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BOR - Papers in Press, published online ahead of print January 23, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.066258
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BIOLOGY OF REPRODUCTION 79, 66–74 (2008)
DOI: 10.1095/biolreprod.107.066258
© 2008 by the Society for the Study of Reproduction, Inc.

Ovary; 

From Hepatopancreas to Ovary: Molecular Characterization of a Shrimp Vitellogenin Receptor Involved in the Processing of Vitellogenin1

Shirley Hiu Kwan Tiu 3, John Benzie 4, and Siu-Ming Chan 2 3

School of Biological Sciences,3 The University of Hong Kong, Hong Kong, People's Republic of China Moana Technologies, LLC,4 Kailua Kona, Hawaii 96740

ABSTRACT

We report the first cloning and characterization of cDNA encoding a putative vitellogenin (Vg) receptor (VgR) from the shrimp, Penaeus monodon. The shrimp VgR cDNA is 6.8 kb; the deduced protein has 1943 amino acids with a molecular weight of 211 kDa. VgR is ovary specific and consists of conserved cysteine-rich domains, epidermal growth factor-like domains, and YWTD motifs similar to the low-density lipoprotein, very low-density lipoprotein, and VgR of insects and vertebrates. VgR expression level in the ovary is low during early vitellogenesis and increases to maximum levels in females with a gonadosomatic index of 3–4, presumably when needed for receptor-mediated endocytosis during the rapid phase of extraovarian Vg production by the hepatopancreas. A peptide from the C-terminal end of VgR was synthesized for antibody production. Anti-VgR antibody recognized an ovarian membrane protein, and the level of this protein was high when extraovarian production of Vg reached peak levels. By immunohistochemical analysis, VgR was detected strongly in the membranes of larger oocytes. VgR expression was knocked down after the shrimp were injected with VgR double-stranded RNA, leading to a decrease in VgR protein content in the ovary, but an increase in the hemolymph level of Vg. This study represents the first report of the functional analysis of a putative VgR in a crustacean.

endocytosis, gene regulation, oocyte development, ovary, receptor, receptor mediated endocytosis, vitellogenesis, vitellogenin, vitellogenin receptor

INTRODUCTION

In oviparous animals, female maturation is characterized by the synthesis of a major yolk protein called vitellin, a glycolipophosphoprotein important for providing nutrition to developing embryos. Vitellin is derived from the yolk precursor, vitellogenin (Vg). In shrimp, extraovarian Vg synthesized in the hepatopancreas is presumably secreted into the hemolymph, where it is sequestered into the developing oocytes by the Vg receptor (VgR) through receptor-mediated endocytosis [1–3]. Thus the VgR plays an important role in oocyte maturation. VgRs have been characterized in many vertebrates, including the chicken, Gallus gallus [4], the frog, Xenopus laevis [5, 6], and the fish, Oncorhynchus mykiss [7] and Oreochromis aureus [8]. In chicken and frog, VgR is a member of the low-density lipoprotein (LDL) receptor (LDLR) superfamily, with eight class-A, cysteine-rich repeated motifs making up its ligand-binding domain (LBD) [4, 5]. Depending on the species, the VgR can be specific or nonspecific for its ligand(s). For example, chicken VgR is multifunctional and is involved in transport of Vg, very low-density lipoprotein (VLDL), {alpha}2-macroglobulin, and lactoferrin into oocytes [4, 9, 10].

In invertebrates, the yolk protein receptor has been described for a number of species, including the fruit fly, Drosophila melanogaster [11], the mosquito, Aedes aegypti [12], the fire ant, Solenopsis invicta [13], and the cockroaches, Blattela germanica, Leucophaea maderae, Periplaneta americana [14]. Although insect VgRs have many similarities to vertebrate VgRs, at 190–214 kDa they are about twice the size of the vertebrate VgRs (95–115 kDa) [5, 10–12]. All insect VgRs described to date contain two LBDs, and each domain comprises five or eight Class A cysteine-rich repeats [14–16]. In contrast, the vertebrate VgRs have a single LBD comprising eight class-A cysteine-rich repeats only [1, 2, 5, 7]. In addition, AaVgR and Dm yolk polypeptide receptor (YPR) contain two epidermal growth factor (EGF) precursor homology domains rather than one, as in the vertebrate VgRs. In both insect and vertebrate VgRs, the binding of Vg is saturable and ovary specific. VgR also shows high Vg affinity/specificity, and is sensitive to changes in pH and Ca2+ concentration [1–3].

As in other oviparous animals, shrimp reproduction is characterized by rapid synthesis of the egg yolk proteins. The major egg yolk protein, vitellin, is synthesized in the ovary from the Vg subunits. Unlike the vertebrates and most insects, both extraovarian and intraovarian sources are responsible for the synthesis of the Vg subunits in shrimp [17–20]. The hepatopancreas is the site of extraovarian Vg production. After its synthesis from the hepatopancreas, Vg is transported to the ovary via the hemolymph, where it is thought to enter the oocyte by receptor-mediated endocytosis. However, information on crustacean VgR is limited to a few species. Limited studies on the VgR and Vg binding have been performed in the fresh water shrimp, Macrobrachium rosenbergii [21], and in the lobster, Homarus americanus [22]. In a more recent study, the estimated molecular weight of the VgR of the crab, Scylla serrata, is 230 kDa, and it exhibited high affinity to the crab Vg in the presence of Ca2+[23]. It also bound to mammal LDL and VLDL, suggesting the presence of conserved receptor-binding sites between the crab Vg and the mammalian LDL and VLDL. So far, VgR has not been characterized at the molecular level in any crustaceans. In recent years, aquaculture has become a key provider of quality shrimp meat, and P. monodon is one of the important species cultured. The development of reliable techniques to manipulate gonad maturation will emerge only once knowledge on reproduction and the processes of Vg production and processing is developed. The main objectives of this study were: 1) to clone the cDNA encoding the putative VgR of the shrimp P. monodon; 2) to study its developmental expression during the gonad maturation cycle; and 3) to perform functional characterization of VgR with double-stranded RNA (dsRNA) interference.

