developed,
Mn-Vg expression in the hepatopancreas peaked 7 days after eyestalk
ablation in the treatment group, whereas it peaked at 9 days in the control group. A similar expression pattern was found in the ovary, but the
expression level in the eyestalk ablation group peaked 4 days in advance
of the control group.

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3.6. Expression of Vg and VgR after RNAi

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Many researchers have reported that injection of double stranded

RNA (dsRNA) leads to gene silencing. When RNA interference (RNAi)
was used in this study, the Vg-dsRNA clearly inhibited development of
the ovary based on the gonadosomatic index (GSI) (Fig. 7a) and expression pattern data. In the ovary, injection of Vg-dsRNA resulted in an 80%
decrease of Vg expression after 3 days (Fig. 7b). Although the expression
level increased in both the test and control groups as the ovary matured,
different patterns were observed. When the control group had completed a normal reproductive cycle, the experimental group was still in the
yolk accumulation phase (stage III). The same expression pattern was
detected in the hepatopancreas (Fig. 7c). These results illustrate that
RNAi was able to delay the development cycle of the ovary.
In order to detect the effectiveness of Vg-dsRNA in inhibiting maturation of the ovary, we injected another 4 g/g of dsRNA in the two injection
group after one week. The delay in maturation in the experimental group
was even more pronounced compared to that in the one injection and
control groups. When the control prawns had begun a new reproductive
cycle, the two injection group was still in the pre-vitellogenesis stage accumulating yolk protein (Fig. 8).
qPCR analysis indicated that injection of Vg-dsRNA resulted in downregulation of VgR. The Mn-VgR gene transcript level declined by 90% in
the ovary, presumably due to decreased Vg expression (Fig. 7d). In the
process of ovarian maturation, the level of Mn-VgR uctuated in the control group, whereas it stayed low in the Vg RNAi group.

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4. Discussion

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In this study, we identied the complete Mn-Vg transcript sequence,

which is approximately 8 kb in size with 2536 aas encoded by its ORF.
The deduced amino acid sequences revealed the common characteristic
sequence of insect Vg (Chen et al., 1997; Sappington and Raikhel, 1998;
Sappington et al., 2002), with considerable conservation, particularly in
the N-terminus. The vitellogenin N-terminal and VYWD domains are

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3.2. Tissue distribution of the Mn-Vg gene

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slowly compared to the expression pattern in hepatopancreas. Howev- 317

er, the relative expression level in the hepatopancreas was much higher 318
than that in the ovary.
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contains conserved domains, including Vitellogenin_N, VWFD, and the

domain of unknown function (DUF) 1943 super family. The rst two
are highly conserved in other insect and crustacean species. A BLAST
search revealed that Mn-Vg was highly similar to the Vg of the following
crustaceans: M. rosenbergii (91% identity), Exopalaemon carinicauda
(76% identity), P. hypsinotus (61% identity), P. japonica (60% identity), Cherax quadricarinatus (39% identity), Homarus americanus
(38% identity), M. japonicus (38% identity), F. chinensis (38% identity),
and P. merguiensis (37% identity).
A neighbor-joining phylogenic tree (Fig. 2) was constructed based
on the Vg amino acid sequences of all reported decapods, three representative copepods, and ve hexapods. The branches of the phylogenic
tree revealed that Mn-Vg was most closely related to M. rosenbergii,
followed by P. japonica and P. hypsinotus. Vgs of all crustaceans clustered
together as a group. However, copepods and hexapods formed a distinctive cluster separated from the other crustacean Vg proteins, and Vgs
from decapods were more closely related to those of copepods than to
those of hexapods.

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Fig. 1. The sketch map of the deduced amino acid sequences of M. nipponense Vg. The conserved domain (Vitellogenin_N, DUF 1943, VWFD) of VG was colored. Putative signal peptide and
subtilisin cleavage sites (RQKR) are highlighted.

