Introduction

Penaeus monodon or black tiger shrimp is one of the most invaluable shrimp in the aquaculture industry contributing to economic prosperity. However, over the past years, the amount of shrimp export has reduced, mainly due to viral disease outbreaks. White spot syndrome virus (WSSV) is a major virus pathogen causing white spot syndrome and accounted for almost 100% mortality within a few weeks after infection1. So far, there is no proven method for 100% preventing WSSV outbreaks. When foreign particles or pattern pathogen-associated molecular patterns (PAMPs) attach to pattern recognition proteins (PRPs) or receptors (PRRs) in shrimp, the innate immune system is activated through intracellular signaling cascades. The humoral immune responses include the melanin synthesis by the prophenoloxidase (proPO) system, blood clotting system and the generation of circulating antimicrobial peptides (AMPs)2. The cellular responses, on the other hand, cause apoptosis, phagocytosis, nodule formation and encapsulation3.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway was first identified in mammalian systems and shown to transduce the various cytokines and growth factor signals4. The JAK/STAT pathway plays a role in haematopoiesis, immune function, cell growth, differentiation and development5 as well as innate immune and adaptive immune systems6. In mammalian, the main components of the JAK/STAT pathway are wide and diverse range of extracellular ligands (i.e. growth factors, interferons, interleukins and colony-stimulating factors), transmembrane receptors (i.e. interferon receptors IFNAR1, IFNAR2, IFNGR1 and IFNGR2), four JAK members (JAK1, JAK2, JAK3 and TYK2) and seven STAT proteins (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6)7. On the contrary, the JAK/STAT pathway of invertebrates is simpler than vertebrates with only a few components necessary for the signal transduction8. In Drosophila, the JAK/STAT pathway consists of three unpaired ligands (UPD1, UPD2 and UPD3)9, a receptor (Domeless or DOME)10, a JAK (Hopscotch or HOP)11 and a STAT (STAT92E)12. The binding of ligand to the Domeless receptor activates the receptor associated JAK tyrosine kinase Hopscotch, leading to phosphorylation of the receptor/JAK complex, which subsequently phosphorylates STAT92E dimer. Once the activated STAT92E dimers are translocated into the nucleus, they can bind to consensus DNA recognition motifs in the promoters, resulting in a transcription activation13. Several genes including TotA, TotM and virus induced RNA-1 (vir-1) contain STAT-binding sites in their promoter and are activated by the JAK/STAT pathway. Moreover, multiple regulatory proteins have been reported in Drosophila such as protein inhibitor of activated STAT (PIAS), protein tyrosine phosphatases (PTPs) and the suppressor of cytokine signaling (SOCS), the gene expression of which is activated by STAT and thus creates feedback inhibition of the pathway14.

In Pacific white shrimp Litopenaeus vannamei, LvDOMELESS enhanced the wsv069 promoter activity by acting on the STAT-binding motif and knocking down the LvDOMELESS, resulting in lower cumulative mortality of shrimp and less WSSV copies15. Meanwhile, LvJAK silencing caused a higher mortality rate and increased the WSSV viral load16. It was reported that shrimp STAT was hijacked by WSSV to enhance the viral immediate early gene expression17.

In this work, we report the full length of Domeless receptor in the black tiger shrimp Penaeus monodon (PmDOME) and investigate the role of the JAK/STAT pathway during WSSV infection. RNA interference techniques and mortality study were employed and the transcription levels of genes in the Toll, Immune Deficiency (Imd), JAK/STAT, interferon regulatory factor (IRF)-Vago, proPO and AMPs were examined under the silencing of PmDOME or PmSTAT during WSSV challenge. This work might lead to a better understanding of the characteristic of PmDOME and the relationship between the JAK/STAT pathway and the immune-related genes in response to WSSV infection in the black tiger shrimp.

Results

Cloning and sequence analysis of PmDOME

The partial nucleotide sequence related to PmDOME (2038 bp) was retrieved from the P. monodon EST database. The complete full length of PmDOME was obtained by PCR amplification using specific primers (Supplementary Table S1) and a cDNA template prepared from healthy shrimp hemocytes. The full length of PmDOME is 5102 bp including 314 bp of the 5′-untranslated region (5′-UTR), 597 bp of 3′-UTR containing a polyA signal sequence (ATTAAA) and 4191 bp of the open reading frame (ORF) that encoded a putative protein of 1396-amino acid residues (Fig. 1). The cDNA sequence was deposited in the NCBI database (MW187497). The calculated molecular weight of PmDOME is 156.05 kDa and the isoelectric point (pI) is 5.81. The ORF of PmDOME shared high sequence identity with Pacific whiteleg shrimp LvDOMELESS (AGY46351.1, 94.70%)15 and DOME from Marsupenaeus japonicus, MjDOME (APA16577.1, 87.12%)18. Meanwhile, PmDOME exhibited 21.92% sequence identity with Drosophila melanogaster domeless (DmDOME, NP_523412.1) and 28.57% sequence identity with human cytokine receptor (CAA41231.1).

