The DESeq2 online software (Version 1.24.0, http://bioconductor.org/packages/stats/bioc/DESeq2/) was used to screen for the differentially expressed genes (DEGs) in our previously published transcriptome data (GenBank accession number: PRJNA385392). This transcriptome data was generated from hemocytes samples collected from shrimp infected with two strains of V. parahaemolyticus, i.e., non-AHPND-causing strain [MCCC1H00058, purchased from Marine Culture Collection of China (MCCC)] and AHPND-causing strain [a gift from Professor Lo Chufang (National Cheng Kung University, Taiwan, China)] (9, 23). A fold change (FC) >= 2 and false discovery rate (FDR) < 0.01 were used for screening the DEGs, where FC represents the ratio of expression of the same gene in two samples, while FDR was obtained by correcting the p-value for the significant difference. The screened DEGs were categorized into two sets, i.e., V. parahaemolyticus _(AHPND) vs. control and V. parahaemolyticus vs. control, designated as Gene Set A1 after removing redundancy. Next, all the predicted transcription factor sequences from the AnimalTFDB (Version 3.0, http://bioinfo.life.hust.edu.cn/AnimalTFDB/#!/) were downloaded in Fasta format and designated as TF-data (24). Finally, Gene Set A1 and TF-data genes were compared using local BLAST+ software (Version 2.9.0, ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.9.0/) to identify differentially expressed transcription factors (25).

Healthy shrimp P. vannamei (weight of 8 ± 2 g each), bought from a local shrimp farm, Shantou Huaxun Aquatic Product Corporation (Shantou, Guangdong, China), were cultured in aerated laboratory tanks containing seawater (1% salinity) at 25°C for 3-5 days laboratory acclimatization before experiments. All animal experiments were performed according to the guidelines and approved by the Animal Research and Ethics Committees of Shantou University, Guangdong, China. Hemolymph was collected into an equal volume of precooled acid citrate dextrose (ACD) anti-coagulant buffer (27 mM C₆H₅O₇Na₃·2 H₂O, 33 mM C₆H₈O₇, 110 mM C₆H₁₂O₆, 140 mM NaCl, pH 6.0) and the hemocytes harvested by centrifugation at 500 g for 10 min at 4°C. Other tissues (i.e., muscle, intestine, gill, hepatopancreas, nerve, heart, stomach, and eyestalk), were collected and processed as previously described (26). Total RNA was extracted from the tissues using the RNAFAST 200 kit (FeiJie, Shanghai, China) following the manufacturer’s instructions. The RNA concentration was determined with a NanoDrop2000 spectrophotometer (Nano-drop Technologies, Wilmington, DE), and the RNA quality was ascertained using the A260/280 ratio (>= 2.0) and on 1% agarose gel electrophoresis. Only good-quality RNA samples were used for further analysis. First-strand cDNA synthesis was carried out with 1.0 μg of total RNA using the EasyScrpt One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China), following the manufacturer’s instructions.

RT-qPCR was performed using Master SYBR Green I system (GenStar, Beijing, China) on the Roche Light-Cycler 480 system (Roche, Switzerland) using the following thermal cycling parameters: one cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 30 s. All the gene-specific primers were listed in Table 1 , the elongation factor 1 alpha gene from P. vannamei (PvEF1α) was used as an internal control. The relative gene expression was calculated using the 2-^ΔΔCT method. Samples were analyzed in triplicates using five shrimps per group.

To examine the response of PvKr-h1 to immune challenge, healthy shrimp were randomly divided into four groups, with 100 shrimps per group, before being intramuscularly injected via the third abdominal segment with V. parahaemolyticus _(AHPND) (1 × 10⁵ CFU), V. parahaemolyticus (1 × 10⁵ CFU), Streptococcus iniae (1 × 10⁵ CFU) or an equal volume of sterile 0.01 M PBS (137 mM NaCl, 8 mM Na₂HPO₄, 2 mM NaH₂PO₄, pH 7.4), respectively. Hemocytes were collected from five randomly selected shrimps per group at 0, 12, 24, 48, and 72 h post-injection (hpi). Total RNA extraction, cDNA synthesis and RT-qPCR analysis were conducted as described in subsection 2.2.