MATERIALS AND METHODS

Experiment Animals

Adult females (200–250 g) were purchased from a local seafood market. They were acclimated to laboratory conditions at 25°C, salinity of 30 ppt, and a 12L:12D photoperiod over 3 days. The ovary maturation stage of the female was determined by gonadosomatic index (GSI; the percentage of gonad weight to total body weight) and the appearance of the ovary.

RNA Extraction

Ovaries or hepatopancreas of females were dissected and homogenized in denaturing solution (6 M guanidium thiocyanate, 0.5% N-lauroylsarcosine in 25 mM sodium citrate, pH 7.0, and 100 mM β-mercaptoethanol). After homogenization, 0.1 volume of 2 M sodium acetate (pH 4.0), an equal volume of phenol (pH 4.0), and 0.2 volume of chloroform were added to the homogenate. After vigorous mixing, the samples were incubated on ice for 15 min and then centrifuged at 10 000 x g for 20 min at 4°C. The upper aqueous phase was transferred to a new tube, and the total RNA was precipitated by adding an equal volume of isopropanol at –20°C for 30 min. After centrifugation, RNA pellets were rinsed thrice with 75% ethanol, air dried, and dissolved in diethyl pyrocarbonate (DEPC)-treated water. The quality and quantity of total RNA was monitored by 1.2% denaturing RNA gel containing formaldehyde and optical density measurement of the absorbance at 260 nm and 280 nm with a spectrophotometer. Poly(A+) RNA was prepared from 1 mg ovarian total RNA with PolyA+Tract mRNA Isolation kit (Promega), and the final Poly(A+) was dissolved in DEPC-treated water following the manufacturer's instructions.

PCR Cloning of the Full-Length VgR cDNA

Ovary total RNA (3 µg) and oligo-(dT)12 primer were denatured in DEPC-treated water at 70°C for 10 min and quickly chilled on ice for 2 min. First-stranded cDNA was synthesized by reverse transcriptase reaction. Reverse transcription reaction (37°C, 3 h) was performed in a final volume of 50 µl containing 1x First strand buffer (0.2 mM dNTP mix; Amersham), 1.25 µg oligo (dT)12, 0.5 µl RNase inhibitor (31750 U/ml; Amersham), and 0.5 µl MMLV-RT (200 U/µl; USB).

For RT-PCR, the degenerate primers, VgRF8 (forward: AVYKANKF; 5'-GC (AT)GT(CG)TATAA(AG)GC (CA)AA(TC)AAATTC-3') and VgRR8 (reverse: MNFDNPVY; 5'-GTACAC(ACTG)GGATTGTC(GA)AAGTTCAT-3'), were designed based on conserved regions of Drosophila yolkless cDNA [11]. The reaction mix consisted of 1x reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTP mix (Amersham), 0.5 µM each of a pair of forward degenerate primers (PmVgF8) and reverse primers (PmVgR8), 0.1 µl Taq DNA polymerase (5 U/µl), and 1 µl of the RT mix was added as template. PCR amplification was performed with denaturation at 95°C for 1 min, followed by 34 cycles of denaturation at 95°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 2 min. At the end of the PCR, the reaction was further incubated at 72°C for 10 min. The amplified PCR product was subcloned into the pGEM-T vector and sequenced from both ends by Big Dye terminator v2.1 cycle sequencing kit (USA Scientific) and ABI Prism 3100 autosequencer (Applied Biosystems). Nucleotide sequences were analyzed by BlastP search database from GenBank. As the sequencing results revealed high similarity of the shrimp partial cDNA to mosquito VgRs, several gene-specific primers were designed for 5' and 3' rapid amplification of cDNA ends (RACE) to clone the full-length VgR (Fig. 1).


Figure 01
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  		FIG. 1 Schematic drawing for the cloning strategies of the P. monodon VgR. A cDNA fragment of 200 bp, corresponding to the second LBD of P. monodon VgR, was obtained by RT-PCR with degenerate primers, Deg.F and Deg.R (black bar). Gene-specific primers were then designed for 5' and 3' RACE and the deduced full-length sequence was obtained from the overlapping RACE clones (grey bars). Furthermore, the primers ORF.F and ORF.R were designed to obtain the full-length cDNA of P. monodon VgR and the untranslated region with the poly-A tail. The small inserted picture represents agarose gel analysis of the PCR amplification for the open reading frame (ORF; arrow) of the shrimp VgR cDNA with primer ORF.F and ORF.R.

The 5' and 3' RACE were performed with a kit (Roche) and the procedure followed the manufacturer's instruction. For 5' RACE, the first-stranded cDNA was produced from 2 µg ovary total RNA with gene-specific primers (5' RACE1–5' RACE 10; for location, see Fig. 1), and PCR was performed with another gene-specific primer (but upstream to the one used in reverse transcription) and the adapter primers provided in the kit. A total of 30 cycles (denaturation at 95°C for 1 min, annealing at 60°C for 30 sec, and extension at 72°C for 3 min). Similarly, 3' RACE was also performed with the same kit. The first-stranded cDNA was produced by reverse transcription with the oligo(dT)-adapter primer provided in the kit. A nested PCR was performed with the forward gene-specific primers. For 5' and 3' RACE, PCR products were analyzed on agarose gel followed by Southern blot hybridization before subcloning into sequencing vector (pGEM-T; Promega) and analyzed by DNA sequencing.

Sequence Comparisons and Phylogenetic Analyses

Representative sequences of VgRs and related proteins were obtained from GenBank. These included VgRs for: the insects, B. germanica Blage_VgR (GenBank ID CAJ19121), L. maderae Leuma_VgR (BAE93218), P. americana Peram_VgR [24] (BAC02725), A. aegypti Aedae_VgR (AAK15810), S. invicta Solin_VgR (AAP92450), Dermacentor variabilis Derva_VgR, (AAZ31260); fish, Morone americana Moram_VgR (AAO92396), O. aureus oreau_VgR (AAO27569); chicken, G. gallus Galga_VgR (P98165); frog, X. laevis Xenla_VgR (BAA22145); and shrimp P. monodon Penmo_VgR (EU024890); the VLDL receptors (VLDLR) of G. gallus Galga_VLDLR (NP990560), Homo sapiens Homsa_VLDLR (NP990560), and Mus musculus Musmu_VLDLR (AAA59384); and the LDLRs of Mus musculus Musmu_LDLR (AAH19207) and Homo sapiens Homsa_LDLR (AAP72971). These sequences were analyzed with Clustal X (EMBL-European Bioinformatics Institute, Cambridge, UK; http://www.ebi.ac.uk/Tools/clustalW2/index.html). Poorly aligned positions and divergent regions were eliminated by using gblocks 0.91b [25]. The resulting alignment was analyzed with the PHYML program based on the maximum-likelihood principle with the amino acid substitution model. Four substitution rate categories with a gamma shape parameter of 1.444 were used. The data were bootstrapped for 100 replicates with PHYML.

