Shrimp Marsupenaeus japonicus (10–12 cm in length) were cultured at 20°C in tanks with seawater. For each treatment, 20 randomly selected individuals were raised in an 80 l aquarium. To ensure that the shrimp were virus-free prior to experiments, the shrimp hemocytes were subjected to PCR detection using WSSV-specific primers (5′-TATTGTCTCTCCTGACGTAC-3′ and 5′-CACATTCTTCACGAGTCTAC-3′). The virus-free shrimp were intramuscularly injected with 0.1 ml of WSSV solution (10⁵ copies/ml) using a syringe with a 29-gauge needle. The WSSV solution was obtained from WSSV-infected shrimp (18). At various times postinfection, the shrimp hemocytes were collected for further analysis.

Total miRNAs were extracted from shrimp hemocytes using a mirVana miRNA isolation kit (Ambion, USA) following the manufacturer’s instructions. The RNAs were separated on a denaturing 15% polyacrylamide gel containing 7 M urea in 1× TBE buffer (90 mM Tris–boric, 2 mM EDTA, pH 8.0) and subsequently transferred to a Hybond-N+ nylon membrane. After UV cross-linking, the membrane was prehybridized in DIG (digoxigenin) Easy Hyb granule buffer (Roche, Basel, Switzerland) for 0.5 h at 42°C and then hybridized with DIG-labeled miR-12 (5′-ACCAGTACCTGATGTAATACTCA-3′) or U6 (5′-GGGCCATGCTAATCTTC TCTGTATCGTT-3′) probe at 42°C overnight. The DIG labeling and detection were performed using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Germany) according to the manufacturer’s instructions.

To overexpress or knock down the expression of miR-12 in shrimp, miR-12 (5′-T GAGTATTACATCAGGTACTGGT-3′) synthesized using the in vitro transcription T7 kit (TaKaRa, Japan) or the synthesized anti-miR-12 oligonucleotide (AMO-miR-12) (5′-ACCAGTACCTGATGTAATACTTCA-3′) was injected into shrimp at 30 μg/shrimp. As controls, the sequence of miR-12 or AMO-miR-12 was randomly scrambled, generating miR-12-scrambled (5′-ACTCATAATGTAGTCCAT GACCA-3′) or AMO-miR-12-scrambled (5′-TGGTCATGGACTACATTATGAGT-3′). All synthesized miRNAs were dissolved in miRNA solution (50 mM Tris–HCl, 100 mM NaCl, pH 7.5) and quantified using NanoDrop ND-100 spectrophotometer. At different times after injection, three shrimp were randomly selected from each treatment, and the selected shrimp hemocytes were collected and mixed for later use. The above assays were biologically repeated three times.

To evaluate the proliferation of WSSV in shrimp, the quantitative real-time PCR was performed using WSSV-specific primers (5′-TTGGTTTCATGCCCGAGATT-3′ and 5′-CCTTGGTCAGCCCCTTGA-3′) and a TaqMan fluorogenic probe (5′-FAM-TGCTGCCGTCTCCAATAMRA-3′). The PCR mixture (10 µl) consisted of 5 µl Premix Ex Taq (TaKaRa, Japan), 200 ng DNA template, 0.2 µl of 10 µM primers, and 0.2 µl of 10 µM TaqMan fluorogenic probe at a final concentration of 0.2 µM. The DNA template was extracted from the shrimp hemocytes using an SQ tissue DNA isolation kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. A plasmid containing a 1,400-bp DNA fragment from the WSSV genome was used as the reference plasmid as previously described (19). The PCR conditions were 95°C for 1 min, followed by 45 cycles at 95°C for 30 s, 52°C for 30 s, and 72°C for 30 s.

To label WSSV virions with FITC, the purified virions were incubated in 1 mg/ml FITC (Sigma, USA) solution (dissolved in 0.1 M NaHCO₃, pH 9.0) for 1 h at room temperature, followed by washing with 0.1 M NaHCO₃. The shrimp hemocytes were rinsed with cold PBS (50 mM Tris–HCl, 100 mM NaCl, pH 7.5). To perform the phagocytosis assay, the FITC-labeled WSSV virions were mixed with shrimp hemocytes at a ratio of 50:1 (WSSV copies: cell numbers) and subsequently incubated at 28°C for 30 min. The hemocytes were rinsed with PBS to remove unphagocytosed FITC-labeled WSSV virions. After centrifugation at 200 × g for 10 min, the hemocytes were resuspended in 1% paraformaldehyde (Sigma, USA). Finally, the hemocytes were analyzed using flow cytometry (Beckman Coulter, USA). For each sample, 10,000–20,000 hemocytes were assessed. The experiments were biologically repeated three times.

