LKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by Vibrio alginolyticus infection.

LKB1 (liver kinase B1) is a key upstream kinase of AMPK and plays an important role in various cellular activities. While the function and mechanism of LKB1 have been widely reported in the study of tumor, there are few reports on its role in bacterial infectious diseases, especially in shrimp. In the present study, molecular characterization revealed that LvLKB1 has an open reading frame (ORF) of 1266 bp encoding 421 amino acids with a molecular weight of about 48 KDa, including the kinase region, N-terminal regulatory domain and C-terminal regulatory domain. LvLKB1 in hepatopancreas and hemocytes was significantly upregulated after infection with Vibrio alginolyticus (V. alginolyticus). After silencing LvLKB1 gene in Litopenaeus vannamei (L. vannamei) and artificially infecting V. alginolyticus, the survival rate of L. vannamei was significantly decreased. Subsequently, it was found that the expression of inflammatory factors in hepatopancreas and hemocytes of shrimp was up-regulated, and the expression of lipid oxidation factors was decreased after silencing LKB1, leading to the phenomenon of lipid accumulation in hepatopancreas. In order to explore the mechanism, autophagy levels of shrimp were detected after silencing LKB1, which showed that autophagy levels in hepatopancreas and hemocytes were significantly reduced. Further studies conclusively showed that silencing LvLKB1 inhibited AMPK phosphorylation induced by V. alginolyticus infection, thereby activating TOR pathway and inhibiting autophagy in shrimp. These results indicate that LvLKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by V. alginolyticus infection.

Effects of dietary Ginkgo biloba leaf extract on growth performance, immunity and environmental stress tolerance of Penaeus vannamei.

Ginkgo biloba leaf extract (GBE) has been extensively used in the treatment of diseases due to its anti-inflammatory, antioxidant, and immunomodulatory effects. In aquaculture, GBE is widely used as a feed additive, which is important to enhance the immunity of aquatic animals. The current study evaluated the effects of adding GBE to the diet of Penaeus vannamei (P. vannamei) under intensive aquaculture. The GBE0 (control group), GBE1, GBE2, and GBE4 groups were fed a commercial feed supplemented with 0.0, 1.0, 2.0, and 4.0 g/kg GBE for 21 days, respectively. The results showed that dietary GBE could alleviate hepatopancreas tissue damage and improve the survival rate of shrimp, and dietary 2 g/kg GBE could significantly increase the total hemocyte count (THC), the hemocyanin content, the antioxidant gene's expression, and the activity of their encoded enzymes in P. vannamei. Furthermore, transcriptome data revealed that immunity-related genes were upregulated in the GBE2 group compared with the GBE0 group after 21 days of culture. Drug metabolism-cytochrome P450, sphingolipid metabolism, linoleic acid metabolism, glycerolipid metabolism, fat digestion and protein digestion and absorption pathways were significantly enriched, according to KEGG results. Surprisingly, all of the above KEGG-enriched pathways were significantly upregulated. These findings demonstrated that supplementing P. vannamei with 2 g/kg GBE improved its environmental adaptability by improving immunity, lipid metabolism, and detoxification. In this study, a comprehensive evaluation of the effects of dietary GBE on the intensive aquaculture of P. vannamei was conducted to provide a reference for the healthy culture of P. vannamei.

Andrographis paniculata improves growth and non-specific immunity of shrimp Litopenaeus vannamei, and protects it from Vibrio alginolyticus by reducing oxidative stress and apoptosis.