Northern Blot Analysis VgR Expression

Poly A+ RNA from the ovaries and other tissues were prepared with a Poly A+ RNA synthesis kit (Promega). RNAs were analyzed by formaldehyde agarose gel electrophoresis and mRNAs from the gel were blotted onto an Immobilon Nylon membrane (Millipore Corp., Bedford, MA). After the RNA was ultraviolet cross-linked, the blot was hybridized in high-SDS hybridization buffer containing a nonradioactive probe at 50°C for overnight. The probe was derived from the 1.2-kb cDNA fragment of VgR. It was synthesized by a nonradioactive digoxigenin (DIG) DNA Labeling Kit (Roche). After hybridization, the membrane was washed in 2x saline-sodium citrate (SSC) with 0.1% SDS twice for 15 min, and then in 0.1x SSC with 0.1% SDS twice for 15 min at 58°C. For signal detection, the membrane was incubated in 1:20 000 anti-DIG-AP conjugate in 1x blocking buffer. For detection, 200 µl CDP-Star (Roche) was added onto the membrane, luminescent signal was exposed on a film, and the film was developed by a Kodak film processor. As for RT-PCR detection of VgR expression, primers (forward, 5'-ATGAGTTCCATCGGTACTGGGTATGAT-3'; reverse, 5'-GCAGACCCATGATAATTCTAGT-3') were designed for PCR amplification. For PCR, 1/10 volume of reverse transcription reaction product was used as template with similar temperature profiles as described previously here. The β-actin (Actb) gene was used as an internal control for normalizing the expression [26]. For quantitative analysis of the expression level of VgR and Vg protein, the software ImageJ was used [27].

Generation of Anti-VgR Antibody and Immunodetection

A synthetic peptide (i.e., QNVLKPPVELRVEWD) derived from the C-terminal end of VgR (i.e., AA1916–1930) was used as antigen to inject rabbits for antibody production. The synthetic peptide was derived from the C-terminal end of the VgR. For antibody production, all procedures and handling of animals described here were reviewed and approved by the university laboratory animal care and use committee, and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals. About 100 µg of the peptide was used to inject a New Zealand white rabbit at three different locations. The injection was repeated twice at a 12-day interval. At 1 wk after the third injection, the antibody titer was monitored and high titer of VgR was prepared from the rabbit whole blood. For Western blot analysis, soluble proteins from ovaries and other tissues were separated by 8% SDS-PAGE and transferred to nitrocellulose membrane. After blocking with 5% nonfat milk in Tris-buffered saline (TBS), the membrane was incubated in primary antibody solution at a dilution of 1:10 000. After washing with 0.3% Tween-20 in TBS (TBST), the membrane was incubated with alkaline phosphate-conjugated goat anti-rabbit immunoglobulin G (1:5000) secondary antibody, and developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. For immunohistochemical detection, ovarian tissues from mature females were dissected in ice-cold PBS, fixed in 4% paraformaldehyde in PBS, pH 7.4, at 4°C for 16 h with gentle shaking. The fixed tissues were dehydrated in ethanol series and embedded in paraffin. Consecutive 10-µm sections were mounted onto 3-aminopropyltriethoxysaline-coated slides and dried at 37°C overnight. Tissue sections were deparaffinized in xylene twice for 5 min each and rehydrated through ethanol series. Endogenous peroxidase was removed by incubating the sections in 3% H2O2 for 5 min and rinsed briefly with PBS. The immumohistochemical staining of sections was performed with a Vectastain Elite ABC kit (Vector Laboratories) according to the manufacturer's instructions. The sections were blocked in 5% normal serum for 1 h at room temperature. After blotting the excess blocking serum, the sections were incubated in anti-VgR antibody (1:10 000 in TBS) overnight at 4°C. The negative control sections were incubated in preimmune rabbit serum instead. After overnight incubation, the sections were washed in a large volume of PBS with gentle agitation and were incubated in a biotinylated secondary antibody solution for 30 min at room temperature. After washing, the sections were further incubated in Vectastain Elite ABC reagent for 30 min at room temperature, and peroxidase substrate solution (1.39 mM 3,3'-diaimnobenzidine [Sigma, St. Louis, MO] in 0.01% H2O2) was added for color development, which was terminated by rinsing the sections in running tap water.

Functional Study of VgR by RNA Interference

To prepare DNA template for dsRNA synthesis, DNA corresponding to the EGF domain of VgR was amplified by PCR with T7 promoter linked primers (VgRT7-EGF.F1: 5'-TAATACGACTCACTATAGGGTACTTATCTACAGTGTCA ACAGGGAGACA-3' [+2170]; reverse: VgRT7.EGF.R1: 5'-TAATACGACTCACTATAGGGTACTTCATTACACATGCCTGACTTGCACT-3' [+3229]-3'). PCR was performed with similar temperature profiles described previously here. The resulting PCR products were analyzed by 2% agarose gel electrophoresis. The target PCR product band was purified by the GENECLEAN II kit (QBioGene). For the synthesis of VgR dsRNA, 1–2 µg of purified T7 promoter-linked VgR cDNA was used as template, and the transcription reaction was performed with T7 Megascript RNAi Kit (Ambion) according to the manufacturer's recommendations. In 20 µl reaction, DNA template was mixed with nuclease-free water, 2 µl 10x T7 reaction buffer, 2 µl ATP solution, 2 µl CTP solution, 2 µl GTP solution, 2 µl UTP solution, and 2 µl T7 enzyme mix. The mixture was incubated at 37°C for 18 h. During the transcription, the two RNA strands were hybridized to form dsRNA. Shrimp (160–200 g) with similar GSIs of 3–4 were used for dsRNA injection: 20 µg of VgR dsRNA (50 µl in 1x PBS) was injected into shrimp through the arthrodial membrane of the periopods by a Hamilton syringe. The controls received either a nonspecific dsRNA (i.e., a Drosophila dsRNA provided by the Megascript RNAi kit; Ambion) or PBS injection. Five microliters of hemolymph was taken from shrimp at 48, 72, and 96 h after the injection. The hemolymph was dispensed into 200 µl of sodium bicarbonate buffer (0.05 M, pH 9.6) and total protein was analyzed by SDS-PAGE and Western blot with P. monodon-specific Vg antibodies. At the end of the experimental period (i.e., 96 h), shrimp were killed for total RNA preparation from different tissues. The relative level of VgR expression in the ovary was used to indicate the comparative effect of RNAi between the treatment and control groups.

