Acute hepatopancreatic necrosis disease (AHPND) caused by toxin-producing Vibrio parahaemolyticus (VpAHPND) has severely affected shrimp production. Long non-coding RNA (lncRNA), a regulatory non-coding RNA, which can play important function in shrimp disease responses. This study aimed to identify and investigate the role of lncRNA involved in VpAHPND infection in Pacific white shrimp, Litopenaeus vannamei. From a total of 368,736 de novo assembled transcripts, 67,559 were identified as putative lncRNAs, and only 72 putative lncRNAs showed differential expression between VpAHPND-infected and normal shrimp. The six candidate lncRNAs were validated for their expression profiles during VpAHPND infection and tissue distribution using RT-qPCR. The role of lnc2088 in response to VpAHPND infection was investigated through RNA interference. The result indicated that the suppression of lnc2088 expression led to an increase in shrimp mortality after VpAHPND infection. To explore the set of genes involved in lnc2088 knockdown, RNA sequencing was performed. A total of 275 differentially expressed transcripts were identified in the hepatopancreas of lnc2088 knockdown shrimp. The expression profiles of five candidate metabolic and immune-related genes were validated in lnc2088 knockdown and VpAHPND-infected shrimp. The result showed that the expression of ChiNAG was significantly increased, while that of NCBP1, WIPF2, and NFKB1 was significantly downregulated in ds2088-injected shrimp. Additionally, the expression of NFKB1, NCBP1 and WIPF2 was significantly increased, whereas that of ChiNAG and CUL5 were significantly decreased after infection with VpAHPND. Our work identified putative lncRNA profiles in L. vannamei in response to VpAHPND infection and investigated the role of lncRNA in shrimp immunity.

The CpG oligodeoxynucleotides (CpG ODNs) reportedly possess the capacity to strengthen immunity in mammals. This experiment was conducted to evaluate the impact of dietary supplementation with 17 types of CpG ODNs on intestinal microbiota diversity, antioxidant capacity, and immune-related gene expression profiles of the shrimp Litopenaeus vannamei. Diets including 50 mg kg-1 CpG ODNs wrapped in egg whites were prepared and divided into 17 different groups, with 2 control groups (normal feed and feed with egg whites). These CpG ODNs supplemented diets and the control diets were fed to L. vannamei (5.15 ± 0.54 g) three times daily at 5%-8% shrimp body weight for three weeks. The results of consecutive detection of intestinal microbiota by 16S rDNA sequencing indicated that 11 of the 17 types of CpG ODNs significantly enhanced intestinal microbiota diversity, increased the populations of several probiotic bacteria, and activated possible mechanisms relevant to diseases. The immune-related genes expression and antioxidant capacity in hepatopancreas further demonstrated that the 11 types of CpG ODNs effectively improved the innate immunity of shrimp. Additionally, histology results showed that the CpG ODNs in the experiment did not damage the tissue structure of hepatopancreas. The results suggest that CpG ODNs could be used as a trace supplement to improve the intestinal health and immunity of shrimp.