Andrographis paniculata (AP) is a traditional medicinal plant with many pharmacological activities, including anti-inflammatory, antimicrobial, immunity stimulation and so on. Several studies have reported that AP plays a strong role in promoting the immune system of aquatic animals to resist several pathogens. In the present study, we investigate the effects of a diet containing AP on the immune responses, growth, and the resistance to Vibrio alginolyticus (V. alginolyticus) in Litopenaeus vannamei (L. vannamei). Four diets were formulated by adding AP at the dosage of 0% (Control), 0.25%, 0.5%, and 1% in the basal diet, respectively. Each diet was randomly fed to one group with three replicates of shrimps in a 28-day feeding trial. The results showed that dietary AP improved the growth performance and non-specific immune function of shrimps. To investigate the effect of AP on disease resistance of L. vannamei, shrimps fed with diet containing AP were challenged with V. alginolyticus. Compared with the control group, the shrimps fed diet containing AP showed significantly higher survival. Furthermore, the hepatopancreas injury in the shrimp fed with AP was less than control group at 6 h after V. alginolyticus infection. However, no difference was observed in the degree of hepatopancreas injury between AP groups and control group at 12 h and 24 h after V. alginolyticus infection. Based on this result, the samples at 6 h after V. alginolyticus infection was selected for subsequent detection. Reactive oxygen species (ROS) accumulation in hemocytes and O2- production in hepatopancreas caused by V. alginolyticus infection was significantly reduced after feeding a diet containing 0.25% and 0.5% AP (p < 0.05). In addition, we found that feeding AP significantly up-regulated the expression of pro-apoptotic genes (Bax, Caspase 3, p53) and down-regulated the expression of anti-apoptotic genes (Bcl-2) in hepatopancreas after V. alginolyticus infection. In conclusion, AP promote the growth and immunity of L. vannamei, and protects shrimps against V. alginolyticus by regulating the oxidative damage and apoptosis. These results provide useful information regarding the effects of AP extracts as a shrimp feed additive for sustainable shrimp culture.

Andrographolide promote the growth and immunity of Litopenaeus vannamei, and protects shrimps against Vibrio alginolyticus by regulating inflammation and apoptosis via a ROS-JNK dependent pathway.

Vibrio alginolyticus (V. alginolyticus) is one of the major pathogens causing mass mortality of shrimps worldwide, affecting energy metabolism, immune response and development of shrimps. In the context of the prohibition of antibiotics, it is necessary to develop a drug that can protect shrimp from V. alginolyticus. Andrographolide (hereinafter called Andr), a traditional drug used in Chinese medicine, which possesses diverse biological effects including anti-bacteria, antioxidant, immune regulation. In this study, we investigated the effect of Andr on growth, immunity, and resistance to V. alginolyticus infection of Litopenaeus vannamei (L. vannamei) and elucidate the underlying molecular mechanisms. Four diets were formulated by adding Andr at the dosage of 0 g/kg (Control), 0.5 g/kg, 1 g/kg, and 2 g/kg in the basal diet, respectively. Each diet was randomly fed to one group with three replicates of shrimps in a 4-week feeding trial. The results showed that dietary Andr improved the growth performance and non-specific immune function of shrimps. L. vannamei fed with Andr diets showed lower mortality after being challenged by V. alginolyticus. After 6 h of V. alginolyticus infection, reactive oxygen species (ROS) production, tissue injury, apoptosis, expression of inflammatory factors (IL-1 ß and TNFα) and apoptosis-related genes (Bax, caspase3 and p53) were increased in hemocytes and hepatopancreas, while feeding diet with 0.5 g/kg Andr could inhibit the increase. Considering that JNK are important mediators of apoptosis, we examined the influence of Andr on JNK activity during V. alginolyticus infection. We found that Andr inhibited JNK activation induced by V. alginolyticus infection on L. vannamei. The ROS scavenger N-acetyl-l-cysteine (NAC) suppressed V. alginolyticus-induced inflammation and apoptosis, suggesting that ROS play an important role in V. alginolyticus-induced inflammation and apoptosis. Treated cells with JNK specific activator anisomycin, the inflammation and apoptosis inhibited by Andr were counteracted. Collectively, Andr promote the growth and immunity of L. vannamei, and protects shrimps against V. alginolyticus by regulating inflammation and apoptosis via a ROS-JNK dependent pathway. These results improve the understanding of the pathogenesis of V. alginolyticus infection and provide clues to the development of effective drugs against V. alginolyticus.

miR-144 and DJ-1/NF-κB regulates UCP4 maintain mitochondrial homeostasis in Penaeus vannamei.