The detection of shrimp hemocyte apoptosis using annexin V (Invitrogen, USA) was conducted following the manufacturer’s instructions. Shrimp hemocytes were washed with cold PBS. Then the hemocytes were incubated in 1× annexin binding buffer at 1 × 10⁶ cells/ml, followed by the addition of 5 µl of Alexa Fluor 488-annexin V and 1 µl of 100 µg/ml propidium iodide (PI) in the dark. After incubation for 15 min, 400 µl of 1× annexin binding buffer was added to each sample to stop the reaction. The sample was analyzed using flow cytometry.

Shrimp hemocytes were collected and separated on poly-l-lysine-coated glass slides (Sigma), followed by standing for 10 min at 4°C. The hemocytes were fixed in 4% paraformaldehyde for 25 min at 4°C. The fixed hemocytes were washed with cold PBS and then subjected to the permeabilization with 0.2% Triton X-100 for 5 min. Next, the hemocytes were equilibrated in 100 µl of equilibration buffer at 4°C for 10 min. The equilibrated hemocytes were counterstained with PI after incubation with rTdT mix in a humid environment for 1 h. Subsequently, 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) was added to the slide to stop the reaction. The slide was covered with antifade solution (Invitrogen) to prevent signal quenching.

The apoptosis of shrimp hemocytes was detected using a Caspase-Glo3/7 assay (Promega, USA) according to the manufacturer’s instructions. Briefly, shrimp hemocytes were harvested and washed with PBS. Subsequently the hemocytes were incubated with Caspase-Glo 3/7 reagent at room temperature for 2 h. The caspase 3/7 activity was measured using a Synergy 2 Multi-Mode microplate reader (BioTek).

To predict the target genes of miR-12, four algorithms including TargetScan, miRanda, Pictar, and miRInspector were used to predict the targeted sites in the 3′UTRs of the WSSV genes and the shrimp genes as previously described (20).

To explore the direct interaction between the predicted targets and miR-12, the 3′UTR of a predicted target gene was cloned into the pIZ/V5-His vector (Invitrogen, USA), generating a recombinant plasmid containing EGFP and the 3′UTR of a predicted target. As a control, the 3′UTR sequence matching the seed sequence of miR-12 was scrambled to produce the mutant construct. All the recombinant plasmids were confirmed through sequencing.

Insect High Five cells (Invitrogen) were cultured in Express Five serum-free medium (Invitrogen) containing l-glutamine (Invitrogen) at 27°C. At 70% confluence, the cells were co-transfected with 2 µg of EGFP, EGFP-target gene 3′UTR or EGFP-Δtarget gene 3′UTR and 100 pM of miR-12 or miR-12-scrambled using Cellfectin transfection reagent (Invitrogen) according to the manufacturer’s instructions. The miRNAs were synthesized at Shanghai GenePharma Co., Ltd. (Shanghai, China). At 48 h after co-transfection, the fluorescence intensity of cells was assessed using a Flex Station II microplate reader (Molecular Devices, USA) at 490/510 nm excitation/emission (Ex/Em). The experiments were biologically repeated three times.

The mRNA levels of wsv024, PTEN, PARP, and BI-1 were quantified with real-time PCR using sequence-specific primers (wsv024, 5′-CATCCTGTTAGAGT TTCCTGTTTC-3′ and 5′-ATATTACTGCCATGTTATCTGTTGC-3′; PTEN, 5′-CCAAAATAACCACAACAACAC-3′ and 5′-CACTTCCTGCTCTCCCTT-3′; PARP1, 5′-GAAGAATTACAACTGCGTCCTG-3′ and 5′-GTTCCTCGAAATGGG CTATG-3′; BI-1, 5′-TGCGGCTTCATTGTTTACG-3′ and 5′-CACGGTTCTTCCTCTTGTTCTG-3′; and β-actin, 5′-CGAGCACGGCATCGTTACTA-3′ and 5′-TTGTAG AAAGTGTGATGCCAGATCT-3′). Shrimp hemocytes were collected and subjected to total RNA extraction using the RNAprep Pure Cell/Bacteria kit (Tiangen Biotech Co. Ltd., Beijing, China). The cDNA was synthesized using the PrimeScript™ 1st strand cDNA synthesis kit (Takara, Japan). Quantitative real-time PCR was performed in a total volume of 25 µl containing 5 µl of SYBR^® Premix Ex Taq, 0.5 µl of 10 µM forward and reverse primers, and 100 ng of cDNA template. The PCR conditions were 95°C for 1 min, followed by 40 cycles at 95°C for 15 s and 60°C for 45 s.

