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Vibrio parahaemolyticus carrying a plasmid encoding the PirAvp and PirBvp toxins is the causative agent of acute hepatopancreatic necrosis disease (AHPND) in penaeid shrimps. In the Pacific white shrimp farming industry, one possible strategy to reduce economic loss due to AHPND is the development of a shrimp line resistant to the disease. In this study, we identified single nucleotide polymorphisms (SNPs) in the Litopenaeus vannamei anti-lipopolysaccharide factor 1 (nLvALF1) and penaeidin 2-1 (PEN2-1) genes, and we analyzed the associations between these SNPs and resistance/susceptibility to V. parahaemolyticus infection in the Pacific white shrimp. Postlarvae (PL20) shrimp from a local hatchery in Prachuap Khiri Khan Province were challenged with an isolate of VPAHPND and mortality was observed for 14 days. DNA was extracted from susceptible (died within 6 days) and resistant (survived the challenge) shrimp (45 individuals/group) and used for PCR amplifications of nLvALF1 (397 bp) and PEN2-1 (637 bp) gene fragments. PCR products were sequenced by direct sequencing and SNPs were identified from sequencing chromatograms. Nine and seven SNPs were identified in nLvALF1 and PEN2-1 gene fragments, respectively. Analyses of allele frequencies in susceptible and resistant samples using Chi-square tests revealed that four and six SNPs in nLvALF1 and PEN2-1, respectively, were associated with resistance/susceptibility to V. parahaemolyticus infection (P

Tyramine beta-hydroxylase (TBH) is needed for the biosynthesis of the octopamine (OA) from tyramine (TA). Both OA and TA act as neurotransmitters, neurohormones, and neuromodulators in the invertebrate nervous system. In this study, TBH was identified in white shrimp, Litopenaeus vannamei, and further investigation on its potential function was conducted after inducing hypothermal stress and Vibrio alginolyticus infection. TBH of L. vannamei (LvTBH) was comprised 2178 nucleotide residues and contained an open reading frame encoding 408 amino acids, belonging to the Copper type II, ascorbate-dependent monooxygenases, was characterized by two Cu2_monooxygen domains and five glycosylation sites. LvTBH expression was especially abundant in muscle, and mainly in brain and thoracic ganglia of nervous system, eyestalk tissues, epithelium, and stomach, as determined by quantitative real-time PCR. The effects of hypothermal stress showed significant increases in LvTBH at 15, 30 and 60 min in brain and at 30 min in haemocyte, accompanied by an increase in OA level in haemolymph from 15 to 60 min. Significant increases in LvTBH occurred at 15, 30 and 60 min in haemocyte and at 60 min in brain tissue, and was proportional to the OA level of haemolymph under Vibrio alginolyticus infection from 30 to 60 min. Here, we demonstrated that LvTBH is functionally responsible for biogenic amine synthesis, suggesting that the increased release of OA in haemolymph for potential modulation of physiological and immunological responses is the consequence of the upregulated LvTBH gene expression in L. vannamei exposed to hypothermal stress and Vibrio alginolyticus infection.

Diseases caused by Vibrio harveyi in shrimps have gradually become one group of the most serious threats to shrimp production, while related molecular mechanisms of infections with Vibrio harveyi are still not known well in shrimps. Here, we performed proteomic sequencing of hepatopancreas in whiteleg shrimps (Litopenaeus vannamei) infected with exogenous Vibrio harveyi, and subsequent functional annotation and calculation of differentially expressed proteins (DEPs) in this study. A total of 145 DEPs were obtained, among them 36 were up-regulated and 109 were down-regulated after the infection. Meanwhile, our results showed that after the infection of Vibrio harveyi, expression levels of a variety of C-type lectins (CTLs) were changed significantly. In-depth functional domain analysis and spatial structure prediction of these CTLs revealed that amino acid sequences and spatial structures of the C-type lectin domain (CTLD) shared by the CTL-S and IML proteins were variant, suggesting differential functions between the two CTLs. In summary, various members of the CTL family have different epidemic responses to Vibrio harveyi infection, which provides a theoretical guidance for deep-going investigations on practical immunity reactions and pathogen infections in shrimps.