UCP4, as an uncoupling protein in mitochondrial intima, is closely related to the resistance to oxidative stress and the function of mitochondria. However, whether and how its antioxidant capacity also works in crustaceans has not been reported in detail. This study showed that the expression of PvUCP4 was negatively correlated with the expression of pva-miR-144. The content of reactive oxygen species (ROS), ATP, and apoptosis was significantly increased, while the mitochondrial membrane potential (MMP) was seriously depolarized, Edema, vacuolation, and ambiguity of cristae and membrane were observed clearly in mitochondria after the knockdown of PvUCP4 induced by V. alginolyticus. The sharp drop in THC and severe damage in the hepatopancreas were all due to the knockout of PvUCP4 under the stress of V. alginolyticus. The co-transfection of pva-miR-144 and PvUCP4 could partially recover MMP compared with the abnormal expression of pva-miR-144. Similarly, co-transfection of pva-miR-144 and PvUCP4 could partially eliminate apoptosis compared with the abnormal expression of pva-miR-144. In addition, PvUCP4 3'-UTR has a pva-miR-144 predicted binding site in 1417-1428, which also was confirmed by the dual luciferase reporter assay. By the way, the results of ROS, MMP, and apoptosis showed that PvDJ-1 regulated the expression of PvUCP4 through PvNF-κB. Altogether, these results indicated that PvUCP4 has the antioxidant function of resisting oxidation reaction and weakening oxidative damage, to protect the normal operation of mitochondrial function and maintaining the cell homeostasis in shrimp.

MYC drives autophagy to adapt to stress in Penaeus vannamei.

MYC proto-oncogene (MYC), a first oncogenic nuclear transcription factor isolated from the human genome, belongs to the helix loop helix/leucine zipper protein family (bHLHzip). MYC plays an important part in the process of various physiological and biochemical of vertebrate, such as cell growth, proliferation, cycle, and autophagy. However, its molecular regulation mechanism and function in invertebrates are still unclear. In this study, a novel transcription factor MYC gene was screened, cloned, and characterized from Penaeus vannamei. The open reading frame of PvMYC was 1593bp, encode a polypeptide of 530 amino acids with molecular weight of 58.5 kDa, and a theoretical PI of 5.75. The results of tissue distribution showed that PvMYC was constitutively expressed in all detected tissues, and highest expression in hepatopancreas. The expression level of PvMYC up-regulated significantly and responded to low temperature stress by nuclear ectopic after low temperature stress. Overexpression of PvMYC in shrimp hemocytes negatively regulated the expression of Beclin-1 and reduced the conversion from LC3I to LC3II, yet p62 was decreased significantly. Meanwhile, RAPA eliminated the inhibition of autophagy caused by overexpression of PvMYC. ROS levels and autophagy flux showed the similar trend under low temperature stress after silencing PvMYC. The expression levels of Beclin-1, key ATG gene and LC3II increased significantly, while p62 decreased significantly under the same conditions. In addition, the Total hemocyte count (THC) decreased sharply, and accelerated the injury of hepatopancreas under low temperature stress after silencing PvMYC. Collectively, these results suggest that PvMYC has vital role in the cold adaptation mechanism of P. vannamei by negatively regulating autophagy.

Nemo-like kinase (NLK) gene regulates apoptosis via the p53 signaling pathway in Litopenaeus vannamei under low-temperature stress.

The Nemo-like kinase (NLK) is an important serine/threonine-protein kinase in many signaling pathways. However, its function in crustaceans, such as shrimps, is still poorly understood and needs to be further explored. In the present study, the full-length cDNA of NLK from Litopenaeus vannamei (LvNLK) was cloned. The full-length LvNLK cDNA has 2497 bp, including an open reading frame (ORF) of 1524 bp encoding a protein with 507 amino acids and a predicted molecular mass of 56.1 kDa. Phylogenetic analysis revealed that LvNLK shared high similarities with NLK from other known species. Low-temperature stress markedly upregulated the expression of LvNLK. Its overexpression in hemocytes suppressed the expression of BCL2-associated X (Bax) and tumor protein P53 (p53) in vitro. Meanwhile, the BCL2 apoptosis regulator (Bcl-2), MDM2 proto-oncogene (MDM2), and Yin Yang 1 (YY1) were upregulated. Moreover, LvNLK silencing in vivo increased the susceptibility of shrimps to low-temperature stress. The generation of ROS and the rate of hemocyte apoptosis also increased when LvNLK was silenced. Additionally, qPCR results indicated that LvNLK might participate in apoptosis via the p53 signaling pathway in vitro and in vivo. These results suggested that LvNLK is indispensable for the environmental adaptation of L. vannamei. Our current findings also demonstrated that NLK is evolutionarily conserved in crustaceans and provided insights into the environmental adaptation of invertebrates.

