Identification of HSF1 as a key factor induced by high temperature.

(A) Diagram illustration of the experimental setup for transcriptome analysis. Group W was injected with WSSV and maintained continuously at 25 °C. In contrast, Group TW, also injected with WSSV, had their culture temperature increased to 32°C at 24 hours post-injection (hpi). (B) Identification of high temperature-induced genes by RNA-Seq. RNA sequencing was performed 12 hours post-temperature increase. Gill samples from 8 shrimp in each group were collected for Illumina sequencing (NCBI SRA database accession number PRJNA1050424). (C) Heatmap showing the differential expression of upregulated genes encoding potential transcription factors. The heatmap displays the fragments per kilobase per transcript per million mapped reads (FPKM) values from three biological replicates, shown after logarithmic transformation.

Upregulation of LvHSF1 in shrimp challenged by WSSV at both low (25 °C) and high (32 °C) temperatures.

(A) Transcriptional expression of LvHSF1 in different tissues of healthy shrimp subjected to low (25 °C) and high (32 °C) temperatures. The pleopod tissue at 25 °C serves as a reference, with its expression level set to 1.0. Expression values were normalized against EF-1α and β-Actin using the Livak (2−ΔΔCT) method. Data represent the mean ± SD from triplicate assays. (B) Protein levels of HSF1 at 25 °C and 32 °C detected by western blotting. (C-E) Expression profiles of LvHSF1 after Poly (I:C) injection in hemocytes (C), gills (D) and intestines (E). Expression levels were measured by qPCR, normalized against EF-1α and β-Actin using the Livak (2−ΔΔCT) method, and presented as mean ± SD from triplicate assays. (F-H) Expression profiles of LvHSF1 after WSSV injection in hemocytes (F), gills (G) and intestines (H). Expression levels were measured by qPCR, normalized against EF-1α and β-Actin using the Livak (2−ΔΔCT) method, and presented as mean ± SD from triplicate assays. Statistical significance was calculated using the Student’s t-test (**p < 0.01, *p < 0.05). All experiments were representative of three biological replicates and yielded similar results.

Role of HSF1 in restricting WSSV replication in shrimp at low (25 °C) temperatures.

(A, B) Silencing Efficiency of dsRNA-LvHSF1. The efficacy of two dsRNA-LvHSF1 constructs (dsLvHSF1-1 and dsLvHSF1-2) was assessed by qPCR in hemocytes (A) and gills (B). Additionally, protein levels of LvHSF1 were evaluated by western blotting in both tissues (panels a and b). Expression values were normalized against EF-1α and β-Actin using the Livak (2−ΔΔCT) method. Data represent the mean ± SD from triplicate assays. (C, D) Quantification of WSSV copies. The quantity of WSSV copies in hemocytes (C) and gills (D) at low temperature (25 °C) post-WSSV infection was determined using absolute quantitative PCR. (E, F) Transcriptional expression of the VP28 gene. The transcriptional expression of the VP28 gene was analyzed by qPCR in hemocytes (E) and gills (F) at low temperature (25 °C) following WSSV infection. (G) Expression of WSSV VP28 protein. Expression of WSSV VP28 protein was detected by western blotting in hemocytes and gills at low temperature (25 °C) after WSSV infection. Quantification and statistical analysis of three independent repeats were performed using ImageJ (panel g). (H) Survival rates of WSSV-infected shrimp post-LvHSF1 knockdown. The survival rates of WSSV-infected shrimp post-LvHSF1 knockdown were monitored at low temperature (25 °C), with recordings made every 4 hours. Statistical analysis was performed using the Kaplan-Meier plot (log-rank χ2 test). All experiments were conducted with three biological replicates, consistently yielding similar results.

Role of HSF1 in restricting WSSV replication in shrimp at high (32 ℃) temperatures.

(A) Survival rates of WSSV-infected shrimp post-LvHSF1 knockdown. The survival rates of WSSV-infected shrimp post-LvHSF1 knockdown were monitored at high temperature (32 °C), with recordings made every 4 hours. Statistical analysis was performed using the Kaplan-Meier plot (log-rank χ² test). (B, C) Quantification of WSSV copies. The quantity of WSSV copies in gills (B) and muscles (C) at high temperature (32 °C) post-WSSV infection was determined using absolute quantitative PCR. (D) Expression of WSSV VP28 protein. Expression of WSSV VP28 protein was detected by western blotting in gills at high temperature (32 °C) after WSSV infection. Quantification and statistical analysis of three independent repeats were performed using ImageJ (panel d). Statistical significance was calculated using the Student’s t-test (**p < 0.01). All experiments were conducted with three biological replicates, consistently yielding similar results.

nSWD is a potential antiviral effector regulated by HSF1.