Figure 1
figure 1

Nucleotide and deduced amino acid sequence of PmDOME. Nucleotides and amino acids are both numbered on the left of the sequence. The open reading frame (ORF) of the nucleotide sequence is shown in upper-case letters, while the 5' and 3' UTRs are in lower-case letters. The signal peptide is shaded in red and the cytokine binding motif is underlined. The fibronectin-type-III like domains are highlighted in yellow. The transmembrane domain is in blue. The poly (A) signal is in the box.

Full size image

Moreover, the phylogenetic tree was performed in MEGA 11.0 software to construct a neighbor-joining (NJ) phylogenetic tree based on the deduced amino acid sequence. The bootstrap sampling was reiterated 1000 times and demonstrated that PmDOME was clustered into the group of invertebrates and closely related to those from L. vannamei and M. japonicus (Fig. 2A). NetNGlyc-1.0 and NetOGlyc-4.0 servers predicted that PmDOME contains 19 possible N-glycosylation sites based on the presence of the N-Xaa-T/S motif, while there are 15 possible O-glycosylation sites in the full-length of PmDOME. Meanwhile, the GlycoEP server estimated 26 potential N-glycosylation sites and 59 O-glycosylation sites for PmDOME.

Figure 2
figure 2

Sequence analysis of PmDOME. (A) Phylogenetic tree of cytokine receptors from invertebrates and vertebrates. PmDOME is shown with a dot. Analyzed proteins are CqDOME, Cherax quadricarinatus (Accession No. QKU38106.1), HaCR, Homarus americanus (Accession No. XP_042241219.1), EsDOME, Eriocheir sinensis (Accession No. QBA18592.1), SpCR, Scylla paramamosain (Accession No. AHH29324.1), MjDOME, Marsupenaeus japonicus (Accession No. APA16577.1), PmDOME, Penaeus monodon (Accession No. MW187497), LvDOME, Litopenaeus vannamei (Accession No. AGY46351.1), CoCR, Chionoecetes opilio (Accession No. KAG0716771.1), GoCR, Galendromus occidentalis (Accession No. XP_003744080.1), CcCR, Cyphomyrmex costatus (Accession No. XP_004518381.1), DmDOME, Drosophila melanogaster (Accession No. NP_523412.1), TpCR, Thrips palmi (Accession No. XP_034251704.1), NvCR, Nasonia vitripennis (Accession No. XP_008202634.1), CfCR Camponotus floridanus (Accession No. XP_ EFN69830.1), AeCR Acromyrmex echinatior (Accession No. EGI63440.1), TsCR Trachymyrmex septentrionalis (Accession No. KYN30898.1), MmCR Mus musculus (Accession No. CAA37810.1), HsCR Homo sapiens (Accession No. CAA41231.1), OmCR Oncorhynchus mykiss (Accession No NP_001268335.1). CR indicates a cytokine receptor. (B) Domain diagrams of PmDOME and cytokine receptors from various species. Regions shaded in red refer to signal peptides. Fibronectin type III like domains and transmembrane regions are shown in yellow and blue, respectively. (C) Multiple sequence alignment of the N-terminal of cytokine receptors. Four conserved cysteines and the tryptophane in a CX-(9–10)-CXWX-(26–32)-CX-(10–15)-C sequence are highlighted in black and gray, respectively. WSXWS motif is present in a yellow box.

Full size image

From protein motif prediction, PmDOME possessed 24 residues of a signal peptide, a cytokine binding motif, four fibronectin type III (FNIII) domains and a transmembrane domain. Figure 2B demonstrates domain diagrams of PmDOME and others from seven closely related species. PmDOME shows four FNIII domains, similar to Pacific whiteleg shrimp LvDOMELESS and Chinese mitten crab EsDOME. In contrast, kuruma shrimp MjDOME displayed five FNIII domains, similar to redclaw crayfish Cherax quadricarinatus DOME (CqDOME), DmDOME and cytokine receptor from mud crab Scylla paramamosain (SpCR). Previously, DmDOME was shown most similarities to the interleukin-6 (IL-6) receptor family10. In vertebrates, IL-6 receptors belong to the type I cytokine receptor family, identified by the cytokine binding homology region (CHR). CHR possesses four conserved cysteine residues arranged in a CX-(9–10)-CXWX-(26–32)-CX-(10–15)-C sequence, forming two disulfide bonds at the N-terminal domain and a WSXWS motif at the C-terminal domain19. In this research, the amino acid sequences of PmDOME and cytokine receptors from 11 species were analyzed using the Clustal Omega tool. Multiple sequence alignment showed that PmDOME contained four cysteine residues at the N-terminal domain and an incomplete WSXWS motif at the C-terminus, similar to other invertebrate cytokine receptors. Meanwhile, the vertebrate cytokine receptors, HsCR, MmCR and OmCR showed four conserved cysteine residues, along with a full WSXWS motif (Fig. 2C). It is worth noting that while PmDOME and other examined cytokine receptors from both invertebrates and vertebrates contain a tryptophane in a CX-(9–10)-CXWX-(26–32)-CX-(10–15)-C sequence, phenylalanine is present in this particular sequence of CcCR and DmDOME. Based on sequence analysis, PmDOME shared several characteristics with the vertebrate cytokine class I receptors and invertebrate DOME receptors, suggesting that PmDOME is a signal-transducing receptor, involved in the JAK/STAT pathway.