According to the gene sequence, siRNAs specifically targeting these genes were synthesized, generating the corresponding siRNAs (wsv024-siRNA, 5′-CGAUGAG UACUUGUCUAGCGUUUAA-3′; PTEN-siRNA, 5′-UAGAGUAGCAGAUGUU UGAAGUGUA-3′; and I-1-siRNA, 5′-GCAAACUGGAGAAAGUGCUUUCUGA-3′). As controls, the siRNA sequences were randomly scrambled, producing the corresponding scrambled siRNAs (wsv024-siRNA-scrambled, 5′-CGAAGUACUUG UCUAGCGUUUGUAA-3′; PTEN-siRNA-scrambled, 5′-UAGGAUGACGUAUUU AAGUGAGGUA-3′; BI-siRNA-scrambled, 5′-GCAGGUCAAGAGUGAUUUCCA AUGA-3′). All siRNAs were synthesized using an in vitro transcription T7 kit (TaKaRa, Japan) according to the manufacturer’s instructions. The synthesized siRNAs were quantified using spectrophotometry and dissolved in siRNA buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl).

The shrimp were injected with siRNA (4 nM) or siRNA-scrambled (4 nM) and WSSV (10⁴ copies/shrimp). At different times after injection, the hemocytes of three randomly selected shrimp from each treatment were collected for later use.

The proteins were analyzed in a 12% SDS-PAGE gel and then transferred onto a nitrocellulose membrane (Bio-Rad, USA). The membrane was blocked in blocking buffer [3% bovine serum albumin in TBST (250 mM NaCl, 10 mM Tris–HCl, 0.5 ml/l Tween 20, pH 7.4)] for 2 h at 4°C. Subsequently the membrane was incubated with a primary antibody (anti-wsv024 IgG, anti-BI-1 IgG, anti-PTEN IgG, or anti-actin IgG) for 2 h at 4°C. After two washes with TBST, the membrane was incubated with HRP (horseradish peroxidase)-conjugated anti-mouse IgG (Sigma, USA) for 1 h at 4°C, followed by chemiluminescence detection with ECL substrate (Thermo Scientific, USA). All of the primary antibodies were prepared in our laboratory.

The 3′UTRs of wsv024, PTEN, and BI-1 mRNAs were amplified with specific primers (wsv024, 5′-TAATACGACTCACTATAGGTCTTCGTTAAAATCAGTTTTACCT-3′ and 5′-GTGCTAATAAACCAACGTCTTTC-3′; PTEN, 5′-TAATACGACTCACTATAGGGCGCACGAAGCTTTGGC-3′ and 5′-TCCTGTACACTACAAATAATAAGCCTC-3′; BI-1-F, 5′-TAATACGACTCACTATAGGGATGCCACA GATGTAATTTAGAAG-3′ and 5′-AATAGGATAATCACATGAAATTCTG-3′), which contained T7 promoter sequence at their 5′-ends of the forward primers, using a T7 transcription kit (TaKaRa, Japan) according to the manufacturer’s instructions. To get the Ago1 complex of shrimp, shrimp muscles were homogenized in lysis buffer [20 mM Tris–HCl, 150 mM NaCl, 1.5 mM MgCl₂, 0.25% NP-40 (Non-idet P-40), and 1 mM PMSF (phenylmethanesulfonyl fluoride), pH 7.5] for 15 min on ice and subsequently centrifuged at 10,000 × g for 10 min at 4°C. The cell supernatants were incubated with the shrimp Ago1-specific antibody, prepared in our laboratory, for 1 h at 4°C, followed by mixing with protein-G-coupled agarose beads for 4 h at 4°C. After washes with cold PBS, the beads were resuspended in reaction buffer [100 mM KOAc, 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5 mM MgCl₂, 2 mM Dithiothreitol, 0.35% Triton X-100, and 1 mM PMSF, pH 7.6]. To investigate the miRNA-mediated degradation of target mRNAs, the co-immunoprecipitated products of shrimp Ago1, 3′UTR (wsv024, PTEN, and/or BI-1) (100 ng), and miR-12 were mixed at different concentrations and supplemented with 2 µl of 10 mM ATP and 2 µl of 2 mM GTP at a total volume of 20 µl. After incubation at 30°C for different times, the RNA samples were separated using 1% agarose gel electrophoresis and then transferred to a nylon membrane (GE Healthcare, USA). The RNAs were detected using a 5′ DIG (digoxigenin)-labeled probe (wsv024, 5′-TCACGGTTATGGCCAGCGCTACGC-3′; PTEN, 5′-GAACACAGAGCAGAG GGGATGATAG-3′; and BI-1, 5′-TCTCAAACTCATCTATATCATTC-3′).