The study investigated the dietary effects of Clostridium autoethanogenum protein (CAP) substituting fish meal on the growth, intestinal histology, serum immune indexes and transcriptome of Pacific white shrimp, Litopenaeus vannamei. Four isonitrogenous and isolipidic diets were designed as the control diet (CON) containing 560 g/kg fish meal, and three fish meal-substituted diets in which 30% (CAP-30), 45% (CAP-45) and 70% (CAP-70) fish meal were replaced with CAP, respectively. The four diets were fed to shrimp with initial body weight of 2.78 ± 0.13 g for 8 weeks. The results showed that the weight gain, feed intake, survival and intestinal villus height in CAP-45 and CAP-70 groups were lower than those of the control and CAP-30 groups (P

To reach the sustainable development goals on waste recycling, cacao pod husk (CPH), produced as an agricultural waste byproduct during the cacao bean processing was applied to manufacture CPH pectin for developing the potential for diverse application in aquaculture, minimizing CPH impact to the environment and bringing benefits to the agriculture and aquaculture industries. In this study, CPH pectin (5 g/kg diet) and Lactobacillus plantarum (LP; 1010 cfu/kg diet) were separately introduced to the diets of Litopenaeus vannamei for a 56-day feeding trial, and two synbiotic combinations of CPH pectin and LP (CPH pectin at 5 g/kg diet + LP at 107 cfu/kg diet or at 1010 cfu/kg diet) were also conducted. After the 56-day feeding trial, significantly elevated percent weight gain, percent length gains and feeding efficiency in L. vannamei were only observed in synbiotic combination of CPH pectin at 5 g/kg diet and LP at 107 cfu/kg diet treatment, and the remainder of the treatments remained consistently similar to the control. Significantly increases in total haemocyte count, granular cells, phenoloxidase activity, and respiratory bursts were observed in L. vannamei fed with synbiotics at 7–28 days of feeding, accompanied by significant promotion of phagocytic activity and clearance efficiency in response to V. alginolyticus challenge during 56 days of feeding trial. Furthermore, at the end of the 56 days of feeding trial, shrimp receiving CPH pectin and/or LP treatments showed a significantly higher survival ratio against V. alginolyticus infection and hypothermal stress. It was therefore concluded that CPH pectin or LP was confirmed as an immunostimulant for L. vannamei to trigger immunocompetence through oral administration without negative effects within 56 days of feeding trial, and the synbiotic combination of CPH pectin and LP exhibited complementary and synergistic effects on growth performance and immunocompetence in L. vannamei.

Cell survival is based on the stability of intracellular state. It was well known that biochemical reactions in cells require specific intracellular environments, such as pH and calcium concentration. While the mechanism of stabilizing the intracellular environment is complex and far from clear. In this study, a Sma and Mad related protein 5 gene (LvSmad5) of Litopenaeus vannamei was cloned. LvSmad5 was located to both cytoplasm and nucleus. And subcellular localization of LvSmad5 was responsed to the changing of cells internal and external environment. Besides, it was found that subcellular localization of LvSmad5 was also regulated by unfolded protein response. Moreover, it was proved that nucleic localization of LvSmad5 could significantly increase the white spot syndrome virus (WSSV) infection in shrimp, and knockdown expression of LvSmad5 decreased the cumulative mortality of WSSV infection shrimp. Further investigation revealed that cytoplasm LvSmad5 could interplay with shrimp hexokinase 1, and contribute to glycolysis. These results indicated that LvSmad5 played a role in L. vannamei environmental stress response, and was used by WSSV for its replication.

Xiao-Chaihu-Decoction (XCHD), a classical traditional Chinese medicine with diverse biological activities, is widely applied to prevent and treat many human diseases. Effects of dietary XCHD on growth performance, immune response, detoxification system, intestinal microbiota and resistance against aflatoxin B1(AFB1) of Litopenaeus vannamei was studied. Four isonitrogenous and isolipidic diets were formulated to contain 0, 1, 2, and 5 g/kg (control, XCHD1, XCHD2 and XCHD3) of XCHD, respectively. Seven hundred and eighty shrimp (1.16 ± 0.09 g) were assigned randomly to 12 tanks (400 L, three tanks each group, 65 shrimp in each tank) for 6 weeks. After sampling, 25 shrimp from each tank were selected for a 2-week AFB1 (2500 μg/kg) challenge experiment. The results indicated that the final weight, weight gain and specific growth rate in XCHD2 and XCHD3 groups were significantly increased compared to control. The protease, amylase, superoxide dismutase (SOD), glutathione s-transferase (GST), sulfotransferase (SULT) activities, total antioxidant capacity (T-AOC) and glutathione (GSH) contents in hepatopancreas were significantly increased in XCHD3 groups and the expressions of immune-related genes (Toll, Dorsal and Cru) in hepatopancreas were significantly up-regulated in XCHD2 and XCHD3 groups. High-throughput sequencing analysis revealed that the abundance of Proteobacteria decreased and the abundances of Bacteroidetes increased in XCHD2 and XCHD3 groups. Additionally, AFB1 challenge experiments showed that AFB1 caused histological damage to the hepatopancreas and significantly increased the levels of malondialdehyde (MDA) and protein carbonylation (PC) in hepatopancreas as well as the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Nevertheless, XCHD could effectively alleviated the growth toxicity, immunosuppression and macromolecular damage caused by AFB1 to shrimp by inhibiting the Phase I enzyme and enhancing Phase II enzyme and antioxidant system.