Tissue distribution of PmDOME, PmJAK and PmSTAT transcripts in shrimp

The distributions of PmDOME, PmJAK and PmSTAT transcripts in various tissues of healthy shrimp were determined by quantitative RT-PCR using the elongation factor-1α gene (EF-1α) as an internal control. PmDOME was expressed at the highest level in lymphoid, followed by eyestalk, hemocyte and gill, respectively, while the lowest expression level of PmDOME was found in hepatopancreas (Fig. 3A). The highest level of PmJAK transcript was found in hemocyte, followed by lymphoid and epipodite, respectively (Fig. 3B). The heart and intestine were the organs that contained the lowest level of PmJAK transcript among the test tissues. Moreover, the highest expression of PmSTAT was detected in eyestalk, followed by hemocyte and lymphoid, respectively. The lowest level of PmSTAT transcript was in gill and heart (Fig. 3C).

Figure 3
figure 3

Tissue distribution of PmDOME, PmJAK and PmSTAT in healthy P. monodon. Expression profiles of PmDOME (A), PmJAK (B) or PmSTAT (C) in shrimp. The expression level in the intestine was used as a control and set as 1.0. Bars indicate the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Duncan’s new multiple range test. The experiment was carried out in triplicate and considered with different letters for statistical differences with significance at p < 0.05.

Full size image

Expression profiles of PmDOME and PmSTAT in response to WSSV infection and their dsRNA efficiency

In this study, the RNA interference (RNAi) technique was used to knock down PmDOME and PmSTAT transcripts in order to investigate the role of the JAK/STAT pathway in shrimp’s innate immune system. PmDOME dsRNA, PmSTAT dsRNA and GFP dsRNA were successfully synthesized with the size of approximately 500 bp, 250 bp and 700 bp, respectively. Shrimp were injected with either 5 or 10 µg of PmDOME dsRNA per 1 g shrimp. Injection of 5 µg of PmDOME dsRNA per 1 g of shrimp could knockdown PmDOME gene expression by approximately 66% at 24 hpi, whereas injection of 10 µg of PmDOME dsRNA per 1 g of shrimp could reduce PmDOME gene expression by about 50% at 24 hpi (Supplementary data, Fig. S1A). Meanwhile, shrimp were received a double injection of either 10 µg + 5 µg PmSTAT dsRNA per 1 g shrimp or 10 µg + 10 µg PmSTAT dsRNA per 1 g shrimp. A set of double injection of 10 µg + 10 µg PmSTAT dsRNA per 1 g shrimp was more efficient than a set of 10 µg + 5 µg PmSTAT dsRNA per 1 g shrimp to lower  the PmSTAT gene expression (Supplementary data, Fig. S1B). Double injection of 10 µg + 10 µg PmSTAT dsRNA per 1 g shrimp decreased PmSTAT transcript by approximately 79% at 24 hpi. As a result, dosages of 5 µg of PmDOME dsRNA per 1 g shrimp and double injection of 10 µg + 10 µg PmSTAT dsRNA per 1 g shrimp were used to knock down the PmDOME and PmSTAT expression, respectively.

As shown in Fig. 4A,B, WSSV infection caused an increase in PmDOME and PmSTAT expression levels. PmDOME mRNA levels in control and GFP dsRNA treated groups were up-regulated around 2.5- and 2-fold at 6 hpi and 24 hpi, respectively, in comparison to the unchallenged group. Similarly, PmSTAT transcription levels in control and GFP dsRNA injected shrimp increased by twofold at 6 h and 24 h after WSSV challenge. Although WSSV infection seemed to stimulate PmDOME and PmSTAT expression, the transcript levels of these genes remained low in the PmDOME and PmSTAT knockdown shrimp upon WSSV infection.

Figure 4
figure 4

Effects of PmDOME and PmSTAT knockdown in P. monodon. Expression profiles of PmDOME (A) or PmSTAT (B) in shrimp after either PmDOME or PmSTAT dsRNA injection and WSSV challenge at 6 and 24 hpi. The gene expression levels were relative to that of the control (PBS groups, in which the gene expression level was set to 1.0 at 0 hpi). Transcription levels of immune-related genes in PmDOME (C,D) and PmSTAT (E,F) silenced shrimp at 6 h and 24 h after WSSV infection. Bars indicate the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Duncan’s new multiple range test. The data were derived from independently triplicate experiments and considered for statistical differences with significance at p < 0.05.