RESULTS

Cloning of the Shrimp Ovary VgR cDNAs

For initial RT-PCR, a cDNA fragment of 130 bp was amplified from the ovary, and DNA sequencing revealed that the cDNA carried coding sequence homologous to the second LBD of the insect VgR (Fig. 1, dark bar). Gene-specific primers were designed to clone the full-length cDNA of the shrimp VgR (Fig. 1, Table 1). For 5' RACE, several pairs of gene-specific reverse primers (i.e., 5' RACE1–5' RACE10) were designed, and four different overlapping 5' cDNA clones were isolated with this primer walking approach. For 3' RACE, the gene-specific primers (i.e., 3' RACE1 and 3' RACE2) were designed and a large 3'-end cDNA clone was obtained (Fig. 1, Table 1). The shrimp full-length VgR cDNA was reconstructed from these overlapping clones. Final confirmation of the coding sequence was performed by RT-PCR with primers ORF.F and ORF.R. The reconstructed VgR cDNA is 6037 bp, with the longest open reading frame of 5823 bp coding for 1941 deduced amino acids (Fig. 2) with an estimated molecular weight of 211 kDa and isoelectric point of 5.27. The shrimp VgR cDNAs consist of a 109 bp 5' noncoding region and a 102 bp 3' noncoding region with the polyadenylation signal site (i.e., ATTAAA) located at 70 bp downstream from the stop codon (GenBank ID EU024890). Using the signal peptide prediction software [28], it was predicted that amino acid residues 1–22 represent the signal peptide region of VgR (Fig. 2).


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  		TABLE 1 Primer sequences used for RT-PCR.


Figure 02
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  		FIG. 2 Deduced amino acid sequence of P. monodon VgR. The signal peptide cleavage site is indicated by a downward arrow. The LDLR class-A domains are AA77–278 and AA981-1328; the YWTD motifs (underlined) in the EGF-like domain consist of the LDLR (type-B repeat). The SDE sequences in each repeat in the LBD are denoted by small boxes. The region AA1768–1782 indicates the O-linked sugar domain (shaded area) and AA1783–2005 is the transmembrane domain of the receptor. The two internalization motifs FXNPX(Y/F) (at AA1835–1840 and AA1874–1879) are shaded and not boxed. The SH3 binding motif, XØPXXP, shown in italics (AA1860–1866), is in the cytoplasmic region of the receptor. The region AA1916–1930 (boxed) indicates the sequence used for synthetic peptide production and antibody generation.

SMART program analysis results revealed that VgR belongs to the LDLR superfamily, as it consists of characteristic modular elements found in members of this group (Fig. 3A). P. monodon VgR consists of two LBDs, and each consists of five or eight class-A cystenine-rich repeats. Each LBD is followed by an EGF homology domain that contains two types of motifs (i.e., the class-B repeats with six-cystenine residues and the YWXD repeats). Following the second EGF homology domain, there is an O-linked sugar domain, a single-pass transmembrane domain, and a cytoplasmic tail, which contains two internalization signal (NPX(Y)F) motifs. Additionally, there is an Src homology 3 (SH3) binding motif (X{Phi}PXXP), located at AA1860–1865 (Fig. 2). The shrimp VgR consists of 13 putative N-linked glycosylation sites with conserved NX(S/T) motif (predicted by NetNGlye 1.0 http://npsa-pbil.ibcp.fr) and 80 putative phosphorylation sites on serine or threonine (as predicted by NetPhos 2.0 software www.cbs.dtu.dk/services/NetPhos/).


Figure 03
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  		FIG. 3 Structural organization of the shrimp VgR and comparison with insect VgR. A) Cysteine-rich regions are represented by letter A for class-A repeats and B for class-B repeats (EGF-like repeat). The LBDs are identified as LBD1 and LBD2. Each LBD consists of an EGF homology domain and several YWXD repeats (rpts). An O-linked sugar domain (OD) very rich in serine, the transmembrane domain (TM), and the cytoplasmic tail (CT) are also annotated. The signal peptide (SP) is located at the N-terminal end of the protein. B) Table summarizing the analysis of amino acid sequence alignment of the different invertebrate VgRs. Sequences are given for the shrimp, P. monodon, mosquito, A. aegypti, the fire ant, S. invicta, and the cockroaches, B. germanica, L. maderae, and P. americana.

Sequence Comparison and Phylogenetic Analysis

Similar to insect VgRs, the P. monodon VgR (211 kDa) is almost twice the size of most vertebrate VgRs. The larger size of P. monodon VgR is due to the additional LBD and the EGF precursor homology domain (Fig. 3A). BLASTX search analysis revealed that the shrimp VgR cDNA was most similar to the cockroach, B. germanica VgR (31% overall identity), P. americana receptor (31% overall), L. maderae VgR (30% overall identity), and mosquito A. aegypti VgR (30% identity overall) (Fig. 3B). Except for the fire ant, S. invicta, the shrimp and most insect deduced VgR also have an O-linked sugar domain and EGF homology domains. The P. monodon VgR shared 27%–29% overall similarity to the VLDL and vitellogenin receptors VLDLR of vertebrate. However, a much higher sequence identity was observed in the conserved in the EGF domain and the cysteine-rich region of the LBDs. The FXNPX(Y/F) sequence, which is indispensable for the LDLR family to cluster in coated pits, was not identified in the shrimp VgR (see Fig. 2). However, two sequences (i.e., FANPGF and FENPFF), which show high sequence identity to FDNPVY, were identified in the C-terminal end (i.e., AA1834–1839 and AA1874–1879) in the shrimp VgR. Maximum-likelihood analysis of the shrimp VgR, with insect VgRs, the VLDLRs, and LDLR of the vertebrates as out-groups, generated a tree, the topology of which follows the current phylogeny of these species (Fig. 4), with the shrimp VgR closer to those of the insects than to those of the vertebrates.