Transcriptome analysis of Pacific white shrimp (Litopenaeus vannamei) hepatopancreas challenged by Vibrio alginolyticus reveals lipid metabolic disturbance.

Vibrio alginolyticus is a devastating bacterial pathogen of Pacific white shrimp (Litopenaeus vannamei), which often causes acute hepatopancreatic necrosis syndrome (AHPNS) and early mortality syndrome (EMS). Elucidation of molecular mechanisms of L. vannamei in responding to infection is essential for controlling the epidemic. In the present study, transcriptomic profiles of L. vannamei hepatopancreas were explored by injecting with PBS or V. alginolyticus. Hepatopancreas morphology of L. vannamei was also assessed. The result reveals that compared with the hepatopancreas of PBS group, the storage cells (R-cell), secretory cells (B-cell) and star-shaped polygonal structures of the lumen were disappeared and necrotic after challenged by V. alginolyticus at 24 h. Transcriptome data showed that a total of 314 differential expression genes were induced by V. alginolyticus, with 133 and 181 genes up- and down-regulated, respectively. These genes were mainly associated with lysosome pathway, glycerophospholipid metabolism, drug metabolism-other enzymes, cysteine and methionine metabolism, aminoacyl-tRNA biosynthesis and PPAR signal pathway. Among these pathways, the lysosome pathway, glycerophospholipid metabolism and PPAR signal pathway were both related with lipid metabolism. Therefore, we detected the lipid accumulation in hepatopancreas by Oil Red O staining, TG and CHOL detection and the relative mRNA expression of several lipid metabolism related genes in the hepatopancreas of shrimp after challenge to V. alginolyticus. The present data reveals that lipids from the L. vannamei are nutrient sources for the V. alginolyticus and define the fate of the infection by modulating lipid homeostasis. These findings may have important implication for understanding the L. vannamei and V. alginolyticus interactions, and provide a substantial dataset for further research and may deliver the basis for preventing the bacterial diseases.

Integrative analysis of the miRNA-mRNA regulation network in hemocytes of Penaeus vannamei following Vibrio alginolyticus infection.

Penaeus vannamei is an important cultured shrimp that has high commercial value in the worldwide. However, the industry suffers heavy economic losses each year due to disease outbreaks caused by pathogenic bacteria. In the present study, after Vibrio alginolyticus infection, DNA damage in the hemocytes of the shrimp markedly increased, and autophagy and apoptosis increased significantly. Subsequently, hemocytes were sampled from the control and infected shrimp and sequenced for mRNA and microRNA (miRNA) 24 h after V. alginolyticus infection to better understand the response mechanism to bacterial infection in P. vannamei. We identified 1,874 and 263 differentially expressed mRNAs (DEGs) and miRNAs (DEMs) respectively, and predicted that 997 DEGs were targeted by DEMs. These DEGs were involved in the regulation of multiple signalling pathways, such as Toll and IMD signalling, TGF-beta signalling, MAPK signalling, and cell apoptosis, during Vibrio alginolyticus infection of the shrimp. We identified numerous mRNA-miRNA interactions, which provide insight into the defense mechanism that occur during the antimicrobial process of P. vannamei.

TBC domain family 7-like enhances the tolerance of Penaeus vannamei to ammonia nitrogen by the up-regulation of autophagy.