Full size image

Effects of PmDOME and PmSTAT silencing on immune-related genes during WSSV infection

To investigate the role of the JAK/STAT signaling pathway during WSSV infection, the mRNA levels of immune-related genes in PmDOME and PmSTAT knockdown shrimp were determined by qRT-PCR genes. These included genes in the signaling pathways (PmDorsal, PmIMD and PmSOCS2), melanization process (ProPO2), IFN-like gene (PmVago5) and AMPs (ALFPm3, Penaeidin3, CrustinPm1 and CrustinPm7). As shown in Fig. 4C–F, expressions of ProPO2, Penaeidin3 and CrustinPm7 expressions significantly increased by 17, 28 and 14-fold at 6 h and 12, 16 and 2.5-fold at 24 h after WSSV challenge, in comparison with unchallenged shrimp. This suggested that ProPO2, Penaeidin3 and CrustinPm7 play an important role in WSSV defense. Other genes including PmSTAT, PmDOME, PmSOCS2, Vago5 and ALFPm3 were also up-regulated (> twofold) at 6 and 24 hpi. It is worth mentioning that PmDorsal and PmIMD expression levels, representing the Toll and Imd pathway, respectively, were insignificantly changed at 6 and 24 hpi. In addition, CrustinPm1 was up-regulated by 2.5-fold at 6 hpi but its transcript level was not substantially different at 24 hpi, compared with that of the control group.

PmDOME-deprived shrimp also showed down regulation of PmSTAT and PmSOCS2 at 6 and 24 hpi (Fig. 4C,D). PmDOME silenced shrimp exhibited much higher levels of ProPO2, Vago5 and ALFPm3 than that in control and GFP knockdown shrimp upon WSSV infection. As shown in Fig. 4D, ProPO2, Vago5 and ALFPm3 in PmDOME silenced shrimp was up-regulated by 26, 16 and 5-fold at 24 hpi, compared with the control group. Suppression of PmDOME lowered the expression of Penaeidin3, CrustinPm1 and CrustinPm7 at 6 hpi but significantly increased these gene transcripts at 24 hpi, compared with that in control and GFP-treated shrimp infected by WSSV. Notably, the expressions of Penaeidin3, CrustinPm1 and CrustinPm7 in PmDOME-depleted shrimp increased by 57, 5 and 12-fold at 24 h after WSSV challenge.

Consistent with PmDOME silencing, the knockdown of PmSTAT also lower expressions of PmDOME and PmSOCS2 (Fig. 4E,F). Suppression of PmSTAT also led to an increase of ProPO2, Vago5 and ALFPm3 at 6 and 24 hpi. As illustrated in Fig. 4F, the expression levels of ProPO2, Vago5 and ALFPm3 in PmSTAT silenced shrimp were increased by 59, 17.5 and 5.6-fold, respectively at 24 hpi. Penaeidin3, CrustinPm1 and CrustinPm7 transcripts were found to be lower in PmSTAT-silenced shrimp at 6 hpi, compared with that of WSSV-infected control and GFP-treated shrimp. Similar to PmDOME knockdown shrimp, PmSTAT depleted shrimp showed an increase of Penaeidin3, CrustinPm1 and CrustinPm7 at 24 hpi by 40, 8 and 17-fold, compared with that of unchallenged shrimp. Clearly, PmDOME and PmSTAT silencings gave similar results and the JAK/STAT signaling pathway significantly affected ProPO2 (melanization), Vago5 (interferon-like protein) and AMP gene expressions.

Survival rate of WSSV-infected shrimp and viral copy number after PmDOME and PmSTAT silencing

To examine functions of the JAK/STAT pathway during WSSV infection, WSSV copy numbers in PmDOME and PmSTAT silenced shrimp were quantified by the detection of conserved VP28 gene using qRT-PCR, in comparison with those in WSSV infected control and GFP treated group. Clearly, WSSV copy numbers in PmDOME and PmSTAT silenced shrimp were significantly lower than that in control and GFP treated shrimp (Fig. 5A). Moreover, knockdown of PmDOME and PmSTAT resulted in a delay of shrimp mortality (Fig. 5B,C). PmDOME-depleted shrimp reached 100% mortality on day 5 after WSSV infection, while PmSTAT knockdown shrimp attained 100% death on day 7.

Figure 5
figure 5

Effects of PmDOME and PmSTAT silencing during WSSV infection. (A) WSSV genome copies in gill tissues (30 µg) of PmDOME, PmSTAT and GFP dsRNA treated shrimps were measured at 24 hpi. Bars indicate the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Duncan’s new multiple range test. The data were derived from independently triplicate experiments and considered for statistical differences with significance at p < 0.05. (B,C) Cumulative mortality of PmDOME or PmSTAT silenced shrimp caused by WSSV infection. Differences in mortality level between treatments were analyzed by the log-rank (p values < 0.0001).

