Healthy adult Pacific whiteleg shrimp cultured in our lab, with a body length of 16.1 ± 2.0 cm and a body weight of 12.1 ± 1.30 g, were used for tissue distribution analysis. Shrimp with a body length of 7.6 ± 0.5 cm and a body weight of 3.5 ± 1.0 g were used for WSSV infection, RNA interference, and recombinant protein injection experiments. All shrimp were acclimated in air-pumped circulating sea water at 25°C before experiment.

For tissue distribution analysis, hemolymph of 12 healthy adult shrimp were obtained by using a sterile syringe preloaded with equal volume of modified anticoagulant Alsever solution and then centrifuged immediately at 800 g at 4°C for 10 min (19). The hemocytes were collected and preserved in liquid nitrogen. Then, the hepatopancreas, heart, muscle, lymphoid organ (Oka), intestine, stomach, gill, epidermis, eyestalk, testis, and ovary were dissected and kept in liquid nitrogen.

For WSSV challenge experiment, virus particles were prepared according to the method described by Sun et al. (20). The extracted WSSV was diluted to 500 copies/μl with sterile phosphate-buffered saline (PBS). Experimental animals were divided into 2 groups including WSSV group and PBS group, with 64 individuals in each group. 20 µl WSSV solution containing 10⁴ copies of WSSV was injected into each shrimp in the WSSV group and an equal volume of PBS was injected into each shrimp in the PBS group. All shrimp were cultured in air-pumped circulating sea water with a temperature of 25 ± 1°C. Hemocytes and lymphoid organ (Oka) of 12 shrimp in each group were collected separately at 1, 3, 6, 12, and 24 hour (h) post WSSV infection (hpi).

Total RNA of each tissue was extracted using RNAiso Plus reagent (Takara, Beijing) following the manufacturer’s protocol. The RNA concentration was assessed by Nanodrop 2000 (Thermo Fisher Scientific, USA) and the RNA quality was assessed by electrophoresis on 1% agarose gel. All cDNA samples were synthesized from 1 µg total RNA with PrimeScript RT Reagent Kit (Takara, Beijing). First, genomic DNA (gDNA) was removed with gDNA Eraser. And then, the first-strand cDNA was synthesized by PrimeScript RT Enzyme following the manufacturer’s instructions with random primers.

The ORF sequence of LvVEGFR2 cDNA was obtained from an Illumina-based transcriptome sequencing database of L. vannamei built in our lab (21). Primers LvVEGFR2-F1/LvVEGFR2-R1 and LvVEGFR2-F2/LvVEGFR2-R2 (Table 1) were designed to validate the sequence. The PCR program for the amplification of LvVEGFR2 was as follows: 1 cycle of denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 45 s, annealing at 57°C for 45 s, and extension at 72°C for 45 s, followed by an extension at 72°C for 7 min. The specific products were assessed by electrophoresis on 1% agarose gel. Then, the amplified products were purified using TIANgel Midi purification kit (Tiangen, China) and cloned into pMD19-T vector (Takara, Beijing) and then transformed into Trans 5α competent cell for sequencing.

The complete ORF region and amino acid sequence of LvVEGFR2 was deduced using ORF finder.¹ The ORF sequence and deduced protein sequence of LvVEGFR2 were analyzed with BLAST algorithm at the National Center for Biotechnology Information² and InterProScan software³ and Simple Modular Architecture Research Tool.⁴ Eight protein sequences of VEGFR family members from different species were used to perform multiple alignments using ClustalW2⁵ and a phylogenic tree was constructed by the neighbor-joining (NJ) algorithm using the MEGA 4 software. The reliability of the tree was tested by bootstrapping using 1,000 replications. The information of VEGFRs used in the present study was shown in Table 2.

SYBR Green-based quantitative real-time PCR (qPCR) was performed to detect the gene expression level of LvVEGFR2. A pair of primers LvVEGFR2-qF and LvVEGFR2-qR (Table 1) was designed to detect the expression level of LvVEGFR2 in different tissues and samples after WSSV infection. Primers 18S-F and 18S-R (Table 1) were designed to detect the expression of the internal reference gene, 18S rRNA. The program was as follows: denaturation at 94°C for 2 min; 40 cycles of 94°C for 15 s, annealing temperature for 20 s, and 72°C for 20 s. The PCR product was denatured to produce melting curve to check the specificity of the PCR product.

A plasmid containing the predicted transmembrane motif (TM) of LvVEGFR2 was constructed to study the subcellular localization of LvVEGFR2 protein in mammalian 293T cells. The plasmid, designated as pEGFP-VR2, was constructed based on the expression plasmid pEGFP-N1 (Clontech, USA). The nucleotide sequence between the restriction sites Age I and Not I on pEGFP-N1 was replaced by a designed nucleotide sequence which encoded a IgK_secretion tag (METDTLLLWVLLLWVPGSTGD), the full length of enhanced green fluorescent protein (EGFP), and the predicted TM of LvVEGFR2 and its flanking sequence (from Val⁶⁴⁴ to Ser⁷⁰²), and flanked with the restriction sites Age I and Not I. The designed nucleotide sequence was synthesized by Sangon Biotech company (Shanghai, China) and sub-cloned into pEGFP-N1 using the restriction sites Age I and Not I. Three micrograms of the plasmids pEGFP-VR2 and pEGFP-N1 were transfected into the mammalian 293T cells with Lipofectamine 3000 Reagent (Thermo Fisher Scientific, USA) following the manufacture’s instruction, respectively. The transfected cells were cultured for 24 h, fixed with 4% paraformaldehyde, and stained with 100 ng/ml DAPI (4′,6-diamidino-2-phenylindole) solution. The green and blue fluorescence signals were detected and merged.