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Please cite this article as: Bai, H., et al., Molecular characterization and developmental expression of vitellogenin in the oriental river prawn
Macrobrachium nipponense and the effects of RNA ..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.008

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Fenneropenaeus merguiensis
Fenneropenaeus chinensis



Penaeus semisulcatus



Litopenaeus vannamei
Penaeus monodon




Marsupenaeus japonicus


Metapenaeus ensis
Cherax quadricarinatus





Homarus americanus

Decapods

Macrobrachium nipponense



Macrobrachium rosenbergii
Pandalopsis japonica




Pandalus hypsinotus

Upogebia major
Callinectes sapidus



Charybdis feriatus






Scylla paramamosain
Tigriopus japonicas

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Lepeophtheirus salmonis
Paracyclopina nana
Bombyx mori




Apis mellifera



Spodoptera litura
Blattella germanica



Copepods

Hexapods

Leucophaea maderae



Portunus trituberculatus


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widely found in insects and vertebrates (Baker, 1988). The GLLG motif
within the VYWD domain is a sequence unique to decapods. Although
its function has not been clearly established, the GL/ICG motif and cysteine residues are necessary for the oligomerization of vertebrate Vg, thus
this motif might contain receptor binding sites (Mayadas and Wagner,
1992; Mouchel et al., 1996). In our study, a DUF also was found in Mn-

Vg. This DUF family is thought to be particularly present in decapods

(e.g., M. rosenbergii, P. japonicus, and P. hypsinotus), but it has rarely
been examined in insects and vertebrates (Smolenaars et al., 2007).
This domain family possesses a structure consisting of several large
open beta sheets (Thompson and Banaszak, 2002), and a thorough
study is needed to identify its exact function.

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Fig. 2. A phylogenetic tree of vitellogenin amino acid sequences. Numbers at branch nodes represent the condence level of posterior probability. The sequences used were as follows:
Fenneropenaeus merguiensis, AAR88442; Fenneropenaeus chinensis, ABC86571; Penaeus semisulcatus, AAL12620; Litopenaeus vannamei, AAP76571; Penaeus monodon, ABB89953;
Marsupenaeus japonicus, BAB01568; Metapenaeus ensis, AAN40701; Cherax quadricarinatus AAG17936; Homarus americanus ABO09863; Macrobrachium rosenbergii, BAB69831;
Pandalopsis japonica, ACU51164; Pandalus hypsinotus BAD11098; Upogebia major, BAF91417; Portunus trituberculatus AAX94762; Callinectes sapidus, ABC41925; Charybdis feriatus,
AAU93694; Scylla paramamosain, ACO36035; Tigriopus japonicus, ABZ915371; Lepeophtheirus salmonis ABU41134; Paracyclopina nana, ADD735511; Bombyx mori NP_001037309.1;
Spodoptera litura, ABU68426.1 Apis mellifera NP_001011578; Blattella germanica, CAA06379; Leucophaea maderae, BAB19327; Macrobrachium nipponense, KJ768657.

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Fig. 3. The expression prole of Mn-Vg in different tissues was revealed by real-time quantitative PCR. The amount of Vg mRNA was normalized to the -actin transcript level. Data are
shown as means SD of three replicates in various tissues. a): Oovary; FLhepatopancreas, FBbrain; Fxhemocytes; FAGabdominal ganglion; FHheart; FEeyestalk. b) Ttestis;
MLhepatopancreas, MBbrain; Mxhemocytes; MAGabdominal ganglion; MHheart; MEeyestalk. Statistical analyses were performed with one-way ANOVA analysis (a and b indicate a signicant difference).

Please cite this article as: Bai, H., et al., Molecular characterization and developmental expression of vitellogenin in the oriental river prawn
Macrobrachium nipponense and the effects of RNA ..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.008

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H. Bai et al. / Gene xxx (2014) xxxxxx