Full size image

Discussion

Innate immune responses serve as the first line of defense against pathogen infections. These occur via signal transduction pathways to activate diverse humoral and cellular processes. The Toll, Imd and JAK/STAT signaling pathways play important roles in antiviral responses. In this study, we aim to investigate the functions of the JAK/STAT signaling pathway during WSSV infection.

In this research, the complete ORF of PmDOME shared high sequence identities with previously reported Pacific whiteleg shrimp LvDOMLESS15 and kuruma shrimp MjDOME18, respectively. The phylogenetic tree revealed that the PmDOME was clustered into the invertebrate group (Crustacea, Arachnida and Insecta) (Fig. 2A). Although the sequence of DOME differed between species, the main functional domains were relatively similar, including the presence of a signal peptide, several fibronectins type III like motifs, a transmembrane domain and the cytokine binding motif which involved in receptor activation20. PmDOME belongs to the crustacean group and is most closely related to LvDOMELESS, followed by MjDOME. PmDOME and LvDOMELESS possess four fibronectin type III like domains, while MjDOME has five fibronectin type III like domains and is similar to DmDOME, the first identified invertebrate interleukin JAK/STAT receptor10 (Fig. 2B). PmDOME might belong to the IL-6 like receptor family, which bind to extracellular cytokines and trigger intracellular signals. Its biological functions are considered similar to DOME from other crustaceans.

Since invertebrates lack adaptive immune response, hemocyte is important for innate immune response to eliminate the invading pathogens21. From tissue distribution, both PmDOME and PmSTAT transcript levels were high in the hemocyte (Fig. 3). As a result, the hemocyte was selected as a target to study the influence of PmDOME and PmSTAT knockdown upon WSSV infection.

As shown in Fig. 4, the JAK/STAT signaling pathway well responded to WSSV infection as PmDOME, PmSTAT and PmSOCS2 expression levels increased (< twofold) at 6 and 24 hpi, while the mRNA levels of Dorsal and PmIMD, representing the Toll and IMD pathway, in unchallenged and WSSV-infected shrimp showed slight differences. In addition, the gene expressions of ProPO2, Vago5, ALFPm3, Penaeidin3 and CrustinPm7 were up-regulated at 6 and 24 hpi in WSSV-infected shrimp, compared with unchallenged shrimp (Fig. 4C–F). Meanwhile, the gene expression of CrustinPm1 also increased at 6 hpi, but then dropped at 24 hpi. This is in agreement with previous reports showing that ALFPm321, Penaeidin322, Vago23,24 and proPO system25 acted against WSSV.

Surprisingly, knockdown of either PmDOME or PmSTAT resulted in lower expression of the JAK/STAT components, including PmDOME, PmSTAT and PmSOCS2, but significantly enhanced ProPO2, Vago5 and ALFPm3 at 6 and 24 hpi (Fig. 4C–F). In addition, the expression of Penaeidin3, CrustinPm1 and CrustinPm7 in PmDOME or PmSTAT-deprived shrimp was also dramatically increased at 24 hpi (Fig. 4D,F). It is evident that knockdown of the JAK/STAT signaling pathway could enhance the proPO system, IFN-like antiviral cytokine and AMPs in response to WSSV infection. In addition, both PmDOME and PmSTAT-silenced shrimp showed lower WSSV copy numbers, compared with control and dsGFP-treated shrimp (Fig. 5A). This result reflects the fact that knockdown of PmDOME or PmSTAT delayed shrimp mortality caused by WSSV (Fig. 5B,C). In previous research, CqDOME-silenced hematopoietic tissue exhibited lower transcription levels of WSSV-immediately early gene (IE1) and a late gene envelope protein, VP2826. Suppression of CqDOME also decreased CqSTAT phosphorylation, which is required for activation of the IE1 transcript17.

The proPO system, involved in the production of superoxides and hydroxyl radicals to kill the invading pathogen, is important in immune defense against WSSV infection27. For example, in red swamp crayfish Procambarus clarkia, injection with the recombinant proPO2 could significantly decrease 65% mortality of WSSV-infected crayfish and reduced the amount of WSSV copy number in hepatopancreas and gill28. In addition, the silencing of the proPO gene in freshwater prawn Macrobrachium rosenbergi makes them susceptible to WSSV29. It was reported that the proPO gene was highly up-regulated after LPS stimulation, while the lack of LvSTAT down-regulated the proPO expression. It was speculated that the JAK/STAT pathway activated the proPO system by controlling the differentiation of hemocytes to promote the humoral immune response30. In this research, PmDOME and PmSTAT silencing significantly enhanced the expression levels of proPO2 by 30–60-fold during WSSV infection (Fig. 4C–F). This massive activation of the proPO system in PmDOME or PmSTAT-deprived shrimp may reduce WSSV infection and delay shrimp mortality.