To better understand the function of LvVEGFR2, we detected the effects of interference of endogenous LvVEGFR2 on the propagation of WSSV in L. vannamei. One pair of primers with T7 promoter sequences, LvVEGFR2-dsF and LvVEGFR2-dsR (Table 1) were designed to amplify a 701-bp cDNA fragments of LvVEGFR2 as the template for dsRNA synthesis. Primers of dsEGFP-F and dsEGFP-R with T7 promoter sequences (Table 1) were used to amplify a 289-bp DNA fragment of EGFP gene as negative control. The PCR amplification program was as follows: denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 59°C for 30 s and extension at 72°C for 30 s; final extension at 72°C for 10 min. The PCR products were purified using TIANgel Midi purification kit (Tiangen, China). The purified products were used to synthesize the corresponding dsRNAs using TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific, USA). Redundant single-strand RNA was digested by RNaseA (Takara, Beijing). The concentration of synthesized dsRNA was assessed by Nanodrop 2000 (Thermo Fisher Scientific, USA).

In order to optimize the silencing efficiency of dsRNA of LvVEGFR2, 30 healthy shrimp were divided into six groups. Shrimp in each group were injected with negative control or dsRNA with different dosages, including 1, 2, and 4 µg for each individual. At 48 h after interference, cephalothoraxes of four individuals in each group were isolated and frozen in liquid nitrogen for total RNA extraction. The transcription level of LvVEGFR2 was detected by qPCR with primers LvVEGFR2-qF and LvVEGFR2-qR as described in Section “Quantitative Real-time PCR Detection of LvVEGFR2 mRNA Expression.”

In order to explore the effect of LvVEGFR2 silencing on WSSV propagation, 40 shrimp were divided into 2 groups including dsEGFP group and dsVR2 group. One microgram of dsRNA was injected into each shrimp after optimization of RNA interference effect. One microgram of dsEGFP and 1 µg dsLvVEGFR2 dissolved in 20-µl PBS was injected into each shrimp in dsEGFP group and dsVR2 group, respectively. At 48 h after dsRNA injection, 10⁴ copies of WSSV dissolved in 10 µl PBS was injected into each individual. The pleopods of 15 individuals from each group were collected at 24 and 36 h after WSSV injection. The samples collected from each group at each time were divided into three subgroups and frozen in liquid nitrogen for DNA extraction and WSSV copy number detection.

DNA was extracted from the frozen pleopods using Genomic DNA Kit (Tiangen, China) following the manufacturer’s instructions. The WSSV copy number in each DNA samples was quantified by qPCR with primers VP28-qF and VP28-qR (Table 1) according to the method described by Sun et al. (20). Briefly, the plasmid DNA containing a 281 bp fragment of VP28 gene from WSSV was constructed and transfected into Escherichia coli. The plasmid was then extracted and quantified, and the copy number was calculated. Standard curve was constructed using 10-fold dilutions of the plasmid DNA ranging from 10⁸ to 10³. The qPCR was performed with the diluted plasmid DNA and the pleopods DNA under the following conditions: denaturation at 94°C for 2 min; 40 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 20 s. The WSSV copy number per nanogram of pleopods DNA was calculated based on the standard curve.

In order to know whether LvVEGFR2 could regulate downstream signaling pathways, we detected the expression levels of LvFAK and LvPI3K after silencing of LvVEGFR2 gene. Primers LvFAK-qF/LvFAK-qR and LvPI3K-qF/LvPI3K-qR (Table 1) were used to detect the expression level LvFAK and LvPI3K genes. The program was as follows: denaturation at 94°C for 2 min; 40 cycles of 94°C for 15 s, annealing temperature for 20 s, and 72°C for 20 s. The PCR product was denatured to produce melting-curve to check the specificity of the PCR product.

All the assays described above were biologically repeated for three times. For quantitative real-time PCR, four replicates were set for each sample. The relative transcription level of LvVEGFR2 were obtained using 2^−ΔΔCt method (22) and the WSSV copy number per nanogram of DNA was obtained according to the standard curve. In addition, the numerical data from each experiment were analyzed to calculate the mean and standard deviation of triplicate assays. An independent sample t-test was used to analyze the difference between two groups by SPSS 16.0. P < 0.05 was considered statistically significant.