An 8-week feeding trial was conducted to evaluate the effects of L-methionine and methionine hydroxy analogue calcium (MHA-Ca) supplements in low-fishmeal diet on growth performance, hepatopancreas morphology, protein metabolism, anti-oxidative capacity, and immunity of Pacific white shrimp (Litopena eus vannamei). Four isonitrogenous and isoenergetic diets were designed: PC (203.3 g/kg fishmeal), NC (100 g/kg fishmeal), MET (100 g/kg fishmeal +3 g/kg L-methionine) and MHA-Ca (100 g/kg fishmeal +3 g/kg MHA-Ca). White shrimp (initial body weight 0.23 ± 0.00 g, 50 shrimp per tank) were allocated to 12 tanks and divided among 4 treatments in triplicates. In response to L-methionine and MHA-Ca supplementations, the shrimp exhibited higher weight gain rate (WGR), specific growth rate (SGR), condition factor (CF), and lower hepatosomatic index (HSI) compared to those fed the NC diet (p < 0.05). The WGR and SGR of shrimp fed L-methionine and MHA-Ca showed no difference with those in the PC diet (p > 0.05). Both of L-methionine and MHA-Ca supplementary diets significantly decreased the malondialdehyde (MDA) levels of shrimp when compared with the NC diet (p < 0.05). L-methionine supplementation improved the lysozyme (LZM) activity and total antioxidant capacity (T-AOC) of shrimp, while the MHA-Ca addition elevated the reduced glutathione (GSH) levels in comparison with those fed the NC diet (p < 0.05). Hypertrophied blister cells in hepatocytes were observed in shrimp fed the NC diet, and alleviated with L-methionine and MHA-Ca supplementations. Shrimp fed the MET and MHA-Ca diets had higher mRNA expression levels of target of rapamycin (tor) than those fed the NC diet (p < 0.05). Compared to the NC group, dietary MHA-Ca supplementation upregulated the expression level of cysteine dioxygenase (cdo) (p < 0.05), while L-methionine supplementation had no significant impact (p > 0.05). The expression levels of superoxide dismutase (sod) and glutathione peroxidase (gpx) were significantly upregulated by L-methionine supplemented diet in comparison with those in the NC group (p < 0.05). Overall, the addition of both L-methionine and MHA-Ca elevated the growth performance, facilitated protein synthesis, and ameliorated hepatopancreatic damage induced by plant-protein enriched diet in L. vannamei. L-methionine and MHA-Ca supplements enhanced anti-oxidants differently.

Enterocytozoon hepatopenaei (EHP) is the pathogen of hepatopancreatic microsporidiosis (HPM) in shrimp. The diseased shrimp Litopenaeus vannamei exhibits a slow growth syndrome, which causes severe economic losses. Herein, 4D label-free quantitative proteomics was employed to analyze the hepatopancreas of L. vannamei with a light (EHPptp2 < 103 copies/50 ng hpDNA, L group) and heavy (EHPptp2 > 104 copies/50 ng hpDNA, H group) load of EHP to better understand the pathogenesis of HPM. Exactly 786 (L group) and 1056 (H group) differentially expressed proteins (DEPs) versus the EHP-free (C group) control were mainly clustered to lipid metabolism, amino acid metabolism, and energy production processing. Compared with the L group, the H group exhibited down-regulation significantly in lipid metabolism, especially in the elongation and degradation of fatty acid, biosynthesis of unsaturated fatty acid, metabolism of α-linolenic acid, sphingolipid, and glycerolipid, as well as juvenile hormone (JH) degradation. Expression pattern analysis showed that the degree of infection was positively correlated with metabolic change. About 479 EHP proteins were detected in infected shrimps, including 95 predicted transporters. These findings suggest that EHP infection induced the consumption of storage lipids and the entire down-regulation of lipid metabolism and the coupling energy production, in addition to the hormone metabolism disorder. These were ultimately responsible for the stunted growth.

Litopenaeus vannamei Smad5 (LvSmad5) in cytoplasm has been proved to be involved in environmental stress response. As LvSmad5 could also locate in nucleus under specific stress, it was conjectured that LvSmad5 might participate in environmental stress response. While, the experimental evidence is still lacking. In this study, cytosolic LvSmad5 mutant or nuclear LvSmad5 mutant was expressed in Drosophila S2 cells, and then transcriptomic analysis of mentioned cells was performed using Illumina HiSeq based RNA-Seq, to reveal the function of LvSmad5 in nucleus. By comparing the two groups of cDNA libraries from S2 cells with cytosolic or nucleus LvSmad5 mutant, 86 differentially expressed genes as well as 765 differentially expressed transcripts were found. It was revealed that genes in the ER-stress response pathway, such as unfolded protein response and ER-associated degradation (ERAD) were enriched. Additionally, some kinds of metabolic reprogramming occurred in S2 cells with over-expressing nuclear LvSmad5, for significant changes in the expression of some metabolism-related genes. To test our infer that nuclear LvSmad5 was engaged in environmental stress response, homologous gene of Drosophila translocation in renal carcinoma on chromosome 8 in L.vannamei (LvTRC8) was chosen for further investigation. And studies about LvTRC8, a member of ERAD showed that it was induced by ER-stress or heat shock treatment. Suppressed the expression of LvTRC8 increased the cumulative mortality of shrimp upon stress. In some degree, these results support our speculation that nuclear LvSmad5 are involved in the environmental stress response of L. vannamei in fact.