(A) Venn diagram showing the downregulated genes by RNA-Seq. RNA-seq was performed 24 hours after WSSV infection (NCBI SRA database accession number PRJNA1110613). (B) Heatmap showing the differential expression of downregulated genes encoding potential effector molecules. The color bar indicates the gradient of normalized expression levels. (C) mRNA transcription levels of nSWD in hemocytes and gills. mRNA transcription levels of nSWD in the hemocytes and gills of LvHSF1-silenced shrimp under WSSV challenge. (D) Dual-luciferase reporter assays. Dual-luciferase reporter assays were performed to analyze the effects of overexpression of LvHSF1 on the promoter activities of nSWD in Drosophila S2 cells in a dose-dependent manner. Protein expression of LvHSF1 was detected with anti-HA Ab, with β-actin used as a protein loading control (panel d). (E) Schematic diagram of the nSWD promoter regions. Schematic diagram of the nSWD promoter regions in the luciferase reporter gene constructs. The HSF1 binding motif sites are shown in red rectangles. (F) Dual-luciferase reporter assays with mutated HSF1 binding motifs. Dual-luciferase reporter assays were performed to analyze the effects of overexpression of LvHSF1 on the promoter activities of nSWD with mutated HSF1 binding motifs. Protein expression of LvHSF1 was detected with anti-HAAb, with β-actin used as a protein loading control (panel f). (G) Analysis of the nSWD promoter. The HSF1 binding site was analyzed using the online JASPAR database. (H) EMSA assay. LvHSF1 protein interaction with HSF1 binding sites from the nSWD promoter was analyzed in vitro by EMSA assay. Competition assays were performed in the presence of excess unlabeled probes. Statistical significance was calculated using the Student’s t-test (**p < 0.01, *p < 0.05). All experiments were conducted with three biological replicates, consistently yielding similar results.

nSWD possessed potent antiviral activities against WSSV.

(A) Expression of nSWD in gills from WSSV-challenged shrimp. (B) Silencing efficiency of nSWD in gills. Silencing efficiency of nSWD in gills 48 hours after WSSV infection. (C) Quantity of WSSV copies in gills at high temperature (32 °C). The quantity of WSSV copies in gills at high temperature (32 °C) was detected by absolute quantitative PCR at 24 and 48 hours after WSSV infection. DsGFP was used as a control. (D) Survival rates of WSSV-infected shrimp post-nSWD knockdown. Survival rates of WSSV-infected shrimp post-nSWD knockdown at high temperature (32 °C) were monitored, with recordings made every 4 hours. Statistical analysis was performed using the Kaplan-Meier plot (log-rank χ² test). (E) SDS-PAGE analysis of recombinant nSWD protein expressed in E. coli. Line 1: Uninduced E. coli transformed with nSWD; Line 2: Induced E. coli transformed with nSWD; Line 3: Supernatant of ultrasonic lysed E. coli expressing nSWD; Line 4: Precipitate of lysed E. coli expressing nSWD. Line 5: Purified recombinant nSWD protein (black arrow). (F) Western blotting of purified rnSWD protein. Purified rnSWD protein was checked by Western blotting with anti-6×-His Ab. (G) Quantity of WSSV copies in shrimp gills following nSWD application post-nSWD knockdown. The quantity of WSSV copies in shrimp gills following 10 μg nSWD application post-nSWD knockdown at high temperature (32 °C) was measured in vivo at 24 and 48 hours using absolute quantitative PCR. (H) Survival rates post-rnSWD application. Shrimp were injected with 10 μg rnSWD or control protein, mixed with WSSV inoculum post-nSWD knockdown at high temperature (32 °C). Survival rates were recorded every 4 hours. (I) Quantity of WSSV copies in shrimp gills following nSWD application post-LvHSF1 knockdown. The quantity of WSSV copies in shrimp gills following 10 μg nSWD application post-LvHSF1 knockdown at high temperature (32 °C) was measured in vivo at 24 and 48 hours using absolute quantitative PCR. (J) Expression of WSSV VP28 protein. Expression of WSSV VP28 protein was detected by Western blotting in gills at high temperature (32 °C) after WSSV infection. Quantification and statistical analysis of three independent repeats were performed using ImageJ (panel j). (K) Survival rates post-rnSWD application. Shrimp were injected with 10 μg rnSWD or control protein, mixed with WSSV inoculum post-LvHSF1 knockdown at high temperature (32 °C). Survival rates were recorded every 4 hours. Statistical significance was calculated using the Student’s t-test (**p < 0.01, *p < 0.05). All experiments were conducted with three biological replicates, consistently yielding similar results.

nSWD interacts with envelope proteins of WSSV.

(A, B) GST pull-down assay for the detection of the interaction between rnSWD with VP19, VP24, VP26, and VP28. The results were shown via staining with Coomassie blue (A) or Western blotting using 6×-His Ab (B). The GST-tag protein was used as a control. (C, D) His pull-down assay for the detection of the interaction of rnSWD with VP19, VP24, VP26, and VP28 via Coomassie blue staining (C) or Western blotting using the GST-tag Ab (D). The GST tag protein was used as a control. (E, F) The colocalization of nSWD with VP24 and VP26 in S2 cells, 24 hours post-plasmid transfection. VP24 and VP26 was detected with rabbit anti-HA antibodies and anti-rabbit Alexa Fluor 488, while nSWD was identified using anti-Flag antibodies and anti-mouse Alexa Fluor 594. DAPI staining highlighted the nuclei. The scale bar represents 10 μm. (e-f) Quantitative analysis of fluorescence colocalization. Colocalization intensity was quantitatively analyzed, with complete colocalization indicated by overlapping peaks and maxima shifted by less than 20 nm. All experiments were representative of three biological replicates and yielded similar results.