Figure 04
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  		FIG. 4 Phylogenetic analysis of VgRs, LDLRs, and VLDLRs. The species name of the VgR and LDLPRs and the GenBank accession numbers can be found in the Materials and Methods section.

Tissue-Specific Expression of the Shrimp VgR during Gonad Maturation Cycle

Northern blot revealed a 7.0-kb VgR transcript detected exclusively in ovaries and specifically not in eyestalk, brain, thoracic ganglion, ventral nerve cord, gill, gut, heart, hepatopancreas, or swimming legs (Fig. 5A). Gel staining revealed that the VgR transcript level was low in the ovary of shrimp with low GSI (i.e., GSI <0.8), increased in shrimp with a GSI of 1.6, reached a maximum in females with a GSI of 3–4 (Fig. 5, B and C), and decreased toward the end of the gonad maturation cycle (GSI = 4–13.3). To determine if there is a correlation in the expression of Vg and the VgR, we also studied the expression of the P. monodon Vg in the hepatopancreas and the ovary of these shrimp. The maximum expression level of Vg in the hepatopancreas was at the early phase (i.e., GSI = 0.8–3.0), and then decreased and remained low in shrimp with a GSI of 4–13.3 (Fig. 5B). Northern blot analysis of Vg transcript expression, however, showed a different pattern to that of the protein product. In the hepatopancreas, the level of Vg transcript was low initially (GSI <3), increasing for shrimp with a GSI of 4–9, and remaining high toward the end of ovarian maturation. When the expression level of Vg in the hepatopancreas started to decrease, an increase of Vg transcript was recorded in the ovary until an advanced gonad maturation stage (i.e., GSI = 13.3) (Fig. 5B). The increases in specific Vg protein level may have been due to the accumulation of Vg from both the extraovarian (hepatopancreas) and intraovarian sources.


Figure 05
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  		FIG. 5 Expression of the shrimp VgR in shrimp ovary. A) Northern blot detection of VgR transcripts in ovary (Ov) and not in eyestalk (ES), brain (Br), thoracic ganglion (Tg), ventral nerve cord (VNC), gill, gut, heart, hepatopancreas (Hp), and swimming leg. The lower panel shows the ethidium bromide staining of the same gel before capillary transfer. B) Expression pattern of Vg and VgR in different reproductive stages (i.e., GSI) of the females. The expression of Vg (Vg/Ov) in the ovary was compared with the expression of the VgR in the ovary (VgR/Ov) and hepatopancreas (Vg/Hp) during the gonad maturation cycle. C) The bar graph shows the relative expression level of (y axis) Vg (in ovary and hepatopancreas) and VgR (in ovary only). The diagram is generated from the plot of hybridization signals from either the Vg expression in the hepatopancreas (hatched bars) and ovary (black bars), and VgR (grey bars) after normalizing with the rRNA. The numbers on the x axis show the corresponding GSI of different females.

Identification of VgR Protein in Ovary

Analysis of tissue extracts probed by antigens to a synthetic peptide derived from the C-terminal end of VgR indicated the presence of Vg protein in the ovarian tissues, but not in the eyestalk, gill, heart, hemolymph, muscles, nerve cord, or swimming legs (Fig. 6A). The amount of VgR detected in the ovary was low in early mature shrimp (i.e., GSI = 0.7), increased to a maximum in shrimp with a GSI of 1.6–3.3, and decreased toward the end of the maturation cycle (Fig. 6, B and D). In contrast, the ovary Vg level was low in the early stages and increased toward the end of gonad maturation (Fig. 6, C and D). Immunohistochemical analysis of the ovary sections showed that Vg was evenly distributed in the cytoplasm of the large and small oocytes (Fig. 7A). In contrast, similar analysis with anti-VgR antibody detected VgR only in the cytoplasm of small oocytes. The VgR immunopositive signals were detected strongly in the plasma membrane and weakly in the cytoplasm of the large oocytes. No signal was detected in the follicle cells of the ovary (Fig. 7B). As a reference, immunohistochemical analysis performed with similar ovary sections showed that Vg, unlike the VgR, was evenly distributed in the cytoplasm of the oocytes (Fig. 7A). The control, with preimmune serum of the rabbit, did not show any signal (Fig. 7C).


Figure 06
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  		FIG. 6 Tissue distribution (A) of the shrimp VgR and correlation of VgR with Vg during the gonad maturation cycle (B–D). A) Western blot showing detection of VgR in the ovary and not in the eyestalk (ES), gill (Gi), heart (Ht), hemolymph (He), muscle (Mu), nerve cord (Nc), and swimming legs (Sl). B and C) Western blot analysis of ovary total protein isolated from shrimp of different GSIs probed by the P. monodon anti-VgR (B) or anti-Vg (C) antibodies. M indicates a size marker (250 kDa). D) The bar graph shows the relative protein content of (y axis) VgR (dotted bars) and Vg protein (solid bars) quantified using the software ImageJ. The relative protein content was obtained after normalization with the same amount of protein (10 µg) loaded onto the well. The numbers on the x axis show the corresponding GSI of different females.


Figure 07
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  		FIG. 7 Immunohistochemical analysis of the shrimp VgR and Vg in P. monodon. Immunostaining of the shrimp Vg in the ovary under low (A) and high (A') magnification. Immunostaining (arrow, brown color) of PmVgR in the ovary under low (B) and high (B') magnification. The broken arrows in A' and B' point to the oocyte that shows the brown color resulting from immunopositive staining with the anti-Vg and anti-VgR, respectively. Staining of the shrimp Vg is throughout the oocyte, and that for VgR is restricted to oocyte membrane. Ovary section incubated with preimmune serum under low (C) and high (C') magnification. No signal was seen in the control sections. CYT, Vacuolated cytoplasm of vitellogenic oocytes; FC, follicle cells; LUM, lumen; NUC, nucleus; PO, previtellogenic oocytes; VO, vitellogenic oocytes. Bars = 75 µm (A–C); 20 µm (A'–C').