It was reported that the interferon (IFN) regulatory factor (IRF)-like gene identified in L. vannamei could be activated by WSSV and bind the 5′-untranslated region of L. vannamei Vago4 gene31. This suggested that IRF-Vago-JAK/STAT might exist in invertebrates, similar to the vertebrates IRF-IFN-JAK/STAT. Suppression of LvVago 4/5 led to an increase in shrimp mortality by WSSV. Obviously, this result is consistent with our research, showing that Vago5 expression was elevated when the JAK/STAT signaling pathway was disrupted (Fig. 4C–F) and perhaps delaying the effects of WSSV on shrimp mortality (Fig. 5B,C). It was shown previously that cholesta-2,5-diene, a lipid of WSSV envelope, was recognized by the lipid-recognition protein, ML1, in M. japonicus, resulting in Dorsal translocation into the nucleus and Vago expression23. In our work, suppression of PmDOME or PmSTAT did not alter the Dorsal transcription level, although the Vago5 expression was enhanced by 8- to 17-fold at 6 and 24 hpi (Fig. 4C–F). It is possible that Vago5 could be activated via alternative pathways. Li and co-workers reported that linoleic acid promoted the expressions of Vago5 and AMPs via ERK-NF-κB against WSSV24.

The elevated levels of AMP expressions in PmDOME and PmSTAT-silenced shrimp may also contribute to delayed mortality. Previously, ALFPm3 has shown anti-WSSV activity via binding to the viral structural proteins and destroying the viral envelope32,33. In L. vannamei, silencing of the Penaeidin family including BigPEN, PEN2, PEN3 and PEN4 resulted in increasing cumulative mortality and WSSV copy numbers, while co-incubation of each recombinant penaeidin with WSSV inhibited the viral internalization into hemocytes22. It was demonstrated that PEN2 competitively bound to the envelope protein VP24, while BigPEN bound to VP28, which could disrupt WSSV infection22. CrustinPm1 and CrustinPm7 showed strong antimicrobial activity by agglutinating bacterial cells to disrupt the physiochemical properties of bacterial surface34. Although the antibacterial activity of crustin in shrimp is well known, its function in the viral infection process has been less well-studied. Recently, Zhang and co-workers reported that knockdown of MnCRU1, MnCRU6 and MnALF1 in the oriental river prawn Macrobrachium nipponenese resulted in increasing VP28 expression and number of WSSV virions, indicating that crustin and ALF may play anti-WSSV roles in shrimp35.

It was previously reported that ALFPm3, Penaeidin3 and CrustinPm7 were regulated by Toll and Imd pathway36,37, whereas CrustinPm1 was regulated by Toll pathway38. In this research, PmDOME and PmSTAT silencing did not affect the expression of PmDorsal and PmIMD, representing the Toll and Imd pathway. We hypothesized that the Vago5 expression was significantly increased in PmDOME and PmSTAT-silenced shrimp, in order to compensate for the downregulation of the JAK/STAT signaling pathway, resulting in the enhancement of AMP production. In kuruma shrimp, MjVago-L was reported to activate the JAK/STAT and induce the downstream effector MjFicolin through integrin during WSSV infection39.

In previous work, we reported that WSSV caused a decrease in the richness and diversity of microbiota40. In addition, WSSV-challenged shrimp showed higher levels of Photobacterium damselae, a pathogenic marine bacterium, while Shewanella algae, a shrimp probiotic, was reduced. Since the microbiota of PmSTAT-silenced shrimp was different from that of the control, STAT might function to maintain host-microbiota interactions. Taken together, the JAK/STAT signaling pathway response to WSSV infection via regulation of immune-related gene expressions and microbial homeostasis.

Methods

Shrimp

Healthy black tiger shrimp (Penaeus monodon) of average 3 g body weight were obtained from a shrimp farm in Chachoengsao Province, Thailand. Shrimp were acclimated in recirculating water tank system filled with air-pumped seawater with a salinity of 20 ppt at the temperature of 28 ± 4 °C. They were fed with a commercial diet twice a day for at least 1 week before the experiments. This study was conducted under the ethical principles and guidelines according to the animal use protocol 1923021 approved by Chulalongkorn University Animal Care and Use Committee (CU-ACUC). The biosafety guidelines were reviewed and approved by the Institutional Biosafety Committee of Chulalongkorn University (SC-CU-IBC-004-2018).