In vivo knockdown of PvKr-h1 was performed using double-stranded RNA (dsRNA) that targets a portion of the ORF sequence of PvKr-h1 and the EGFP gene as a control. The specific primers ( Table 1 ) used to synthesize the dsRNA were designed with Primer Premier 5 and synthesized using the HiScribe™T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA) following the manufacturer’s protocol. For the knockdown experiments, 300 healthy shrimp were randomly divided into two groups (experimental and control groups), with the experimental group shrimp each intramuscularly injected via the third abdominal segment with 10 μg dsPvKr-h1, whereas the control group shrimp were each injected with an equal amount of dsEGFP. Hemocytes were collected from five randomly selected shrimp per group at 24, 48, and 72 h post-injection for total RNA extraction, cDNA synthesis, and RT-qPCR analysis to determine knockdown efficiency. To determine shrimp mortality rate after PvKr-h1 knockdown followed by pathogen challenge, shrimp injected with dsPvKr-h1 (experimental group) and dsEGFP (control group) for 48 h were then further divided into three subgroups (35 shrimp each), and injected with V. parahaemolyticus _(AHPND) (1 × 10⁵ CFU), V. parahaemolyticus (1 × 10⁵ CFU) or an equal volume of sterile 0.01 M PBS, respectively. Shrimp mortality was recorded at 0, 12, 18, 24, 36, 48, 60, and 72 h post-bacteria challenge, and the cumulative mortality rate was calculated. The mortality graph was plotted using GraphPad Prism 8 software. Statistical significance was determined by Kaplan-Meier survival analysis, and the p-values indicated.

Healthy shrimps were randomly divided into two groups, with one group injected with 10 μg of dsPvKr-h1 while the other group was injected with an equal amount of dsEGFP. At 48 h post-injection, shrimps from each group were further divided into two subgroups, with each subgroup injected with V. parahaemolyticus _(AHPND) (1 × 10⁵ CFU) or with an equal volume of sterile 0.01 M PBS. At 72 h post the first injection, hemolymph was collected from five shrimp per group and centrifuged at 500g for 10 min at 4°C to collect the supernatant. The supernatant was further centrifuged at 16,000 g for 10 min at 4°C to collect the pellet followed by aseptic extraction of genomic DNA (gDNA) using the TIANamp Marine Animals DNA Kit (TIANGEN, Beijing, China) following the manufacturer’s protocols. To identify bacterial (V. parahaemolyticus _(AHPND)) abundance in the hemolymph samples, RT-qPCR-based methods were used. First, a standard curve was prepared using V. parahaemolyticus _(AHPND) strains and gene-specific primers (PirB, Table 1 ) as previously described (27). Next, RT-qPCR assay was performed using primers specific to the V. parahaemolyticus _(AHPND) strain (PirB, Table 1 ) and the DNA extracted above. Finally, the RT-qPCR results and the prepared standard curve were used to calculate the bacterial abundance.

Hemocytes collected from shrimp injected with dsPvKr-h1 or dsEGFP followed by injection with V. parahaemolyticus _(AHPND) or PBS (i.e., dsPvKr-h1 + V. parahaemolyticus _(AHPND), dsEGFP + V. parahaemolyticus _(AHPND), dsPvKr-h1 + PBS, or dsEGFP + PBS) as described in subsection 2.4 were used for total RNA extracted using the TRIzol Plus (Invitrogen, Carlsbad, CA) following the manufacturer’s protocols. Next, the high-quality RNA samples were used to construct cDNA libraries followed by RNA sequencing on the Illumina Novaseq 6000 platform by a commercial company (Majorbio Biotech, Shanghai, China). The data obtained was then analyzed as described previously (9). The data has been submitted to GenBank under accession number PRJNA875156. After the RNAseq data analysis, the significant DEGs in the various groups (i.e., dsPvKr-h1 + V. parahaemolyticus _(AHPND) vs dsEGFP + V. parahaemolyticus _(AHPND) and dsPvKr-h1 + PBS vs dsEGFP + PBS) were selected for functional annotation using Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis on the KEGG database (http://www.genome.jp/kegg/) as described previously (9). Based on the functional annotation results and previous studies on shrimp, key immune-related genes were chosen from the DEGs in group dsPvKr-h1 + V. parahaemolyticus _(AHPND) vs dsEGFP + V. parahaemolyticus _(AHPND). These DEGs were then validated using RT-qPCR with gene-specific primers ( Table 1 ) as described in subsection 2.2.

ROS levels in hemocytes were determined using the Reactive Oxygen Species Assay Kit (Beyotime biotechnology, Shanghai, China) which uses fluorescent compound, DCFH-DA, to measure hydroxyl, peroxyl, and other ROS activity in the cell. Briefly, freshly isolated hemocytes from five shrimp were incubated with 10mM DCFH-DA in PBS for 20 min at 37°C (1 × 10⁶ cells were used per group). Next, cells were harvested by centrifugation and suspended in PBS, then and measured at 485-nm (excitation) and 527-nm (emission) wavelengths on a microplate reader (Synergy HTX; BioTek, Winooski,VT). The level of ROS was calculated as fluorescence intensity. Triplicate samples were measured per treatment for at least three independent experiments. Statistical significance was determined using the Student’s t-test relative to control.

