Fig 1.
WSSV infection damages the mechanical performance of the shrimp cuticle and decreases the chitin content.
(A) Disruption of the lamella integrity of the shrimp cuticle, as shown by SEM image. The red arrow indicates the scattered lamella, while the yellow arrow indicates the peeling fragment. Scale bar, 10 μm. (B) Decrease in stiffness after WSSV infection, as shown by a texture analysis with the P/2N probe. Left panel, texture profile of a representative test; right panel, decrease in cuticle stiffness. n = 15 shrimp; the line shows the median (unpaired Student’s t-test, ***p < 0.001). (C) Decrease in the chitin content in the shrimp cuticle after WSSV infection, as shown by Raman characterization. Left panel, Raman spectra (excitation source laser at 532 nm) in the range of 0–3200 cm-1 displaying the major characteristic peaks of carbonate, carotenoids, and chitin; right panel, Raman spectra focused on the peaks corresponding to chitin. (D) Decrease in the chitin content in the shrimp cuticle after WSSV infection, as determined by an immunohistochemical analysis. The shrimp cuticle was collected for frozen sectioning. The recombinant chitin binding domain (CBD) from Bacillus circulans WL-12 Chitinase A1 was used to mark chitin, and FITC-labeled antibodies were used to visualize the bound CBD; scale bar, 20 μm. (E) Decrease in the chitin density in the shrimp cuticle after WSSV infection. The dimension of shrimp cuticle was determined on the coordinate paper. The cuticle was treated by boiling in 10% NaOH and 3.6% HCl to remove the protein and carbonate. The obtained chitin was dried and weighed. n = 15 shrimp. The line shows the median; unpaired Student’s t-test, ***p < 0.001. (F–G) Impairment in shrimp motor ability after WSSV infection. Shrimp were infected with WSSV. After 72 h, the shrimp were transferred into a new tank, behind which a coordinate paper was placed. The swimming trajectory of the shrimp after a slight prod was recorded using a camera, and the video was analyzed using the Anima Tracker plugin in ImageJ to fit the movement trajectory (F) and to determine the escape distance (G). n = 6 shrimp. The image of movement trajectory was representative of these independent replicates. SEM images, Raman spectra, and immunohistochemical figures are representative of three independent replicates.
Fig 2.
WSSV infection induces Chi2 expression and thereby decreases chitin.
(A) Increase in chitinase activity in the sub-cuticle epidermis after WSSV infection. The sub-cuticular tissue was collected and homogenized in PBS to obtain the supernatant. Chitinase activity was determined by monitoring the ability of tissue homogenate to convert colloidal chitin to N-acetyl glucosamine. n = 9 shrimp. Mean ± SD (unpaired Student’s t-test, ***p < 0.001). (B) Inhibition of the WSSV-induced chitinase increase by Chi2 knockdown. RNAi was performed at 24 h after WSSV infection. Chitinase activity in the sub-cuticle epidermis was determined another 48 later. n = 9 shrimp. Mean ± SD (unpaired Student’s t-test, ***p < 0.001). (C) Inhibition of the WSSV-induced chitin decrease by Chi2 knockdown, as analyzed using Raman spectroscopy. RNAi was performed at 24 h after WSSV infection. The cuticle was collected for he Raman analysis (excitation at 532 nm) after 48 h. (D) Inhibition of the WSSV-induced chitin decrease by Chi2 knockdown, as analyzed using an immunohistochemical analysis. Scale bar, 20 μm. (E–F) Improvement in shrimp motor ability after WSSV infection with Chi2 knockdown. Shrimp were infected with WSSV and injected with dsRNA 24 h later. The movement trajectory(E) and escape distance (F) were determined after 48 h. n = 6 shrimp. The image of movement trajectory was representative of these independent replicates. Images of Raman spectra and immunohistochemical analysis are representative of three independent replicates.
Fig 3.
WSSV infection induces Chi2 expression through E75.
(A) Sequential transcription factors essential for cuticle biosynthesis. The expression of these factors in the epidermis was determined using RT-PCR. The 5′-untranslated regions of Chi2 were obtained from the shrimp genome (GenBank GCA_017312705.1) and potential transcription factor binding sites were predicted using the online tool JASPAR. (B) Effect of the knockdown of selected candidate transcription factors on WSSV-caused Chi2 induction. RNAi was performed at 24 h after WSSV infection. Chi2 expression was detected after 24 h. Results are presented as the mean ± SD from three replicates. (C) Inhibition of the WSSV-caused chitin decrease by E75 knockdown, as analyzed by Raman spectroscopy (excitation at 532 nm). RNAi was performed at 24 h after WSSV infection. The cuticle was collected for the Raman analysis after another 48 h. (D–E) Improvement in shrimp motor ability after WSSV infection with E75 knockdown. Shrimp were infected with WSSV and injected with dsRNA 24 h later. After another 48 h, the shrimp were transferred into new tank to trace their movement trajectory (D) and to determine the escape distance (E) after slight prodding. n = 6 shrimp. The image of movement trajectory was representative of these independent replicates. (F) Illustration of the E75 binding sites in the promoter of Chi2. (G) Interaction of recombinant E75 with biotin-labeled oligonucleotides encoding the E75-binding site, as analyzed using EMSA. Competition assays were performed in the presence of excess unlabeled oligonucleotides, as indicated. (H) Binding of E75 to the promoter fragments of Chi2 after WSSV infection, as analyzed using a ChIP assay. The sub-cuticle epidermis was collected at 48 h after WSSV infection as a pool for the ChIP assay. Images of Raman spectra, EMSA, and ChIP analysis are representative of three independent replicates.
Fig 4.
WSSV-induced saturated LCFAs activate Chi2 expression.