High temperature-induced DmHSF1-AMPs axis restricts DCV replication in Drosophila.

(A) Transcriptional expression of DmHSF1. Transcriptional expression of DmHSF1 in S2 cells with or without DCV infection at low (27 °C) and high (30 °C) temperatures. (B) Transcriptional expression of DmAMPs. Transcriptional expression of DmAMPs in S2 cells at low (27 °C) and high (30 °C) temperatures. (C) Transcriptional expression of DmAMPs with or without DCV infection. Transcriptional expression of DmAMPs in S2 cells with or without DCV infection. (D) Silencing efficiency of DmHSF1. Silencing efficiency of DmHSF1 in S2 cells 24 hours after dsRNA application with or without DCV infection. (E) mRNA expression of DCV. mRNA expression of DCV at low (27 °C) and high (30 °C) temperatures after DmHSF1 knockdown, with dsGFP used as a control. (e) Protein expression of DCV at low (27 °C) and high (30 °C) temperatures after DmHSF1 knockdown. (F) mRNA expression of DmAMPs. mRNA expression of DmAMPs at low (27 °C) and high (30 °C) temperatures after DmHSF1 knockdown and DCV infection. (G) mRNA expression of DCV after DmHSF1 overexpression. mRNA expression of DCV at low (27 °C) and high (30 °C) temperatures after DmHSF1 overexpression. (g) Protein expression of DCV at low (27 °C) and high (30 °C) temperatures after DmHSF1 overexpression. (H) mRNA expression of DmAMPs after DmHSF1 overexpression. mRNA expression of DmAMPs at low (27 °C) and high (30 °C) temperatures after DmHSF1 overexpression and DCV infection. (I) Schematic Diagram of DmAMPs Promoter Regions. Schematic diagram of the DmAMPs promoter regions in the luciferase reporter gene constructs. The HSF1 binding motif sites are shown in red rectangles. (J) Dual-luciferase reporter assays. Dual-luciferase reporter assays were performed to analyze the effects of DmHSF1 overexpression on the promoter activities of DmAMPs with or without mutated HSF1 binding motifs. (K) mRNA expression of DCV following DmHSF1 overexpression post-DmAtta Knockdown. mRNA expression of DCV at high (30 °C) temperatures following DmHSF1 overexpression post-DmAtta knockdown. (k) Protein expression of DCV at high (30°C) temperatures following DmHSF1 overexpression post-DmAtta knockdown. Statistical significance was calculated using the Student’s t-test (**p < 0.01, *p < 0.05). All experiments were conducted with three biological replicates, consistently yielding similar results.

Model for the HSF1-AMPs-mediated high temperature-induced resistance to viruses in arthropods.

Elevated temperature induces a robust expression of LvHSF1, which in turn specifically induces the expression of the antimicrobial peptide (nSWD) in shrimp. The nSWD directly binds to WSSV envelope proteins and inhibits WSSV replication (left panel). Additionally, elevated temperature induces the expression of DmHSF1, which upregulates the expression of Atta, CecA, and Def in Drosophila, subsequently restricting the replication of DCV (right panel). These findings highlight the roles of HSF1 beyond the classical heat shock response, mediating the thermal regulation of immunity and facilitating the innate immune system’s response against viruses.

The expression of transcription factors as determined by qPCR.

The expression of effector molecules as determined by qPCR.

SDS-PAGE and western blotting analyses of the recombinant LvHSF1 protein expressed in E. coli.

(A) SDS-PAGE analysis of the recombinant LvHSF1 protein. Line 1, uninduced E. coli transformed with LvHSF1; line 2, induced E. coli transformed with LvHSF1; line 3, supernatant of ultrasonic lysed E. coli expressing LvHSF1; line 4, precipitate of lysed E. coli expressing LvHSF1; line 5, purified recombinant LvHSF1 protein (black arrow). (B) Western blotting analysis of the recombinant LvHSF1 protein. Line 1, rLvHSF1-GST; Line 2, GST (black arrow). Purified proteins were checked by Western blotting with anti-GST.

High temperature restricts DCV replication in Drosophila S2 cells.

(A) Drosophila S2 cells were seeded in 6-well plate and incubated at 27 ℃ or 30 ℃ for 24 h, then the cells were infected with DCV at a multiplicity of infection (MOI) of 10 at 27 ℃ or 30 ℃ for another 24 h. The cytopathic signs of S2 cells were observed in inverted fluorescence microscope. (B) The transcriptional expression of DCV in S2 cells with or without DCV infection (black arrow). (C) The protein expression of DCV in S2 cells with or without DCV infection.

Transfection in S2 cells.

(A) The protein expression level of DmHSF1 was detected by western blotting at 48 h post-transfection in S2 cells. (B) The silencing efficiency of DmAtta in S2 cells 24 h after dsRNA application with or without DCV infection.