Functional Analysis of VgR by RNA Interference

Relative to the control, only a slight change in VgR expression level was recorded in dsRNA-injected shrimp after 24 h. However, significant VgR gene knockdown was detected in shrimp after 48, 72, and 96 h (Fig. 8A). The decrease in VgR transcript level represented a drop of 90%. The degree of gene knockdown also varied from individual to individual. For example, VgR transcript was detected in some shrimp at 48 and 72 h after injection, but was undetectable in other individuals at those times (Fig. 8A). Western blot analysis of ovarian VgR protein of these shrimp confirmed that the VgR protein level also dropped significantly (Fig. 8B). The SDS-PAGE and Western blot analysis of the ovarian protein of these animals showed that the ovary Vg level of the injected shrimp dropped significantly (Fig. 9A). Although Coomassie blue staining of the gel did not reveal dramatic changes in specific ovarian protein, Western blot revealed a significantly higher level of Vg present in the hemolymph of these dsRNA-injected females (Fig. 9B). For example, the ovary Vg protein levels in the dsRNA-injected shrimp at 48 h were about four times less than the control. The sizes of these proteins corresponded to the Vg subunit identified in our previous studies [17, 18]. Unlike the ovary, Vg-specific proteins detected in the hemolymph also increased in shrimp injected with dsRNA for VgR. The results from analyzing the Vg gene expression in the hepatopancreas and the ovary (Fig. 9, C and D) of the VgR dsRNA-injected shrimp showed that there were no significant changes in the shrimp Vg transcript level in the hepatopancreas (Fig. 9C) or ovary (Fig. 9D) of the dsRNA-injected animals as compared to the control.


Figure 08
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  		FIG. 8 Effect of the shrimp VgR dsRNA injection on VgR expression. A) RT-PCR detection of VgR transcript at 24, 48, 72, and 96 h after the injection of VgR dsRNA. The controls represent shrimp samples collected at 72 h after receiving either a PBS injection (PBSa) or a negative nonspecific (Drosophila) dsRNA in PBS (PBSb). The lower panel shows the ethidium bromide staining of the β actin gene (Actb) of the corresponding shrimp samples. The label "–ve ctrl" indicates a control with no template, with the objective of monitoring the PCR reaction. B) Western blot detection of VgR in the dsRNA-treated (+) and untreated (–) females. Each lane consists of ovary proteins extracted from an individual. Individual lanes represent either RNA (A) or protein (B) levels of P. monodon VgR.


Figure 09
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  		FIG. 9 Effect of P. monodon VgR dsRNA injection on Vg protein level and gene expression in females. A and B) Western blot detection of Vg protein in ovary (A) and hemolymph (B) of shrimp after injection with PBS (–) and VgR dsRNA (+). The bar graph shows the relative Vg protein level of the corresponding shrimp after normalization with respect to total protein. The upper panels show the Western blot detection of Vg in the ovary (A) and hemolymph (B) of the shrimp. Each lane represents a sample obtained from an individual. C and D) Detection of Vg transcript in hepatopancreas (C) and ovary (D) of shrimp injected with PBS (–) or Vg dsRNA (+) after 48, 72, and 96 h. The bar graph shows the relative Vg protein level in the corresponding shrimp after normalization with β-actin transcript (Actb). The upper panels show the Northern blot (C) and slot blot (D) of the Vg transcript. Each lane represents a sample obtained from an individual. Ctrl, Control; ds, dsRNA.

DISCUSSION

The present study has identified and characterized a cDNA that encodes an ovarian VgR in the shrimp, P. monodon. Until now, the only VgR reported for a crustacean was that of the crab, S. serrata, where VgR was purified by gel filtration and HPLC, and its molecular weight was estimated to be 230 kDa. Although the sequence was unknown, the affinity of crab VgR increased in the presence of Ca2+ and the binding was inhibited by suramin, suggesting similarities between crab VgR and the LDLR protein superfamily. Moreover, the crab VgR also showed significant binding to mammalian atherogenic lipoproteins, such as LDL and VLDL [23]. The determination of the nucleotide and amino acid sequences for P. monodon VgR allowed direct comparison of the homology of crustaceans with that of other species. This analysis confirmed significant conservation of the amino acid sequence with the insects and vertebrates. It also suggested conservation of receptor binding sites between crustacean and insect VgR and vertebrate VgR, LDL, and VLDL. The shrimp VgR has a molecular weight (211 kDa) similar to that of the crab (230 kDa) and insects (190–214 kDa), and much larger than that of the vertebrates. The greater number of amino acids reflects the presence of additional LBD and the EGF precursor homology domains in VgR.

Once the Vg and VgR form a complex and have been transported into the oocytes, the entry of the complex into yolk vesicle is controlled by internalization signals [3]. Vertebrate LDLR family members, including the GgVgR/LR8, contain one or more "tight-turn tyrosine" internalization signals (FXNPXY). Although A. aegypti VgR and Drosophila YPR share a region of homology with the vertebrate signal, their motifs lack the critical Tyr residue necessary for internalization [3]. Instead, both insect receptors contain dileucine and/or leucine isoleucine motifs, recently identified as alternative internalization signals in some receptors. For example, Roehrkasten and Ferenz [29] studied the role of the lysine and arginine residues of Vg in high-affinity binding to VgRs in locust oocyte and yolk vesicle membranes. P. monodon has two putative internalization signals (i.e., FANPGFG and FENPFF) and several IL and LI sites (Fig. 2), suggesting that it may use either one or both of these two types as internalization signals.