TBC domain family 7 (TBC1D7) is one of the subunits of tuberous sclerosis complex (TSC) and an important regulator of autophagosome biogenesis. However, the function of TBC1D7 is not fully understood in crustaceans. In the present study, TBC1D7 was identified from Penaeus vannamei. The complete coding sequence of PvTBC1D7 was of 960 bp encoding a predicted polypeptide of 319 amino acids with one conserved TBC domain, which shared high similarity with TBC1D7 of that other species. The mRNA of PvTBC1D7 was highly expressed in hemocyte and hepatopancreas, and the PvTBC1D7 protein was localized specifically in the cytoplasm of hemocyte of shrimp. Besides, PvTBC1D7 was co-localized with PvTSC1 in the cytoplasm of shrimp, indicating that there might existed a binding relationship between PvTBC1D7 and PvTSC1. During the ammonia nitrogen stress, the mRNA transcripts of PvTBC1D7 were significantly upregulated in hemocyte, hepatopancreas, and gill. Functionally, overexpression of PvTBC1D7 in vitro restored the inhibition to autophagy caused by chloroquine (CLQ) and increased the autophagy level, while the silencing of PvTBC1D7 could inhibit the autophagy. More importantly, after interfering with PvTBC1D7, the autophagy level decreased significantly both in hepatopancreas and hemocyte of P. vannamei, the mRNA expression of PvmTOR was increased remarkably with the significantly decrease of autophagy-related genes (PvATG12 and PvATG14). And the reduction of PvTBC1D7 remarkably exacerbated the damage of hepatopancreas, increased the accumulation of ROS, and reduced the survival proportion of shrimp under ammonia nitrogen stress. Altogether, these results indicated that PvTBC1D7 might positively regulate the autophagy by stabilizing the negative regulation of mTOR by TSC complex, reduce the oxidative stress damage and improve shrimp ammonia nitrogen tolerance.

Pva-miR-252 participates in ammonia nitrogen-induced oxidative stress by modulating autophagy in Penaeus vannamei.

MicroRNAs (miRNAs) are critical post-transcriptional regulators, which play a crucial role in resistance to adverse environmental stress by regulating autophagy. However, the mechanism of miRNA involved in the autophagy regulation of shrimp under ammonia nitrogen stress is still limited. In the present study, ammonia nitrogen could induce hepatopancreas injury and oxidative stress of P. vannamei, and significantly increase the content of ROS in hemocytes by flow cytometry. Simultaneously, it is accompanied by autophagy occurred in the hemocytes and hepatopancreas. Furthermore, the qRT-PCR analysis revealed that the expression of pva-miR-252 in P. vannamei decreased significantly after ammonia nitrogen stress, and pva-miR-252 negatively regulated PvPI3K by binding to 3'UTR of PvPI3K by double-luciferase assay. Pva-miR-252 overexpression could significantly increase the level of autophagy, and restore the autophagy inhibition caused by Chloroquine in vitro , whereas silencing of pva-miR-252 resulted in the opposite effect. More importantly, overexpression of pva-miR-252 could enhance the activity of antioxidant enzymes and reduced the production of ROS of shrimp under ammonia nitrogen stress. In conclusion, pva-miR-252 could positively regulate autophagy through PvPI3K and improve the antioxidant enzyme activity of P. vannamei under ammonia nitrogen stress, and our study provides a novel theoretical molecular mechanism for further understanding the shrimp cope with a high ammonia nitrogen environment.

Determining the function of LvSmad3 on Litopenaeus vannamei in response to acute low temperature stress.

Smad3 is a key mediator of the canonical TGF-ß signaling pathway and plays an important role in TGF-ß1-mediated transcriptional regulation. However, the function of Smad3 in crustaceans such as shrimp, is still poorly understood and needs to be further explored. We characterized Litopenaeus vannamei Smad3 (LvSmad3) and its biological functions were investigated in response low temperature stress. Full-length LvSmad3 cDNA was 2341bp and contained an open reading frame (ORF) of 1326 bp that encoded a 441 amino acid long protein, with a predicted molecular mass of 48.35 kDa. Phylogenetic analysis revealed that LvSmad3 has a high degree of similarity with other known species. LvSmad3 mRNA was detected in all the tested tissues and highest transcription occurred mostly in gills. Further research showed that suppressing the expression of Smad3 could reduce ROS production, DNA damage and the apoptosis rate in shrimp hemocyte under low temperature compared with the dsGFP group. Thus, we speculated that Smad3 could promote the apoptosis of hemocytes. We confirmed that Smad3 could inhibit apoptosis in the hepatopancreas by suppressing the expression of pro-apoptotic genes. Taken together, the silencing of Smad3 can reduce ROS production induced by low temperature stress, weaken the damage to hemocytes and the hepatopancreas by inhibit the apoptosis.