Most decapod crustacean Vg pre-propeptides are cleaved into three

fragments at two processing sites. Mn-Vg contains two cleavage sites
(R-X-K/R-R) that are targeted by enzymes of the subtilisin family;
these sites are also present in other crustacean species (P. japonica, M.
rosenbergii, H. americanus, and C. quadricarinatus). The RXRR cleavage
site commonly occurs at aa residues 707730. However, the cleavage
mechanism at the second processing site [(R/K) XX (R/K)] is not consistent and shows more sequence variability. The second site could not be
examined in the brachyuran and penaeid (Jeon et al., 2010; Kang et al.,
2008). Vgs can be divided into two or more peptides from the position
of these cleavage sites.
To the best of our knowledge, the variability of the Vn level was investigated by enzyme-linked immunosorbent assay (ELISA) in Procambarus
clarkii (Xie, 2009). The expression of Vn uctuated as the embryo developed and exhibited a rest period around the gastrula stage. Vn expression
then decreased after the rupture of the embryonic membrane. In our
study of M. nipponense, qPCR analysis indicated that Mn-Vg expression
also uctuated during embryonic development. It was signicantly highly
expressed and peaked at day 1 of larval development, then declined and
stayed at a low level. This expression pattern is consistent with that of
Vn in P. clarkii. These results indicate that Vg is necessary for embryonic
and larval development, which suggests that Vg provides a stream of energy, carbohydrates, and vitamins to fuel embryogenesis and early larval
development. The gastrula stage is critical to embryonic development because massive quantities of nutrients are synthesized and consumed during this stage. During the larval stage, however, the endogenous nutrients
from Vn gradually are replaced by exogenous feeding (Li, 2004). In addition, expression of Vg was detected in juvenile females at 20 days postlarval stage when the primordia of sex gland began to differentiation (in
press). This pattern suggests that the Mn-Vg gene plays a role in the process of sex gland differentiation. Vg also showed sex-dependent expression in the silkworm Bombyx mori (Yano et al., 1994) and the honeybee
Apis mellifera (Guidugli et al., 2005). Our study is the rst to report the involvement of Vg in the development of embryonic and postembryonic
Palaemonidae.
Vgs are commonly expressed in a sex- and tissue-specic manner in
many species. In many decapod species, the female hepatopancreas is
considered to be the major site of Vg gene expression. Vg was only
detected in the female hepatopancreas and hemolymph in species
such as M. rosenbergii (Okuno et al., 2002), P. hypsinotus (Tsutsui et al.,
2004), and C. quadricarinatus (Abdu et al., 2002). In Penaeids such as
M. japonicus (Okumura et al., 2007) and F. merguiensis (Phiriyangkul

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As in the Vgs of other crustaceans (Abdu et al., 2002; Okuno et al.,

2002; Phiriyangkul and Utarabhand, 2006; Zmora et al., 2007; Jia
et al., 2013), the Vg in M. nipponense had no phosvitin or polyserine domains, which contain tandem serine repeats; in contrast, these domains
are present in many vertebrate and insect Vgs. Multiple serine residues
strung together could be a sites for casein kinase II phosphorylation
(Kuenzel et al., 1987; Meggio and Pinna, 1988), which plays a role in receptor binging during receptor-mediated endocytosis in insects (Wahli,
1988). The absence of phosvitin and polyserine domains in crustaceans implies that another mechanism is responsible for Vg receptor
binding during endocytosis of Vg. The Mn-Vg protein includes six Nglycosylation sites, which may be involved in protein folding and
transport and in recognition between Vg and its target, the oocyte
(Roth et al., 2010).

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Fig. 4. The temporal expression of Mn-Vg in the different development larvae before the
metamorphosis and post-larvae after the metamorphosis was revealed by real-time quantitative PCR. The amount of Mn-Vg mRNA was normalized to the -actin transcript level.
Data are shown as means SD of three repeated samples during the larvae and post-larvae.
CScleavage stage; BSblastula stage; GSgastrula stage; NSnauplius stage; ZSzoea
stage; L1the rst day larvae after hatching; P1the rst day post-larvae after metamorphosis, and so on. Statistical analyses were performed with one-way ANOVA analysis (a, b, c, d,
and e indicate a signicant difference with each other).

Q7

Fig. 5. Quantitative analysis of Mn-Vg transcripts using real-time PCR in ovary (a) and hepatopancreas (b) in different development stages of ovaries. Iproliferation stage, IIfusion
nucleolus stage; IIIoil globules stage, IVyolk granule stage, Vmaturation stage, VIparacmasis stage. Each data point represents the mean and standard deviation (n 3 samples).
Statistical analyses were performed with one-way ANOVA analysis (a, b, c, and d indicate a signicant difference with each other).