The mRNA 3′UTR of the miR-12 target gene was incubated with miR-12 (20 nM) and Ago1 complex at 30°C for 2 h. Subsequently, the degraded fragments were separated using 1% agarose electrophoresis. After recovery from the agarose gel, the RNAs were reverse transcribed into cDNAs using the PrimeScript™ II 1st strand cDNA synthesis kit with random 6 primer (TaKaRa, Japan). Then, the single-stranded cDNAs were transformed into double-stranded cDNAs using the second strand cDNA synthesis kit (Beyotime Biotechnology, China). The cDNAs were cloned into the pEASY^®-Blunt simple cloning vector (Transgen Biotech, China) and subjected to sequencing.

All numerical data presented in the present study were analyzed using one-way analysis of variance to calculate the means and SDs of triplicate assays.

To explore the role of host miR-12 in virus–host interactions, the expression level of miR-12 in WSSV-challenged shrimp was characterized. Northern blot analysis indicated that miR-12 was significantly upregulated in shrimp in response to WSSV infection, suggesting that miR-12 played important roles in host–virus interactions (Figure 1A).

To assess the roles of miR-12 in virus–host interactions, miR-12 expression was overexpressed or knocked down in shrimp, followed by the evaluation of virus infection. Northern blot analysis indicated that miR-12 expression in shrimp hemocytes was significantly upregulated from 24 to 48 h after the miR-12 injection compared with the controls (Figure 1B). The results showed that miR-12 overexpression led to significant decreases in WSSV copies in shrimp compared with the controls (WSSV, WSSV + miR-12-scrambled and WSSV + AMO-miR-12-scrambled) (Figure 1C). However, when miR-12 expression was inhibited by AMO-miR-12 (Figure 1B), the WSSV copies were dramatically increased compared with the controls (Figure 1C), indicating that miR-12 played a negative role in the virus infection. The results revealed that the miR-12 expression level in PBS-treated shrimp was identical to those of miR-12-scrambled and AMO-miR-12-scrambled-treated shrimp (Figure 1B), showing that there was no miR-12 or AMO-miR-12 toxicity in shrimp.

The above data implied that miR-12 possesses antiviral activity in shrimp.

To reveal the mechanism of miR-12-mediated antiviral immunity in shrimp, the WSSV genes targeted by miR-12 were characterized. The prediction results obtained using TargetScan, miRanda, Pictar and miRInspector algorithms showed that wsv024 was a potential target of miR-12 (Figure S1A in Supplementary Material).

To assess the interaction between miR-12 and wsv024, a recombinant plasmid containing EGFP and wsv024 3′UTR (pIZ/EGFP-wsv024-3′UTR) was co-transfected with miR-12 into insect High Five cells (Figure S1B in Supplementary Material). The results indicated that the fluorescence intensity of the insect cells co-transfected with miR-12 and pIZ/EGFP-wsv024-3′UTR was remarkably decreased compared with the controls (Figure 2A), showing that miR-12 could directly target the wsv024 gene.

To evaluate the interaction between miR-12 and wsv024 in vivo, miR-12 expression was upregulated or knocked down in WSSV-infected shrimp, followed by the detection of wsv024 expression levels. The results indicated the miR-12 overexpression decreased wsv024 transcript levels in contrast with the controls (WSSV + miR-12-scrambled and WSSV alone) (Figure 2B). When the expression of endogenous miR-12 was reduced by AMO-miR-12, the wsv024 gene expression was significantly upregulated compared with the controls (WSSV + AMO-miR-12-scrambled and WSSV alone) (Figure 2B). These data showed that miR-12 interacts with wsv024 in vivo.

To evaluate the influence of the interaction between miR-12 and wsv024 on virus infection, the expression of wsv024 was knocked down using wsv024-specific siRNA (wsv024-siRNA) in shrimp, followed by an assessment of WSSV proliferation. The results indicated that the expression of wsv024 was efficiently silenced by wsv024-siRNA compared with the controls (Figures 2C,D). In addition, wsv024 silencing resulted in remarkable inhibition of WSSV proliferation in shrimp (Figure 2E), indicating that wsv024 plays a positive role in WSSV infection.