Cloning of the full-length PmDOME

The ORF of PmDOME was obtained from the hemocyte cDNA by PCR amplification using specific primers (Supplementary data, Table S1) based on a partial sequence of PmDOME (accession no. PM 89949) in the expressed sequence tag (EST) database of P. monodon (http://pmonodon.biotec.or.th)3. The full length of PmDOME gene was amplified from the hemocyte cDNA of P. monodon using the 5′ upstream region sequence (UPDOME-F1 primer) and 3′ downstream region sequence (DOME-R primer). The PCR conditions were as followed; (1) 96 °C for 5 min, (2) 25 cycles of 96 °C for 20 s, (3) 56 °C for 30 s, (4) 68 °C for 5 min, (5) a final extension at 68 °C for 7 min. The PmDOME PCR product was ligated into pGEX 6P-3 vector using an In-Fusion cloning kit (Takara) with the following conditions; 37 °C for 15 min, 50 °C for 15 min and 4 °C for 5 min; and then transformed into Escherichia coli TOP10 (Invitrogen). The recombinant plasmid named pGEX 6P-3-PmDOME was verified by sequencing. The full-length PmDOME gene was deposited in the NCBI GenBank (GenBank accession No. MW187497).

Bioinformatics analysis of PmDOME

The amino acid sequence of PmDOME was determined by ExPASy-Translate tool (https://web.expasy.org/translate/) and the protein motif was analyzed by SMART (http://smart.embl-heidelberg.de/) and GenomeNet (https://www.genome.jp/tools/motif/). The polyadenylation site in PmDOME sequences was predicted by Poly(A) Signal Miner (http://dnafsminer.bic.nus.edu.sg/PolyA.html). The predicted molecular weight of PmDOME was calculated by Compute pI/MW (https://web.expasy.org/compute_pi). Clustal Omega program was used to analyze the multiple sequence alignments (https://www.ebi.ac.uk/Tools/msa/clustalo/). The phylogenetic analysis was performed using MEGA 11 software to construct a neighbor-joining phylogenetic tree based on the deduced amino acid sequences. The bootstrap sampling was reiterated 1000 times. The N- or O-glycosylation sites of PmDOME was predicted by NetNGlyc-1.041 or NetOGlyc-4.042 and by GlycoEP server43.

Preparation of WSSV stock

WSSV was prepared from the gill tissue of WSSV-infected moribund shrimp using ultracentrifugation and membrane filtration as described in Jaturontakul et al.44.

Double strands RNA (dsRNA) synthesis

PmDOME dsRNA, PmSTAT dsRNA and GFP dsRNA were synthesized using T7 RiboMAX™ Express Large Scale RNA Production System (Promega). Two sets of specific primers each for PmDOME, PmSTAT and GFP genes were designed as shown in Supplementary Table S1. One of the specific primer pairs contained the T7 promoter sequence at the 5′ end. The hemocyte cDNA of P. monodon was used as a template to amplify PmDOME dsRNA and PmSTAT dsRNA, while the recombinant GFP plasmid was used as a template to amplify GFP dsRNA. The two PCR products each of PmDOME, PmSTAT and GFP genes were separately amplified by the specific primer pairs using the following conditions; 94 °C for 2 min, followed by 35 cycles of 98 °C for 10 s, 58 °C for 30 s and 68 °C for 30 s and a final extension at 68 °C for 7 min. Subsequently, the dsRNA of PmDOME, PmSTAT and GFP was produced in vitro using T7 RiboMAX™ Express Large Scale RNA Production System (Promega). The quality of synthesized dsRNAs was examined by 2% agarose gel electrophoresis and the concentration was determined by measuring absorbance at 260 nm and stored at − 80 °C for further use.

Total RNA isolation and first-stranded cDNA synthesis

Tissue samples from healthy and pathogen-infected shrimp were collected and homogenized in FARB buffer (Tissue Total RNA mini kit, Favorgen, Taiwan). Total RNA was extracted by following the manufacturer’s protocol. Five hundred nanograms of total RNA were converted to the cDNA by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA). The cDNA was kept at − 20 °C for further use.

Analysis of PmDOME, PmJAK and PmSTAT gene expressions in shrimp

Shrimp tissues including hemocyte, eyestalk, epipodite, gill, heart, lymphoid, intestine and hepatopancreas were collected from 9 healthy shrimp and total RNA was extracted by Tissue Total RNA mini kit (Favorgen), followed by cDNA synthesis using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher). PmDOME, PmJAK and PmSTAT gene expression levels in each tissue were identified by quantitative RT-PCR using 2 µL of tenfold diluted cDNA template and specific primers shown in Supplementary Table S1. Elongation factor-1α (EF-1α) gene was used as an internal control. The expression level in the intestine was used as a control and set as 1.0.

The quantitative RT-PCR (qRT-PCR) was carried out using an equal amount of cDNAs in the CFX96 Touch™ Real-Time PCR System and the Luna® Universal qPCR Master Mix (NEB) in the following conditions: one cycle at 95 °C for 1 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 30 s, using specific primers as shown in Supplementary Table S1. The expression of the elongation factor-1α gene (EF-1α) was used as an internal control. Melt curve analysis was performed at the end of the PCR thermal cycle to determine the specificity of amplification. The relative expressions of PmDOME and PmSTAT were calculated using a comparative CT method with the 2−ΔΔCT45. The data were shown as means ± standard deviations (SD). Statistical analysis was performed using one-way ANOVA followed by Duncan’s new multiple range test. The data were considered for statistical differences with the significance at p < 0.05.