Please cite this article as: Bai, H., et al., Molecular characterization and developmental expression of vitellogenin in the oriental river prawn
Macrobrachium nipponense and the effects of RNA ..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.008

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by the ovary, and the hemocytes, and expressed at very low levels in male 438
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tissues. These results imply the presence of the following extra-ovarian Vg 439
synthesis pathway in mature M. nipponense females: Vg is generally 440

et al., 2007), Vg is thought to be expressed only in the female hepatopancreas, hemolymph, and ovary. In our study, qPCR results indicated
that Mn-Vg was highly expressed in the female hepatopancreas, followed

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Fig. 6. Expression level of Mn-Vg after eyestalk ablation in ovary (a) and hepatopancreas (b); each data point represents the mean and standard deviation (n 3 samples). Statistical analyses were performed with one-way ANOVA analysis. 113d represent the days after eyestalk ablation (a, b, c, d, e, f, g, and x indicate a signicant difference with each other).

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H. Bai et al. / Gene xxx (2014) xxxxxx

Fig. 7. Real-time PCR analysis of injection with vg dsRNA (4 g/g). a) represents the effects of Mn-vg knock-down on gonad stimulation index (GSI) of prawns; b) represents the relative Mn-vg
expression levels in the ovary after RNA inference; c) represents the relative Mn-vg expression levels in the hepatopancreas after RNA inference; d) represents the relative Mn-VgR
expression levels in the ovary after inject with vg-dsRNA. Each data point represents the mean and standard deviation (n 3 samples). Statistical analyses were performed with oneway ANOVA analysis (a, b, c, d, e, f, g, and h indicate a signicant difference with each other).

Please cite this article as: Bai, H., et al., Molecular characterization and developmental expression of vitellogenin in the oriental river prawn
Macrobrachium nipponense and the effects of RNA ..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.008

H. Bai et al. / Gene xxx (2014) xxxxxx

(Quackenbush, 1989), P. semisulcatus (Fainzilber et al., 1992), and

M. rosenbergii (Lee and Chang, 1999; Soroka et al., 2000). However,
446
we cannot rule out the possibility that an auto-synthesis pathway
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also exists in the ovary.
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Vg in known to exhibit a variable transcript level during the course
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Q27 of an oogenic cycle (Phiriyangkul et al., 2007; Raviv et al., 2006). In crus450
taceans, Vg gene synthesis activity increases from the primary vitello451
genesis stage to the nal maturation of the ovary and then declines
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after oviposition; this pattern has been documented in M. japonicus
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(Okumura et al., 2007) and Scylla paramamosain (Jia et al., 2013). In
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the current study, this uctuation also was detected in both the ovary
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and the hepatopancreas of M. nipponense. In the ovary, the expression
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level increased from the pre-vitellogenesis stage and peaked at the mat457
uration stage. In contrast, the rise in the hepatopancreas was sharp and
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delayed. On the whole, the relative expression level of Vg was much
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higher in the hepatopancreas than in the ovary. Based on the Mn-Vg tis460
sue distribution and expression pattern during development of the
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ovary, we conclude that the hepatopancreas and ovary are both sites
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of Mn-Vg synthesis, but the hepatopancreas is the main synthesis site.
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This premise is consistent with previous studies of L. vannamei (Raviv
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et al., 2006), M. japonicus (Okumura et al., 2007), M. ensis (Tsang et al.,
465
2003), and F. merguiensis (Phiriyangkul et al., 2007). However, Vg was
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only synthesized in the hepatopancreas in M. rosenbergii (Okuno et al.,
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2002).
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Eyestalk ablation is thought to cut off the source of GIH and result in
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rapid vitellogenesis, as was reported in the giant freshwater prawn
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M. rosenbergii (Murmu et al., 2007). Furthermore, eyestalk ablation in471
duced an increase of Vg mRNA in the ovary but not in the hepatopancre472
Q28 as in M. japonicus (Tsutsui et al., 2005; Okumura et al., 2007) and in
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L. vannamei (Raviv et al., 2006). In our study, the expression pattern of
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Mn-Vg after eyestalk ablation was evaluated in the ovary and hepato475
pancreas. Eyestalk ablation accelerated maturation of the ovary. Mn476
Vg was dramatically up-regulated in both the hepatopancreas and
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ovary 3 days after eyestalk removal in comparison with intact females.
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The accumulation of Vg in the hepatopancreas was sharply, but accu479
mulation in the ovary was more stable; this result suggests that eyestalk
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hormone particularly regulates the process of Vg translation, release
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from the hepatopancreas, and then incorporation into the oocytes. Our
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ndings also indicated that the target tissues for hormone inhibition
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are the hepatopancreas and the ovary and the hepatopancreas was
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the main effect sites.