The full-length ORF of LvVEGFR2 cDNA was 3,594 bp encoding 1,197 amino acid residues (accession number: MF417824). As shown in Figure 1, the deduced amino acids sequence of LvVEGFR2 contained five immunoglobulin subtype domains (Ser⁴⁸ to Phe¹⁴¹, Phe¹⁵⁶ to Lys²³⁰, Ala²⁴³ to Lys³³¹, Thr³⁴⁹ to Leu⁴³⁰, and Asp⁵⁵⁵ to Val⁶⁴⁶) and one immunoglobulin-like domain (Pro⁴¹³ to Gln⁵⁵²) were predicted in the extracellular region. TM of 21 aa was located from Asn⁶⁴⁹ to Gly⁶⁷¹ followed by a protein kinase (PK) domain from Ile⁷²⁶ to Leu¹⁰⁰⁷. Moreover, a tyrosine-PK active site (Val⁹⁷¹ to Leu⁹⁸³) existed in the PK domain. Phylogenetic analysis showed that LvVEGFR2 was first clustered together with LvVEGFR1 and then with VEGFR members from insects, while VEGFRs from vertebrates were clustered into another branch (Figure 2). Sequence alignment showed a 55% similarity between LvVEGFR1 and LvVEGFR2 amino acid sequences (data not shown).

The expression levels of LvVEGFR2 in different tissues were shown in Figure 3. The transcript of LvVEGFR2 was mainly detected in hemocytes, followed by Oka and heart. The expression level of LvVEGFR2 was relatively lower in other tissues.

Subcellular localization analysis showed that the fluorescence signals expressed by the constructed plasmid pEGFP-VR2 that contained the predicted transmembrane motif of LvVEGFR2 were mainly detected on the cell membrane of 293T cells, while the fluorescence signals expressed the control plasmid pEGFP-N1 were mainly detected in the cytoplasm of 293T cells (Figure 4).

The expression profiles of LvVEGFR2 in hemocytes and Oka of shrimp after WSSV challenge were detected. The expression levels of LvVEGFR2 in hemocytes (Figure 5A) and Oka (Figure 5B) were significantly upregulated at 24 hour post WSSV infection (hpi).

RNA interference based on dsRNA was performed to study the function of LvVEGFR2 gene. First, the silencing efficiency of LvVEGFR2 was detected under different dsRNA dosages, including 1, 2, and 4 µg for each shrimp. After optimization to the dsRNA dosage for interference, the dose of 4 µg dsRNA per shrimp, with 52.5% inhibition efficiency (Figure 6), was used for further RNAi experiment.

The WSSV copy number was detected in shrimp after dsRNA and WSSV injection. At 48 and 72 hpi, the WSSV copy number in shrimp from dsEGFP group was about 4 × 10⁵ copies/ng DNA, while it was about 1 × 10⁵ copies/ng DNA in shrimp from dsVR2 group, which was significantly lower (P < 0.05) than that of dsEGFP group (Figure 7).

In order to know whether LvVEGFs could regulate the transcription of LvVEGFR2, LvVEGF1, LvVEGF2, and LvVEGF3 were silenced by dsRNA and then the transcriptional level of LvVEGFR2 was detected. After silencing of LvVEGF1 by 48.6%, the transcriptional level of LvVEGFR2 had no obvious change (Figure 8A). After silencing of LvVEGF2 by 87.0%, the transcriptional level of LvVEGFR2 was downregulated by 55.3% (Figure 8B). After silencing of LvVEGF3 by 86.6%, the transcriptional level of LvVEGFR2 was downregulated by 59.3% (Figure 8C).

Injection of the recombinant protein pVF1, pVF2, and pVF3 could also influence the transcriptional level of LvVEGFR2. As shown in Figure 9, injection of pVF1 did not upregulate the transcriptional level of LvVEGFR2 when compared to pET30A injection and PBS control. After injection of pVF2 or pVF3, the transcriptional level of LvVEGFR2 was obviously upregulated by 44 and 106% compared to pET30A injection and by 104 and 192% when compared with PBS control, respectively.

In order to further investigate the interaction between LvVEGFs and LvVEGFR2, the yeast two-hybrid system was used. Expression of reporter genes in the positive control, which was co-transformed with pGBK-p53 and pGAD-T antigen plasmids, could generate blue color in the yeast cells (Figures 10A–C, zone 1). When pGAD-VF2 and pGBK-VR2(1–4) plasmids or pGAD-VF3 and pGBK-VR2(1–4) plasmids were co-expressed in the yeast cells, the blue color was also detected (Figures 10B,C, zone 2), while co-transformation of pGAD-VF1 and pGBK-VR2(1–4) plasmids into the yeast cells did not generate blue color (Figure 10A, zone 2). These data indicated that LvVEGF2 and LvVEGF3 could interact with the Ig domains region of LvVEGFR2. Self-activation and negative controls were set and no blue signal was detected (Figures 10A–C, zones 3–5).

In order to know whether LvVEGFR2 had impact on other signaling pathways, we detected the expression levels of LvFAK and LvPI3K in shrimp after LvVEGFR2 silencing. After silencing of LvVEGFR2 gene, the expression level of LvFAK and LvPI3K was downregulated by about 38 and 32%, respectively (Figure 11).