Immunofluorescence analysis was used to analyze the translocation of PvRelish into the nucleus as described previously (28). Briefly, hemocytes were collected from shrimp injected with dsPvKr-h1 or dsEGFP followed by injection with V. parahaemolyticus _(AHPND) or PBS (i.e., dsPvKr-h1 + V. parahaemolyticus _(AHPND), dsEGFP + V. parahaemolyticus _(AHPND), dsPvKr-h1 + PBS, and dsEGFP + PBS) as described in subsection 2.5 The collected cells were then resuspended in Insect-XPRESS™ media (LONZA, Basel, Switzerland) followed by seeding 2×10⁶ cells per well onto glass-bottom plates, before being incubated at 28°C for 2 h. Next, cells were fixed with 4% paraformaldehyde at room temperature for 15 min before being washed three times with 0.01 M PBS, followed by incubation with 0.5% Triton X-100 in 0.01 M PBS at room temperature for 20 min. Samples were then washed and incubated with 5% BSA (bovine serum albumin) for 30 min, and then with rabbit anti-PvRelish antisera (1:100, prepared inhouse) overnight at 4°C. After being washed three times with PBST (137 mM NaCl, 8 mM Na₂HPO_4, 2 mM NaH₂PO₄, 0.5% Tween-20, pH 7.4), samples were incubated with goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Invitrogen, Carlsbad, CA, 1:1000) for 1 h at room temperature in the dark. Samples were washed three times with PBST, stained with Hochest 33342 (Beyotime, Shanghai, China) for 10 min at room temperature protected from light, followed by washing six times, before being examined under a Confocal Microscope LSM800 (Carl ZEISS, Heidenheim, Germany).

Screening of our previously published shrimp hemocytes RNA-Seq data (PRJNA385392) revealed the dysregulation of thirty putative transcription factor (TF) genes, among which LOC113823218, LOC113809796, LOC113812137, and LOC113822590 were highly expressed ( Figure 1A ). Validation of the expression of these four genes using RT-qPCR after the infection of shrimps with V. parahaemolyticus and V. parahaemolyticus _(AHPND) ( Figure 1B ) revealed LOC113822590 as the most upregulated gene ( Figure 1B ). LOC113822590 was identified as the P. vannamei homolog of the Kruppel homolog 1-like gene (PvKr-h1) after cloning and sequence alignment ( Figure S1 ). Tissue profiling of PvKr-h1 mRNA transcripts revealed that it was constitutively expressed in all tested tissues i.e., intestine, stomach, eyestalk, heart, hepatopancreas, hemocytes, nerve, gill, and muscle ( Figure 2A ), with muscle expressing the highest. When shrimp were challenged with V. parahaemolyticus and V. parahaemolyticus _(AHPND), mRNA transcript levels were significantly induced in shrimp hemocytes ( Figure 2B ); hence hemocytes were used in all subsequent experiments.

The mRNA transcript levels were not only induced in hemocytes by Gram-negative (V. parahaemolyticus and V. parahaemolyticus _(AHPND)) bacteria ( Figures 2C, D ) but also by Gram-positive (S. iniae) bacteria at different time points ( Figure 2E ). For instance, after challenging with V. parahaemolyticus _(AHPND), the mRNA transcript levels of PvKr-h1 increased steadily from 12 h and peaked at 72 h ( Figure 2C ). A similar significant increase in PvKr-h1 expression was observed upon challenge with V. parahaemolyticus, with peak expression also found at 72 h ( Figure 2D ). The mRNA transcript levels of PvKr-h1 after S. iniae challenge, significantly at 12 h followed by a gradual decreased to baseline at 48 h ( Figure 3E ). Thus, the three bacteria pathogens could induce PvKr-h1 expression in shrimp hemocyates, with V. parahaemolyticus _(AHPND) inducing the strongest.