Currently, there is no information on VgR expression in other crustaceans. However, like most insects, the shrimp VgR was expressed exclusively in the ovary (Fig. 5). During shrimp gonad maturation cycle, VgR expression levels were high at early vitellogenic stage (i.e., GSI = 3–4) and declined thereafter. VgR transcript was also present in small oocytes less than 100 µm in diameter (previtellogenic oocytes), and the transcript level increased at the onset of Vg uptake into the oocytes. Notice also that VgR transcript level is high in the fully matured female (GSI = 12.4). This may be due to the need of new VgR proteins for another cycle of Vg uptake. This pattern is similar to that of the insects. In the cockroaches, P. americanus and B. germanica, VgR mRNA levels are high at previtellogenic period, and decline thereafter during the remaining vitellogenic phase, consistent with this interpretation. VgR transcripts can be detected in previtellogenic oocytes of less than 0.3 mm in diameter up to oocytes in early vitellogenic development measuring up to 1 mm [30]. In rainbow trout, VgR mRNA was expressed at high levels in previtellogenic ovary (i.e., small follicle cells <0.3 mm in diameter), and maximal levels of expression were reached in follicles initiating vitellogenic development [30]. The previtellogenic stage appears to be a period of development during which there is a significant increase in the number of mRNAs coding for receptors and enzymes (e.g., cathepsin D, which mediates the proteolytic processing of Vg into yolk that are required for vitellogenic development). Vg sequestration in rainbow trout occurs until a period shortly before ovulation, when oocytes measure up to 5 µm in diameter. Transcription of Vg receptor mRNA ceases well before this time [31].

The cellular distribution of the shrimp VgR protein, as determined by immunocytochemical techniques, was also consistent with the interpretation that this protein is a VgR. In early vitellogenic ovary of the shrimp, VgR was distributed evenly throughout the oocyte cytoplasm, with a dense accumulation in the membrane of the larger oocytes (Fig. 7B). In the previtellogenic fruit fly, D. melanogaster, the mosquito, A. aegypti, and the cockroaches, P. americana and B. germanica, VgR is spread over the oocyte cytoplasm and accumulates in the cortex (i.e., oocyte membrane) only after vitellogenesis begins [11, 30]. Thus, these VgR (including the shrimp VgR) proteins are recruited to the oocyte surface before Vg uptake is initiated, as has also been reported in fish, frog, and chicken [32, 33]. In insects and shrimp, such relocalization of VgR to oocyte membrane suggests that protein sorting in the oocyte membrane is spontaneous and follows the universal pathway involving the exocyte complex [34].

RNAi with dsRNA successfully knocked down VgR expression in the cockroach, B. germanica, and the tick, D. variabilis [30, 34]. The loss of ligand binding function led to inability of oocytes to enter the rapid growth phase, and in the cockroach, accumulation of Vg in the hemolymph was so dramatic that the protein was processed as in the ovary [24, 30]. Thus, in vivo RNAi experiments were attempted to knock down VgR gene function. The shrimp VgR dsRNA efficiently and specifically disrupted the function of Vg uptake by the oocyte as the amount of extraovarian Vg accumulated in the hemolymph and the amount of Vg in the oocyte in the dsRNA-treated shrimp decreased. The absence of any significant effect on Vg transcript level in ovary and hepatopancreas suggested that the lower amount of ovarian Vg is due to the lack of Vg uptake into the oocyte because of the lower amount of functional VgR on the oocyte membrane.

In conclusion, a full-length cDNA that encodes for a protein with the characteristics of an LDLR or VgR has been cloned and identified in the shrimp, P. monodon. Biochemical, molecular, and RNAi analyses indicate that this receptor is likely to be involved in the endocytosis of extraovarian Vg into developing egg cells, and that the cDNA most likely encodes a shrimp VgR.

FOOTNOTES

1Supported by RGC grant HKU 7484/06M of the Hong Kong Special Administrative Region government to S.-M.C. The Penaeus monodon vitellogenin receptor cDNA sequence has been submitted to the GenBank database with accession number EU024890. Back

Correspondence: 2Siu-Ming Chan, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, PR China. FAX: 852 2857 4672; e-mail: chansm{at}hkucc.hku.hk

Received: 26 October 2007.

First decision: 20 November 2007.

Accepted: 8 January 2008.

