Transcriptomic and morphological analyses of Litopenaeus vannamei intestinal barrier in response to Vibrio paraheamolyticus infection reveals immune response signatures and structural disruption.

The white shrimp Litopenaeus vannamei has been greatly impacted by Vibrio infection. In this study, we investigated the intestinal barrier response of L vannamei following challenge with Vibrio parahaemolyticus E1, by examining morphological changes and transcriptome expression levels. A total of 16,4420 unigenes were obtained from RNAseq data after quality control and assembly, and 4646 differentially expressed genes (DEGs) were identified following Vibrio challenge, of which 2469 unigenes were significantly up-regulated and 2177 were significantly down-regulated. DEGs were determined to be involved in various physical, chemical and immunological intestinal barrier functions, including peritrophin, cytoskeleton and cell junction, pattern recognition receptors, antimicrobial peptide and immune signaling pathways, serine protease/protease inhibitor and prophenoloxidase system, apoptosis and phagocytosis, and antioxidant systems. Fifteen DEGs were randomly selected for validation by real-time quantitative PCR (RT-qPCR) and showed results consistent with the RNA-seq data. Intestinal epithelial cell morphology was also affected by Vibrio challenge, showing epithelial detachment, nuclear pyknosis, and destruction of cell junctions. These results improve our current understanding of the intestinal barrier function in the shrimp response to bacterial infection.

Qi C ，Wang L ，Liu M ，Jiang K ，Wang M ，Zhao W ，Wang B ... -  《-》

Transcriptome analysis of Pacific white shrimp (Litopenaeus vannamei) challenged by Vibrio parahaemolyticus reveals unique immune-related genes.

Pacific white shrimp (Litopenaeus vannamei) is an important cultural species worldwide. However, Vibrio spp. infections have caused a great economic loss in Pacific white shrimp culture industry. The immune responses of Pacific white shrimp to the Vibrio spp. is not fully characterized. In this study, the transcriptomic profiles of L. vannamei hemocytes were explored by injecting with or without Vibrio parahaemolyticus. Totally, 42,632 high-quality unigenes were obtained from RNAseq data. Comparative genome analysis showed 2258 differentially expressed genes (DEGs) following the Vibrio challenge, including 1017 up-regulated and 1241 down-regulated genes. Eight DEGs were randomly selected for further validation by quantitative real-time RT-PCR (qRT-PCR) and the results showed that are consistent with the RNA-seq data. Due to the lack of predictable adaptive immunity, shrimps rely on an innate immune system to defend themselves against invading microbes by recognizing and clearing them through humoral and cellular immune responses. Here we focused our studies on the humoral immunity, five genes (SR, MNK, CTL3, GILT, and ALFP) were selected from the transcriptomic data, which were significantly up-regulated by V. parahaemolyticus infection. These genes were widely expressed in six different tissues and were up-regulated by both Gram negative bacteria (V. parahaemolyticus) and Gram positive bacteria (Staphylococcus aureus). To further extend our studies, we knock-down those five genes by dsRNA in L. vannamei and analyzed the functions of specific genes against V. parahaemolyticus and S. aureus by bacterial clearance analysis. We found that the ability of L. vannamei was significantly reduced in bacterial clearance when treated with those specific dsRNA. These results indicate that those five genes play essential roles in antibacterial immunity and have its specific functions against different types of pathogens. The obtained data will shed a new light on the immunity of L. vannamei and pave a new way for fighting against the bacterial infection in Pacific white shrimp.

Qin Z ，Babu VS ，Wan Q ，Zhou M ，Liang R ，Muhammad A ，Zhao L ，Li J ，Lan J ，Lin L ... -  《-》

The effect of rearing density on immune responses of hepatopancreas and intestine in Litopenaeus vananmei against Vibrio paraheamolyticus E1 challenge.

Rearing density and disease management are considered as pivotal factors determining shrimp farm productivity and profitability. To systematically investigate the potential mechanisms for density-related differences between disease susceptibility and rearing densities, we conducted comparative transcriptome analysis of the molecular differences between hepatopancreas and intestine of Litopenaeus vannamei under two different rearing densities (800- and 400- shrimp/m3) for 15 d and further analyzed the differences in immune response to Vibrio parahaemolyticus E1 (VPE1) raised under two density conditions. Totally 45 different expression genes (DEGs) were identified in the hepatopancreas under two different rearing densities, the DEGs were grouped into four processes or pathways related to animal immune system. Then, exposure to the VPE1 resulted in 639 DEGs, involved into fourteen immune related processes or pathways. In the intestine, seventeen processes or pathways related to the immune system were identified among the 5470 DEGs under two different rearing densities. 279 DEGs were identified post VPE1 challenge, classified into five processes or pathways associated with the immune system. Meanwhile, the results of growth performance, histopathology and the activities of antioxidant enzymes in the hepatopancreas and intestines of shrimp showed that high density decreased weight gain rate (63.20 ± 1.67% and 18.73 ± 3.35% in the high and low rearing density groups, respectively), severely destroyed the histopathology and inhibited the antioxidant enzymes activities. This study demonstrated that rearing density in L. vannamei significantly impacts susceptibility to the VPE1, via altered transcriptional challenge responses, and thus higher mortality due to disease.

