Functions of LysC in maintaining intestinal microbiota homeostasis.

(A) RNAi efficiency of LysC as determined using qRT-PCR or western blotting. dsRNA was injected into shrimp (5 μg/g body weight). LysC mRNA expression and protein level in intestine was detected at indicated time. Bar chart shows the mean ± SD from three replicates, and the blotting images are representative of three independent replicates. Gray values were analyzed using ImageJ software. Each sample originated from five shrimp. (B) Shrimp mortality analysis after LysC knockdown. Shrimp death was recorded each day after dsLysC application. (C) Morphological analysis of shrimp intestines after LysC knockdown. Intestines were collected at 6 d after dsRNA application, sectioned and stained with H&E. Scale bar = 20 μm. The data are representative of two independent repeats. (D) Influence of the abundance of culturable intestinal bacteria by LysC knockdown. Intestines were homogenized at 4 d after dsRNA treatment, and the bacterial load was determined using the plate-counting method. The data are representative of two independent repeats. (E) Influence of the abundance of total bacteria by LysC knockdown, as determined using qPCR. Total DNA was extracted after RNAi from the intestine homogenate or hemolymph. The abundance of 16S rDNA was determined using qPCR, and calibrated to host β-actin. (F) Influence of the abundance of total bacteria after LysC neutralization. Native LysC in intestine lumen was neutralized by 5 μg of purified LysC antibody. An antibody which did not recognize any shrimp protein was used as control. The bacterial abundance was determined 3 d later. (G) Variation in the composition of intestinal microbiota after LysC knockdown at the species level, as assessed using 16S high-throughput sequencing. Each sample originated from at least 30 animals. (H) Influence of P. damselae abundance by LysC neutralization. Total DNA was extracted at 3 d after LysC neutralization to determine P. damselae abundance by detecting the ToxR gene. (I) Influence of total bacterial abundance by rLysC overexpression. The bacterial abundance in intestine was determined by detecting 16S rDNA using qPCR every day after rLysC (2 μg) or control tag administration. (J-K) Rescue of LysC knockdown by rLysC. rLysC or rTag (2 μg) was administered at 24 h after dsRNA application. Total bacterial abundance was determined another 3 d later (J), and the survival rate was recorded (K). For scatter graphs, one dot represents one shrimp. The survival data are representative of two independent repeats. Survival assay was analyzed by log-rank (Mantel–Cox) test, while other statistical analysis was performed using the Students’ t test. *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < 0.001.