REFERENCES

  1. Schneider WJ. Vitellogenin receptors: oocyte-specific members of the low-density lipoprotein receptor supergene family [review]. Int Rev Cytol 1996; 166:103–137.[Medline]
  2. Willnow TE. The low-density lipoprotein receptor gene family: multiple roles in lipid metabolism [review]. J Mol Med 1999; 77:306–315.[CrossRef][Medline]
  3. Sappington TW, Raikhel AS. Molecular characteristics of insect vitellogenins and vitellogenin receptors [review]. Insect Biochem Mol Biol 1998; 28:277–300.[CrossRef][Medline]
  4. Bujo H, Hermann M, Kaderli MO, Jacobsen L, Sugawara S, Nimpf J, Yamamoto T, Schneider WJ. Chicken oocyte growth is mediated by an eight ligand binding repeat member of the LDL receptor family. EMBO J 1994; 13:5165–5175.[Medline]
  5. Okabayashi K, Shoji H, Nakamura T, Hashimoto O, Asashima M, Sugino H. cDNA cloning and expression of the Xenopus laevis vitellogenin receptor. Biochem Biophys Res Commun 1996; 224:406–413.[CrossRef][Medline]
  6. Stifani S, Nimpf J, Schneider WJ. Vitellogenesis in Xenopus laevis and chicken: cognate ligands and oocyte receptors. The binding site for vitellogenin is located on lipovitellin. J Biol Chem 1990; 265:882–888.[Abstract/Free Full Text]
  7. Davail B, Pakdel F, Bujo H, Perazzolo LM, Waclawek M, Schneider WJ, Le Menn F. Solution of oogenesis: the receptor for vitellogenin from the rainbow trout. J Lipid Res 1998; 39:1929–1937.[Abstract/Free Full Text]
  8. Li A, Sadasivam M, Ding JL. Receptor-ligand interaction between vitellogenin receptor (VtgR) and vitellogenin (Vtg), implications on low density lipoprotein receptor and apolipoprotein B/E. The first three ligand-binding repeats of VtgR interact with the amino-terminal region of Vtg. J Biol Chem 2003; 278:2799–2806.[Abstract/Free Full Text]
  9. Barber DL, Sanders EJ, Aebersold R, Schneider WJ. The receptor for yolk lipoprotein deposition in the chicken oocyte. J Biol Chem 1991; 266:18761–18770.[Abstract/Free Full Text]
  10. Bujo H, Lindstedt KA, Hermann M, Mola Dalmau L, Nimpf J, Schneider WJ. Chicken oocytes and somatic cells express different splice variants of a multifunctional receptor. J Biol Chem 1995; 270:23546–23551.[Abstract/Free Full Text]
  11. Schonbaum CP, Lee S, Mahowald AP. The Drosophila yolkless gene encodes a vitellogenin receptor belonging to the low density lipoprotein receptor superfamily. Proc Natl Acad Sci U S A 1995; 92:1485–1489.[Abstract/Free Full Text]
  12. Cheon HM, Seo SJ, Sun J, Sappington TW, Raikhel AS. Molecular characterization of the VLDL receptor homologue mediating binding of lipophorin in oocyte of mosquito Aedes aegypti. Insect Mol. Biol 2001; 31:753–760.
  13. Chen ME, Lewis DK, Keeley LL, Pietrantonio PV. cDNA cloning and transcriptional regulation of the vitellogenin receptor from the imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae). Insect Mol Biol 2004; 13:195–204.[CrossRef][Medline]
  14. Tufail M, Takeda M. Molecular cloning, characterization and regulation of the cockroach vitellogenin receptor during oogenesis. Insect Mol Biol 2005; 14:389–401.[CrossRef][Medline]
  15. Cho KH, Raikhel AS. Organization and developmental expression of the mosquito vitellogenin receptor gene. Insect Mol Biol 2001; 10:465–474.[CrossRef][Medline]
  16. Sappington TW, Kokoza VA, Cho WL, Raikhel AS. Molecular characterization of the mosquito vitellogenin receptor reveals unexpected high homology to the Drosophila yolk protein receptor. Proc Natl Acad Sci U S A 1996; 93:8934–8939.[Abstract/Free Full Text]
  17. Kung SY, Chan SM, Hui JH, Tsang WS, Mak A, He JG. Vitellogenesis in the sand shrimp, Metapenaeus ensis: the contribution from the hepatopancreas-specific vitellogenin gene (MeVg2). Biol Reprod 2004; 71:863–870.[Abstract/Free Full Text]
  18. Tiu SH, Hui JH, He JG, Tobe SS, Chan S-M. Characterization of vitellogenin in the shrimp Metapenaeus ensis: expression studies and hormonal regulation of MeVg1 transcription in vitro. Mol Reprod Dev 2006; 73:424–436.[CrossRef][Medline]
  19. Tiu SHK, J Hui HL, Mak ASC, He JG, Chan S-M. Equal contribution of hepatopancreas and ovary to the production of vitellogenin (PmVg1) transcripts in the tiger shrimp, Penaeus monodon. Aquaculture 2006; 254:666–674.[CrossRef]
  20. Tseng DY, Chen YN, Kou GH, Lo CF, Kuo CM. Hepatopancreas is the extraovarian site of vitellogenin synthesis in black tiger shrimp, Penaeus monodon. Comp Biochem Physiol A Mol Integr Physiol 2001; 129:909–917.[CrossRef][Medline]
  21. Jugan P, Soyez D. Demonstration in vitro de l'inhibition de l'endocytose ovocytaire par un extrait de glande de sinus chez la crevette Macrobrachium roenbergii. CR Acad Sci Paris Ser III 1985; 300:705–709.
  22. Laverdure AM, Soyez D. Vitellogenin receptor from lobster oocyte membrane: solubilization and characterization by solid phase binding assay. Int J Invert Reprod Develop 1988; 13:251–266.
  23. Warrier S, Subramoniam T. Receptor mediated yolk protein uptake in the crab Scylla serrata: crustacean vitellogenin receptor recognizes related mammalian serum lipoproteins. Mol Reprod Dev 2002; 61:536–548.[CrossRef][Medline]
  24. Tufail M, Takeda M. Molecular cloning, characterization and regulation of the cockroach vitellogenin receptor during oogenesis. Insect Mol Biol 2005; 14:389–401.[CrossRef][Medline]
  25. Castresana J. Selection of conserved blocks from multiple alignment intended for their use in phylogenetic analysis. Mol Biol Evol 2000; 17:540–522.[Abstract/Free Full Text]
  26. Chow JFC, Chan KM. Isolation of two actin gene fragments from Chlorella vulgaris. J Mar Biotech 1997; 5:58–72.
  27. Rasband WS. Image J. Bethesda MD: >US National Institute of Health 1997–2007. World Wide Web (URL: http://rsb.info.nih.gov/ij/). (May 7, 2007).
  28. Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004; 340:783–795.[CrossRef][Medline]
  29. Roehrkasten A, Ferenz HJ. Role of the lysine and arginine residues of vitellogenin in high affinity binding to vitellogenin receptors in locust oocyte membranes. Biochim Biophys Acta 1992; 1133:160–166.[Medline]
  30. Ciudad L, Piulachs MD, Belles X. Systemic RNAi of the cockroach vitellogenin receptor results in a phenotype similar to that of the Drosophila yolkless mutant. FEBS J 2006; 273:325–335 Erratum in FEBS J 2006; 273:671.[CrossRef][Medline]
  31. Prat F, Coward K, Sumpter JP, Tyler CR. Molecular characterization and expression of two ovarian lipoprotein receptors in the rainbow trout, Oncorhynchus mykiss. Biol Reprod 1998; 58:1146–1153.[Abstract/Free Full Text]
  32. Stifani S, Nimpf J, Schneider WJ. Vitellogenesis in Xenopus laevis and chicken: cognate ligands and oocyte receptors. The binding site for vitellogenin is located on lipovitellin I. J Biol Chem 1990; 265:882–888.[Abstract/Free Full Text]
  33. Stifani S, Le Menn F, Rodriguez JN, Schneider WJ. Regulation of oogenesis: the piscine receptor for vitellogenin. Biochim Biophys Acta 1990; 1045:271–279.[Medline]
  34. Mitchell RD III, Ross E, Osgood C, Sonenshine DE, Donohue KV, Khalil SM, Thompson DM, Roe RM. Molecular characterization, tissue-specific expression and RNAi knockdown of the first vitellogenin receptor from a tick. Insect Biochem Mol Biol 2007; 37:375–388.[CrossRef][Medline]




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