Taken together, these findings showed that miR-12 functioned in the antiviral immunity of shrimp by targeting the WSSV wsv024 gene.

In a previous study, we revealed that shrimp miR-12 was implicated in antiviral phagocytosis and antiviral apoptosis in shrimp (17). To investigate the mechanism of antiviral phagocytosis and antiviral apoptosis mediated through miR-12, the shrimp genes targeted by miR-12 were predicted. As predicted, PARP1 [poly (ADP-ribose) polymerase 1], PTEN (phosphatase and tensin homolog), and BI-1 (transmembrane BAX inhibitor motif containing 6) genes were potential targets of miR-12 (Figure S2A in Supplementary Material). BI-1 and PARP1 are inhibitors of apoptosis (21, 22), while PTEN is a negative regulator of phagocytosis (23). In this context, the role of miR-12 in the antiviral phagocytosis of shrimp was characterized.

As shown in Figure 3A, when miR-12 was overexpressed, the phagocytic activity of shrimp hemocytes was significantly enhanced. However, miR-12 silencing remarkably decreased shrimp hemocyte phagocytic activity (Figure 3A). These results revealed that miR-12 exerted a positive effect on the antiviral phagocytosis of shrimp to fight against virus infection.

To reveal the mechanism of miR-12-mediated antiviral phagocytosis in shrimp, the interaction between miR-12 and its potential target PTEN gene was explored. Recombinant plasmids containing EGFP and PTEN 3′UTR or ΔPTEN 3′UTR were co-transfected with miR-12 or miR-12-scrambled into insect cells (Figure S2B in Supplementary Material). The results indicated that the fluorescence intensity of cells co-transfected with miR-12 and pIZ/EGFP-PTEN-3′UTR remarkably decreased compared with the controls (Figure 3B), showing that miR-12 directly interacted with PTEN. To evaluate the interaction between miR-12 and PTEN in vivo, miR-12 was overexpressed or knocked down, followed by the assessment of PTEN mRNA and protein levels. The data revealed that miR-12 overexpression led to a significant reduction in PTEN transcript and protein levels, while miR-12 silencing significantly increased the PTEN mRNA and protein levels (Figure 3C). These results showed that miR-12 could target PTEN.

To characterize the role of PTEN in phagocytosis, the expression of PTEN was knocked down, followed by an assessment of the antiviral phagocytic activity of shrimp hemocytes. The results showed that the injection of PTEN-siRNA resulted in a significant reduction of the PTEN transcript level (Figure 3D), indicating that PTEN expression was silenced by PTEN-siRNA. Western blots yielded similar results (Figure 3D). When PTEN was silenced, the shrimp phagocytic activity against WSSV was dramatically strengthened compared with controls (Figure 3E), indicating that PTEN was required in shrimp phagocytosis. Further results showed that PTEN silencing led to the remarkable suppression of WSSV replication in contrast with the controls (Figure 3F). These data revealed that PTEN exerted negative effects on the antiviral phagocytosis of shrimp.

The above findings showed that miR-12 could enhance the antiviral phagocytosis of shrimp by directly targeting PTEN, a phagocytosis negative regulation gene.

To evaluate the effects of miR-12 on apoptosis, miR-12 was overexpressed or knocked down in shrimp, and then the apoptotic activity of shrimp hemocytes was assessed. The results of annexin V analysis showed more annexin V-positive hemocytes in WSSV-infected shrimp than in PBS-treated shrimp (Figure 4A). The miR-12 overexpression augmented the percentage of apoptotic hemocytes in contrast with that of shrimp injected with miR-12-scrambled and WSSV or WSSV alone (Figure 4A), suggesting that miR-12 played a positive role in host cell apoptosis. Contrarily, miR-12 silencing diminished the abundance of the annexin V-positive hemocytes (Figure 4A). TUNEL assays and the detection of caspase 3/7 of shrimp hemocytes yielded results similar to those obtained in the annexin V analysis (Figures 4B,C). These results showed that miR-12 was involved in the positive regulation of apoptosis in shrimp.

As predicted, the PARP1 and BI-1 genes encoding inhibitors of apoptosis were potential targets of miR-12 (Figure S2A in Supplementary Material). To explore the underlying mechanism of miR-12-mediated antiviral apoptosis, the interactions between miR-12 and PARP1 and BI-1 were characterized.