To further explore the role of Kr-h1 in shrimp response to V. parahaemolyticus _(AHPND) infection, shrimp were depleted of Kr-h1 followed by V. parahaemolyticus _(AHPND) challenge. We observed that after PvKr-h1 knockdown ( Figure 2F ) followed by challenge with V. parahaemolyticus, V. parahaemolyticus _(AHPND), or PBS as control, shrimp mortality was significantly higher (p<0.01) in the Kr-h1 depleted groups (i.e., dsPvKr-h1 + V. parahaemolyticus or dsPvKr-h1 + V. parahaemolyticus _(AHPND)) compared with the control groups (i.e., dsEGFP + V. parahaemolyticus or dsEGFP + V. parahaemolyticus _(AHPND)) ( Figure 2G ). When hemolymph total bacterial abundance was analyzed before and after PvKr-h1 knockdown with V. parahaemolyticus _(AHPND) challenge, a significant increase in total bacteria abundance was observed at 12 hpi (p<0.05), 24 hpi (p<0.01), and 36 hpi (p<0.01) in the knockdown challenged group (dsPvKr-h1 + V. parahaemolyticus _(AHPND)) compared with the control challenged group ( Figure 2H ). These results further indicate the role of PvKr-h1 in shrimp antimicrobial response to AHPND.

To further explore how PvKr-h1 modulates shrimp immune response to V. parahaemolyticus _(AHPND) infection, we performed RNAseq using shrimp hemocytes after PvKr-h1 knockdown with PBS challenge. A total of 1014 differentially expressed genes (DEGs) were identified, among which 387 were upregulated while 627 were downregulated ( Figures 3A, B ). Further analysis of the DEGs using KEGG pathway enrichment analysis revealed that the significantly enriched KEGG pathways include signal transduction, cancers-related, carbohydrate metabolism, transport and catabolism, endocrine system, and digestive system ( Figure 3C ). Signal transduction category was the most enriched with 25 upregulated and 40 downregulated genes. Besides, 24 DEGs were enriched in the immune system category ( Figure 3C ).

When the gene expression pattern in the PvKr-h1 knockdown followed by V. parahaemolyticus _(AHPND) challenge group was compared with the control group (i.e., dsEGFP + V. parahaemolyticus _(AHPND)), 1349 DEGs were identified, out of which 760 were upregulated and 589 downregulated ( Figures 3D, E ). KEGG analysis of the 1349 DEGs revealed that these they were enriched in several categories, including immune system, cell growth and death, lipid metabolism, amino acid metabolism and signal transduction ( Figure 3F ). Among the DEGs enriched in the immune system category ( Tables S1 , S2 ), antioxidant-related enzymes, such as Copper-zinc superoxide dismutase (Cu-Zn SOD), glutathione peroxidase (GPX), and glutathione-S-transferase (GST), were significantly upregulated, while antimicrobial peptide genes, such as crustins (CRUs) and penaeidins (PENs) were significantly downregulated.

After observing that antioxidant-related enzymes were dysregulated after PvKr-h1 knockdown followed by V. parahaemolyticus _(AHPND) challenge, we examined whether this could affect reactive oxygen species (ROS) production. When shrimp were depleted of PvKr-h1 followed by V. parahaemolyticus _(AHPND) challenge, the mRNA transcript levels of antioxidant-related genes in hemocytes, i.e., Cu-Zn SOD, GPX, and GST were significantly increased compared with control ( Figure 4A ). The level of ROS in shrimp hemocytes decreased significantly at 12 and 24 h in the PvKr-h1 knockdown followed by V. parahaemolyticus _(AHPND) challenge group compared the control ( Figure 4B ). These results suggest that PvKr-h1 mediate ROS production in shrimp in hemocytes by modulating the expression of antioxidant-related enzymes during V. parahaemolyticus _(AHPND) challenge.

During bacterial infections, the IMD pathway in shrimp is activated through a series of reaction cascades that cleave Relish to enable the entry of the Rel homology domain (RHD) to regulate AMPs expression (29). When shrimp were depleted of PvKr-h1, the mRNA transcript levels of AMPs genes, i.e., CRUs, PENs decreased significantly ( Figure 5A ), suggesting that PvKr-h1 could regulate the expression of AMPs, probably through the IMD pathway.

Given that during pathogenic bacteria infection in shrimp, Relish is reported to regulate the expression of AMPs by targeting their promoters (11) and that Relish is downstream of IMD in innate immune signaling, we determined whether PvKr-h1 knockdown affects the nuclei entry of PvRelish. Using immunofluorescence analysis, we found that after PvKr-h1 knockdown followed by V. parahaemolyticus _(AHPND) challenge, the number of hemocytes with PvRelish fluorescence signals in the nucleus was 75% less than the control ( Figure 5B ). These results indicate that PvKr-h1 could promote AMPs expression during V. parahaemolyticus _(AHPND) challenge through the IMD/Relish pathway.