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Few studies of Vg RNAi have been published. In this study, we detected the expression pattern and GSI changes after Vg-dsRNA injection
into the hepatopancreas or ovary of M. nipponense. Our results show
that Mn-vg RNAi treatment effectively inhibited the development of
ovary in M. nipponense. The expression level of Mn-Vg was signicantly
decreased in both tissues compared with the control group. In addition,
the control group completed oviposition and reached stage I within 13
days, whereas the Vg-dsRNA group took 17 or more days. Thus, injection of 4 g/g Vg-dsRNA delayed oocyte development and extended
the ovarian cycle. This critical discovery can be used to develop a new
method to solve the sexual precocity problem that is widely prevalent
in M. nipponense and Eriocheir sinensis culture areas (Xu and Jiang,
2001; Yang, 2007). To demonstrate the feasibility of using RNAi technology in practice, we conducted the two injection experiment. Our results
indicate that two injections of Mn-Vg RNAi effectively inhibited ovary
development, as the ovary remained in the state of un-development
under the inuence of RNAi. These results will be helpful for further research on sexual precocity and to increase commercially production in
this species.
Upon its release into the hemolymph, Vg is taken up by the oocytes
through receptor-mediated endocytosis, which is an essential reproductive process ubiquitous in all eukaryotes (Roth and Porter, 1964;
Warrier and Subramoniam, 2002). Under the inuence of RNAi, the
down-regulated expression of the VgR transcript in the ovary was synchronized with the expression of Vg, which suggests that the Vg gene
can effectively control the expression of VgR during the process of vitellogenesis. In a previous study, Mekuchi et al. (2008) found that
M. japonicus VgR dsRNA injection leading to a decrease in the Vn content of the ovary. However, our study is the rst report to investigate
the relationship between these two genes through Vg-dsRNA injection in a crustacean.
In summary, we cloned the complete Vg cDNA sequence in
M. nipponense and identied the domain organization and expression
patterns in different tissues, different developmental stages, and
during the ovary development cycle. The main sites of Vg synthesis
in M. nipponense are the ovary and hepatopancreas. Eyestalk ablation
accelerated ovary maturation by removing the hormone inhibition of
Mn-Vg. RNAi inhibited maturation of the ovary, which established a
theoretical basis for solving the sexual precocity issue in aquaculture
practice. In addition, Vg RNAi down-regulated Mn-VgR gene expression in the ovary, which implies a close relationship between Vg and
VgR in the process of vitellogenesis.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.gene.2014.12.008.

synthesized in the hepatopancreas, released into hemolymph, and then

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deposited in developing oocytes through receptor-mediated endocytosis.
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Fig. 8. Real-time PCR analysis of relative Mn-VgR expression levels in the ovary after inject with twice Vg dsRNA (4 ug/g); a) represent the effects of twice-injection effect on gonad stimulation index (GSI); b) represent the relative Mn-Vg expression levels in the ovary after twice RNA inference. Each data point represents the mean and standard deviation (n 3 samples).
Statistical analyses were performed with one-way ANOVA analysis (a, b, c, d, e, and f indicate a signicant difference with each other).

Please cite this article as: Bai, H., et al., Molecular characterization and developmental expression of vitellogenin in the oriental river prawn
Macrobrachium nipponense and the effects of RNA ..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.008

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National Science & Technology Supporting Program of the 12th Fiveyear Plan of China (Grant No. 2012BAD26B04), Jiangsu Provincial Natural
Science Foundation for Young Scholars of China (Grant No. BK2012091),
the Science & Technology Supporting Program of Jiangsu Province
(Grant No. BE2012334), and the three aquatic projects of Jiangsu Province
(D2013-6).

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The project was supported by the Freshwater Fisheries Research
531
Center, China Central Governmental Research Institutional Basic Special

Please cite this article as: Bai, H., et al., Molecular characterization and developmental expression of vitellogenin in the oriental river prawn
Macrobrachium nipponense and the effects of RNA ..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.008

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Please cite this article as: Bai, H., et al., Molecular characterization and developmental expression of vitellogenin in the oriental river prawn
Macrobrachium nipponense and the effects of RNA ..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.008

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