Recombinant pIZ/EGFP-BI-1-3′UTR and pIZ/EGFP-ΔBI-1-3′UTR were constructed (Figure S3 in Supplementary Material), and the recombinant plasmids were co-transfected with miR-12 or miR-12-scrambled into insect cells. The results indicated that the fluorescence intensity of the cells co-transfected with pIZ/EGFP-BI-1-3′UTR and miR-12 was significantly decreased compared with the controls (Figure 4D), showing that miR-12 directly interacted with BI-1. To determine whether miR-12 interacted with BI-1 in vivo, miR-12 was overexpressed or silenced in shrimp, followed by an assessment of the BI-1 expression level. The results showed that miR-12 overexpression significantly reduced BI-1 expression levels compared with the controls, while knocking down miR-12 expression by injection with AMO-miR-12 significantly upregulated BI-1 expression in shrimp (Figure 4E). These data indicated that miR-12 interacted with BI-1 in vivo.

To investigate the interaction between PARP1 and miR-12, the synthesized miR-12 and the pIZ/EGFP-PARP1-3′UTR construct were co-transfected into insect cells. The results showed no significant difference in the fluorescence intensity between various treatments (Figure S4 in Supplementary Material). The in vivo assays revealed that the PARP1 expression level did not change in shrimp when miR-12 was overexpressed or silenced (Figure S4 in Supplementary Material). These results indicated that miR-12 did not interact with PARP1. Therefore, miR-12 could promote apoptosis by targeting the BI-1 gene.

To further reveal the role of BI-1 in apoptosis, BI-1 expression was knocked down by BI-1-siRNA, followed by evaluation of apoptosis. The results showed that the BI-1 expression in shrimp was silenced using BI-1-siRNA (Figure 4F), indicating the specificity of BI-1-siRNA. BI-1 silencing significantly increased caspase3/7 activity in shrimp hemocytes compared with the controls (Figure 4G), showing that BI-1 exerted an inhibitory effect on apoptosis. The data from the annexin V and TUNEL assays yielded results similar to that of caspase 3/7 activity detection (Figures 4H,I). These findings revealed that BI-1 was an inhibitor of apoptosis.

To explore the role of BI-1 in virus infection, the expression of BI-1 was silenced by BI-1-siRNA, and then the WSSV copy number in shrimp was assessed. The results revealed that BI-1 silencing significantly suppressed the proliferation of WSSV compared with the controls (Figure 4J). These data indicated that BI-1 exerted a positive effect on WSSV infection in shrimp.

The above findings suggested that miR-12 could trigger the antiviral apoptosis of shrimp by downregulating expression of the BI-1 gene, which encodes an inhibitor of apoptosis.

The above data showed that shrimp miR-12 promoted antiviral phagocytosis and antiviral apoptosis and inhibited virus infection by targeting the shrimp genes PTEN and BI-1 and the viral gene wsv024 in shrimp. To further explore the mechanism of the miR-12-mediated simultaneous triggering of multiple antiviral pathways in shrimp, the miR-12-mediated degradation of three different target genes was evaluated. The results of time-course experiments showed that the amount of degraded fragments of three target mRNAs increased with extended reaction time (Figure 5A), indicating that miR-12 mediated the degradation of its target mRNAs in the Ago1 complex. Additional data revealed that miR-12 mediated the degradation of three target mRNAs in a miRNA-concentration-dependent manner (Figure 5B).

To evaluate whether miR-12 mediated the synchronous degradation of multiple target mRNAs, the 3′UTRs of PTEN, BI-1, and wsv024 mRNAs were mixed at equivalent amounts and then incubated with miR-12 and the Ago1 complex. Northern blots indicated that the three mRNAs were simultaneously degraded (Figure 5C). These data showed that miR-12 could mediate the synchronous degradation of multiple target genes. To confirm the miR-12-mediated degradation of target mRNAs, the degraded 3′UTR fragments of wsv024, PTEN, and BI-1 mRNAs were sequenced. The results indicated that miR-12 could mediate the 5′–3′ exonucleolytic degradation of its target mRNAs, and the miR-12-mediated degradation terminated at a vicinity of the 3′UTR sequence complementary to the seed sequence of miR-12 (Figure 5D).

Taken together, the findings showed that miR-12 simultaneously triggered phagocytosis, apoptosis, and antiviral immunity through synchronous targeting of shrimp genes (PTEN and BI-1) and a viral gene (wsv024) (Figure 5E).