Wang Y ，Wang B ，Shao X ，Shao J ，Liu M ，Wang M ，Wang L ... -  《-》

Exploring the influence of the surface proteins on probiotic effects performed by Lactobacillus pentosus HC-2 using transcriptome analysis in Litopenaeus vannamei midgut.

In order to understand the mediation function of surface proteins in probiotic effects executed by Lactobacillus pentosus HC-2 in midgut of Litopenaeus vannamei, the immune and digestion related enzymes and the transcriptome expression were analyzed after shrimp fed with normal HC-2 or with stripped surface proteins HC-2 by lithium chloride (LiCl) treatment. The results showed that the shrimp fed with normal HC-2 produced much higher immune and digestion related enzymes than the control group or LiCl-treated HC-2 group to defense the Vibrio parahaemolyticus E1 infection. We obtained total over 275,099 unigenes from L. vannamei midgut, 981 genes were significant differentially expressed in normal HC-2 group compared with control, 1314 genes were significant differentially expressed in LiCl-treated HC-2 group compared with control, and 1689 genes were significant differentially expressed in LiCl-treated HC-2 group compared with normal HC-2 group. The GO/KEGG enrichment analysis of the significantly different genes demonstrated that L. vannamei fed with normal HC-2 induced immune-related, signal transduction, ion homeostasis, cell-cell adhesion, response stress/stimulus, vascular endothelial growth factor and peritrophin genes up-regulation, which were important genes involved in improving the shrimp intestine immune response, nutrition and growth performance, and bacteria adhesion and colonization, but these genes were suppressed in the midgut of shrimp fed with deprived surface proteins bacteria. Taken together, these results indicated that the surface proteins were essential for HC-2 executing probiotic effects in midgut of shrimp. Our data contribute to improve the current understanding of host - Lactobacillus interaction and the probiotic mechanisms in shrimps.

Du Y ，Wang B ，Jiang K ，Wang M ，Zhou S ，Liu M ，Wang L ... -  《-》

Transcriptomics reveals different response mechanisms of Litopenaeus vannamei hemocytes to injection of Vibrio parahaemolyticus and WSSV.

As the most important cultural crustacean species worldwide, studies about Pacific white shrimp (Litopenaeus vannamei) have received more attention. It has been well-documented that various pathogens could infect L. vannamei, resulting in huge economic losses. The studies about the responding mechanism of L. vannamei to sole pathogens such as Vibrio parahaemolyticus and white spot virus (WSSV) have been extensively reported, while the studies about the differently responding mechanisms remain unclear. In the present study, we identified the differently expressed genes (DEGs) of L. vannamei hemocytes post V. parahaemolyticus and WSSV infection with RNA-seq technology and compared the DEGs between the two groups. The results showed 2672 DEGs post the V. parahaemolyticus challenge (1079 up-regulated and 1593 down-regulated genes), while 1146 DEGs post the WSSV challenge (1067 up-regulated and 513 down-regulated genes). In addition, we screened the genes that simultaneously respond to WSSV and V. parahaemolyticus (434), solely respond to WSSV (1146), and V. parahaemolyticus challenge (2238), respectively. Six DEGs involved in innate immunity were quantified to validate the RNA-seq results, and the results confirmed the high consistency of both methods. Furthermore, we found plenty of innate immunity-related genes that responded to V. parahaemolyticus and WSSV infection, including pattern recognition receptors (PRRs), the proPO activating system, antimicrobial peptides (AMPs), and other immunity-related proteins. The results revealed that they were differently expressed after different pathogen challenges, demonstrating the complex and specific recognition systems involved in defending against the invasion of different pathogens in the environment. The present study improved our understanding of the molecular response of hemocytes of L. vannamei to V. parahaemolyticus and WSSV stimulation.

Lin J ，Wan H ，Xue H ，He Y ，Peng B ，Zhang Z ，Wang Y ... -  《-》