(A) Increase in the relative abundance of differential metabolites in the epidermis, as shown using a heatmap. The sub-cuticle epidermis was collected at 24 h after WSSV infection or PBS injection for the metabolomics analysis. The color bar indicates the gradient of normalized abundance. Differential metabolites were identified with VIP > 1.0, p < 0.05, and fold change > 2. Each group consisted of 10 independent replicates. (B) Increase in the relative abundance of three saturated LCFAs after WSSV infection. Mean ± SD, unpaired Student’s t-test, *p < 0.05, **p < 0. 01, ***p < 0.001. (C) Induction of Chi2 by three saturated LCFAs. The saturated LCFAs were administered to the shrimp hemocoel at 5 μg per shrimp. DMSO was used as a control. Chi2 expression was detected 12 h later using qRT-PCR. Mean ± SD, unpaired Student’s t-test, ***p < 0.001. (D) Induction of chitinase activity by MA in the sub-cuticular epidermis. Chitinase activity was detected at 24 h after MA administration. n = 9 shrimp. Mean ± SD, unpaired Student’s t-test, ***p < 0.001. (E) Decrease in the cuticle chitin content after MA application, as analyzed using Raman spectroscopy (excitation at 532 nm). The cuticle was collected for the Raman analysis at 48 h after MA injection. (F) Decrease in the cuticle chitin content after MA application, as analyzed using an immunohistochemical analysis. Scale bar, 20 μm. (G–H) Impairment of shrimp motor ability by MA application. Shrimp were transferred into a new tank at 48 h after MA administration to trace their movement trajectory(G) and to determine the escape distance (H) after slight prodding. n = 6 shrimp. The image of movement trajectory was representative of these independent replicates. Images of Raman characterization and immunofluorescent assay are representative of three independent replicates.
Fig 5.
The LCFA MA acts as a ligand for E75 to induce Chi2 expression.
(A) Induction of the E75-mediated transcriptional regulation of Chi2 by MA. A ChIP assay was performed at 12 h after MA (5 μg) or DMSO administration. (B) Inhibition of the MA-induced Chi2 expression by E75 knockdown. MA was administered to shrimp after E75 silencing and the expression of Chi2 was detected 12 h later using qRT-PCR. Mean ± SD, unpaired Student’s t-test, ***p < 0.001. (C) Inhibition of the MA-induced increase in sub-cuticular epidermis chitinase activity by E75 knockdown. MA was administered to shrimp after E75 silencing and chitinase activity was determined 24 h later. Mean ± SD, unpaired Student’s t-test, ***p < 0.001. (D) Inhibition of the MA-induced decrease in the cuticle chitin content by E75 knockdown. MA was administered to shrimp after E75 silencing. The cuticle was collected for Raman spectroscopy (excitation at 532 nm) at 48 h after MA injection. (E) Molecular docking of the interaction between E75 LBD and three saturated LCFAs. The modeled structures of the complexes formed by E75-LBD and each fatty acid were overlaid for display. The experimentally determined structure of human PPARα-PA (PDB: 6KAX) is shown as a reference. The structure of E75 LBD was predicted using AlphaFold2. Docking was performed using AutoDock Vina 1.1.2, and the results were visualized using PyMOL version 2.4.1. (F) Characterization of the interaction between MA and rE75-LBD using an ITC assay. MA (800 μM) in the syringe was injected into cells containing rE75-LBD or the control rTag (10 μM) and stirred at 750 rpm for 150 s; 18 injections were performed. Raman spectra and immunofluorescent and ITC assays results are representative of three independent replicates.
Fig 6.
Blocking of WSSV-caused lipogenesis restores the chitin content and mechanical performance of the shrimp cuticle.
(A) Inhibition of WSSV-caused Chi2 induction by C75 application. C75 was administered to the shrimp hemocoel (5 μg) at 12 h after WSSV infection. Chi2 expression was detected another 24 h later. Mean ± SD, unpaired Student’s t-test, ***p < 0.001. (B) Inhibition of the WSSV-caused increase in chitinase activity by C75 application in the sub-cuticular epidermis. C75 was administered into shrimp at 12 h after WSSV infection. Chitinase activity was detected another 36 h later. n = 9 shrimp. Mean ± SD, unpaired Student’s t-test, ***p < 0.001. (C) Inhibition of the WSSV-caused chitin decrease by C75 application, as analyzed using Raman spectroscopy (excitation at 532 nm). The cuticle was collected at 72 h after WSSV infection and 60 h after C75 application. (D) Inhibition of the WSSV-mediated chitin decrease by C75 application, as analyzed using an immunohistochemical analysis. Scale bar, 20 μm. (E) Inhibition of the WSSV-caused decrease in the chitin density in the shrimp cuticle by C75 application. n = 8 shrimp. The line shows the median. Unpaired Student’s t-test, ***p < 0.001. (F) Restoration of the microstructure of the shrimp cuticle by C75 application, as shown by SEM analysis. Scale bar, 10 μm. (G) Restoring the stiffness of the shrimp cuticle by C75 application, as shown by a texture analysis. Left panel, texture profile of a representative test; right panel, decrease in cuticle stiffness. n = 15 shrimp; the line shows the median (unpaired Student’s t-test, ***p < 0.001). (H–I) Restoration of shrimp motor ability by C75 application. Shrimp were transferred into a new tank to trace their movement trajectory(H) and to determine the escape distance (I) after slight prodding. n = 6 shrimp. The image of movement trajectory was representative of these independent replicates. SEM images, Raman spectra, and immunohistochemical figures are representative of three independent replicates.
Fig 7.
WSSV infection causes an increase in saturated LCFAs, which activate E75 to increase the transcription and production of Chi2. Then, Chi2 digests chitin in the cuticle and thus damages the cuticle integrity.