Anti-lipopolysaccharide factors (ALFs) are a family of common innate immune effectors in crustaceans, and they exhibit broad spectrum antimicrobial activity. In this study, we identified and characterized five novel ALF genes (designated as LvALF1-5) from the Pacific white shrimp (Litopenaeus vannamei) to investigate their potential immune functions. The amino acid sequence alignments showed that LvALFs contained two conserved cysteine residues, a hydrophobic N-terminal region, and the conserved signature sequence W(T/K)CPG(S)WT(A). They all shared high similarity with previously reported ALFs and were clearly novel members of the ALF family. The mRNA transcripts of LvALFs were most highly expressed in hemocytes and the hepatopancreas. After shrimp were stimulated with Vibrio parahaemolyticus or white spot syndrome virus, expression of the LvALFs was significantly induced in hemocytes and the hepatopancreas with various expression profiles. Recombinant proteins of LvALFs exhibited potent bacteriostatic activity in vitro. Together, these results suggest that LvALF1-5 participate in the immune response of Pacific white shrimp against invading pathogens.

Ammonia nitrogen tolerance is an economically important trait of the farmed penaeid shrimp Litopenaeus vannamei. To identify the genes associated with ammonia nitrogen tolerance, we performed an extreme phenotype genome-wide association study method (XP-GWAS) on a population of 200 individuals. The single nucleotide polymorphism (SNP) genotyping array method was used to construct the libraries and 36,048 SNPs were genotyped. Using the MLM, FarmCPU and Blink models, six different SNPs, located on SEQ3, SEQ4, SEQ5, SEQ7 and SEQ8, were determined to be significantly associated with ammonia nitrogen tolerance. By integrating the results of the GWAS and the biological functions of the genes, seven candidate genes (PDI, OZF, UPF2, VPS16, TMEM19, MYCBP2, and HOX7) were found to be associated with ammonia nitrogen tolerance in L. vannamei. These genes are involved in cell transcription, cell division, metabolism, and immunity, providing the basis for further study of the genetic mechanisms of ammonia nitrogen tolerance in L. vannamei. Further candidate gene association analysis in the offspring population revealed that the SNPs in the genes zinc finger protein OZF-like (OZF) and homeobox protein Hox-B7-like (HOX7) were significantly associated with ammonia nitrogen tolerance trait of L. vannamei. Our results provide fundamental genetic information that will be useful for further investigation of the molecular mechanisms of ammonia nitrogen tolerance. These associated SNPs may also be promising candidates for improving ammonia nitrogen tolerance in L. vannamei.