Effects of PmDOME and PmSTAT silencing on immune-related genes after WSSV infection

To verify the silencing efficiency of dsRNA in vivo, shrimp with an average body weight of 3 g were injected with different dosages of either PmDOME dsRNA or PmSTAT dsRNA through intramuscular injection. A single injection of PmDOME dsRNA contained either 5 or 10 µg dsRNA per 1 g shrimp. Meanwhile, either a set of 10 µg dsRNA, followed by 10 µg dsRNA or a set of 10 µg dsRNA, followed by 5 µg dsRNA per 1 g shrimp was used in a double injection of PmSTAT dsRNA with 24 h interval between each injection. The dsRNAs were dissolved in 1X-PBS, pH 7.4 and the experiments were carried out in triplicate. The control group was injected with 1 × PBS (pH 7.4). Three shrimp hemolymphs in each group were collected at 24 h post injection. The hemolymph was collected using a sterile 1-mL syringe with 500 μL of anticoagulant, pH 7.0 (27 mM sodium citrate, 336 mM sodium chloride, 115 mM glucose, 9 mM ethylenediaminetetraacetic (EDTA). The hemolymph-anticoagulant mixture was then centrifuged at 800×g for 15 min at 4 °C to separate the hemocytes from the plasma.

To examine the effects of PmDOME and PmSTAT silencing on immune-related genes upon WSSV infection, shrimp with an average body weight of 3 g were divided into ten groups with three shrimps per group. The experiment was carried out in triplicate. Shrimp in groups 1 and 2 were injected with 1 × PBS (pH 7.4), while shrimp in groups 3 and 4 were injected with 5 µg of PmDOME dsRNA per 1 g of shrimp; and shrimp in groups 5 and 6 received 5 µg of GFP dsRNA per 1 g of shrimp as a control of PmDOME silenced group. Shrimp in groups 7 and 8 were double injected with 10 µg of PmSTAT dsRNA per 1 g of shrimp and shrimp in groups 9 and 10 were double injected with 10 µg of GFP dsRNA per 1 g of shrimp as a control of  the PmSTAT silenced group. After 24 h post dsRNA injection, shrimp in the even-numbered groups were infected with ~ 6 × 106 viral copies of WSSV. The hemolymph in each group was collected at different time points, 6 and 24 h post-WSSV infection, before the total RNA was extracted and converted to cDNA. The expression profiles of immune-related genes including PmSOCS2, Dorsal, PmIMD, Vago5, ProPO2, ALFPm3, Penaeidin3, CrustinPm1 and CrustinPm7 were determined by qRT-PCR using gene specific primers in Supplementary Table S1.

Quantification of WSSV copy number in shrimp

To study the effect of PmDOME and PmSTAT suppression on WSSV replication, the WSSV copy number in shrimp was determined. The shrimp’s gill samples were collected in parallel with the WSSV challenge experiment. Shrimp genomic DNA was extracted using FavorPrep Tissue Genomic DNA Extraction Mini Kit (Favorgen, Taiwan) and quantified by NanoDrop™ 2000c Spectrophotometer (Thermo Scientific). The extracted sample was used as a DNA template for viral copy number analysis. qRT-PCR was performed in triplicate using Luna® Universal qPCR Master Mix (NEB) with 2 μL genomic DNA (15 ng/μL) and VP28 primers (Supplementary Table S1). The cycling condition was previously mentioned. The recombinant plasmid containing a conserved region of WSSV VP28 gene was used to prepare the standard curve.

Mortality assays of PmDOME and PmSTAT silencing shrimp upon WSSV infection

Shrimps were separated into six groups and each contained 10 shrimps per group. The assay was conducted in triplicate. Shrimp in groups 1 and 2 were injected with 1 × PBS (pH 7.4), while those in groups 3 and 4 were either single injected with 5 µg of PmDOME dsRNA per 1 g of shrimp or double injected with 10 µg of PmSTAT dsRNA per 1 g of shrimp. Groups 5 and 6 were control groups; and the animals were either single injected with 5 µg of GFP dsRNA per 1 g of shrimp or double injected with 10 µg of GFP dsRNA per 1 g of shrimp. Shrimp in the even-numbered group were then injected with ~ 6 × 106 viral copies of WSSV at 24 h after dsRNA infection. The cumulative mortalities were recorded every 12 h after WSSV infection up to 7 days. The cumulative mortality experiment of PmDOME and PmSTAT silenced shrimp were performed separately. Data were analyzed using GraphPad Prism 8 and presented as percent survival with the p values (< 0.0001) calculated by the logrank test.