Nanoplastic is recognized as an emerging environmental pollutant due to the anticipated ubiquitous distribution, increasing concentration in the ocean, and potential adverse health effects. While our understanding of the ecological impacts of nanoplastics is still limited, we benefit from relatively rich toxicological studies on other nanoparticles such as nano metal oxides. However, the similarity and difference in the toxicokinetic and toxicodynamic aspects of plastic and metallic nanoparticles remain largely unknown. In this study, juvenile Pacific white shrimp Litopenaeus vannamei was exposed to two types of nanoparticles at environmentally relative low and high concentrations, i.e., 100 nm polystyrene nanoplastics (nano-PS) and titanium dioxide nanoparticles (nano-TiO2) via dietary exposure for 28 days. The systematic toxicological evaluation aimed to quantitatively compare the accumulation, excretion, and toxic effects of nano-PS and nano-TiO2. Our results demonstrated that both nanoparticles were ingested by L. vannamei with lower egestion of nano-TiO2 than nano-PS. Both nanoparticles inhibited the growth of shrimps, damaged tissue structures of the intestine and hepatopancreas, disrupted expression of immune-related genes, and induced intestinal microbiota dysbiosis. Nano-PS exposure caused proliferative cells in the intestinal tissue, and the disturbance to the intestinal microbes was also more serious than that of nano-TiO2. The results indicated that the effect of nano-PS on the intestinal tissue of L. vannamei was more severe than that of nano-TiO2 with the same particle size. The study provides new theoretical basis of the similarity and differences of their toxicity, and highlights the current lack of knowledge on various aspects of absorption, distribution, metabolism, and excretion (ADME) pathways of nanoplastics.

This study applied bisindolylmaleimide I (BSM), a pharmacological competitive inhibitor of protein kinase C (PKC) enzymatic activity, at 1.25 pmol shrimp-1 for 60 min to investigate the potential involvement of PKC in signal transduction pathways in the hemocytes of Litopenaeus vannamei. A novel PKC in L. vannamei (LvnPKC) was identified and characterized and was determined to be involved in mediating the neuroendocrine-immune regulatory network. The hemocytes of L. vannamei that receive BSM exhibit significantly decreased PKC activity and LvnPKC gene and protein expression levels. Furthermore, the total hemocyte count, hyaline cells, and semigranular cells increased significantly along with significant decreases in granular cells, and meanwhile, the significantly increased phenoloxidase activity, respiratory bursts, superoxide dismutase (SOD) activity, phagocytic activity, and neutrophil extracellular trap were observed; however, phagocytic activity decreased significantly. In a molecular model, the gene expressions of lipopolysaccharide- and β-1,3-glucan-binding protein, peroxinectin, cytosolic manganese SOD, mitochondrial manganese SOD, and copper/zinc SOD in the hemocytes of L. vannamei that had received BSM decreased significantly, but prophenoloxidase I increased significantly. In catecholamine biosynthesis, tyrosine, dopamine, and norepinephrine decreased significantly in the hemocytes of L. vannamei that had received BSM, and l-dihydroxyphenylalanine increased. Moreover, tyrosine hydroxylase (TH) activity increased significantly, whereas TH and dihydroxyphenylalanine decarboxylase gene expression decreased significantly. These findings suggest that BSM inhibits PKC activity in hemocytes in which LvnPKC gene and protein expression are also inhibited. Additionally, the hemocytes\' immunocompetence, including their prophenoloxidase and antioxidant systems, phagocytic activity, and catecholamine biosynthesis, was disrupted, confirming the roles of LvnPKC in mediating the neuroendocrine-immune regulatory network in hemocytes.

The Pacific white shrimp (Litopenaeus vannamei) is the most widely cultured shrimp in the world. A great attention has been paid to improve its body weight (BW) at harvest through genetic selection for decades. Genome-wide association study (GWAS) is a tool to dissect the genetic basis of the traits. In this study, a GWAS approach was conducted to find genes related to BW through genotyping 94,113 single nucleotide polymorphisms (SNPs) in 200 individuals from a breeding population. Four BW-related SNPs located in LG19 and LG39 were identified. Through further candidate gene association analysis, the SNPs in two candidate genes, deoxycytidylate deaminase and non-receptor protein tyrosine kinase, were found to be related with the body weight of the shrimp. Marker-assisted best linear unbiased prediction (MA-BLUP) based on the SNPs in these two genes was used to estimate the breeding values, and the result showed that the highest prediction accuracy of MA-BLUP was increased by 9.4% than traditional BLUP. These results will provide useful information for the marker-assisted breeding in L. vannamei.