This study investigates the effects of Phyllanthus amarus extract (PAE) on immune responses, growth, and resistance to Vibrio alginolyticus in white shrimp (Litopenaeus vannamei). In vitro PAE treatment did not alter the cell viability of haemocytes and significantly enhanced immune parameters such as phenoloxidase (PO) activity, phagocytic activity, and superoxide anion (O2−) production. We conducted two feeding trials to examine the effects of PAE on the growth, disease resistance, and innate immune parameters of white shrimp. In the first in vivo trial, shrimps (4.01 ± 0.03 g) were fed a diet containing 0 g (control), 10 g (PAE10), 20 g (PAE20), or 40 g (PAE40) of PAE per kilogram of feed for 56 days. After the feeding period, the PAE20 group showed a significantly higher weight gain and specific growth rate than shrimp fed the control diet. Furthermore, after challenge with V. alginolyticus, shrimp fed a diet containing PAE showed significantly higher survival than those fed the control diet. The second in vivo trial (28 days) was performed to identify the mechanisms of enhanced immunity in PAE-fed shrimp. Shrimp fed the PAE20 diet generally had the highest total haemocyte count, PO activity, phagocytic activity, and O2− production, followed by the PAE40 and PAE10 groups. Thus, our results suggest that administration of 20 g of PAE per kilogram of feed can enhance immunity, growth, and resistance to V. alginolyticus in white shrimp.

To investigate shrimp immunoregulation and tissue self-repair mechanism during temperature fluctuation stage, Litopenaeus vannamei (L. vannamei) was treated under conditions of gradual cooling from an acclimation temperature (28 °C, C group) to 13 °C (T group) in 2 days with a cooling rate of 7.5 °C/d and then rewarmed to 28 °C (R group) with the same rate. Tandem mass tags (TMT) -based proteomics technology was used to investigate the protein abundance changes of intestine in L. vannamei during temperature fluctuation. The results showed that a total of 5796 proteins with function annotation were identified. Of which, the abundances of 1978 proteins (34%) decreased after cooling and then increased after rewarming, 1498 proteins (26%) increased during the whole stage, 1263 proteins (22%) increased after cooling and then decreased after rewarming and 1057 proteins (18%) decreased during the whole stage. Differentially expressed proteins such as C-lectin, NFκBIA and Caspase may contributed to the regulation of immunity and tissue repair of shrimp intestine during the temperature fluctuation stage. These findings contribute to the better understanding of shrimp’ regulatory mechanism against adverse environment.

The effect of fully deacetylated chitosan (FDCH) edible coating combined with kojic acid (KA) and clove essential oil (CEO) as natural preservatives was investigated on the shelf-life of white prawn shrimp (Litopenaeus vannamei) during 15 days at cold storage. The results indicated that FDCH1:KA0.25:CEO0.25 coating significantly inhibited the increase in total aerobic plate count, total volatile basic nitrogen content and pH of shrimp in comparison with the control. Moreover, compared with the control, the changes of total color difference (ΔE values) and melanosis were significantly retarded, and the texture parameters and sensory scores were significantly improved in shrimp treated by FDCH1:KA0.25:CEO0.25, suggesting that there is a synergistic effect between FDCH, KA, and CEO. In addition, a significant (P

p70S6K is involved in cellular response, such as tumor metastases, the immune response and tissue repair in vertebrates. The role of p70S6K in these physiological processes in crustaceans remains, however, unknown. In this study, the Lvp70S6K was identified, containing a 5' UTR of 294 bp, an ORF of 1494 bp ad a 3' UTR of 211 bp, encoding 497 amino acids with a theoretical molecular weight of 70 kDa and an estimated isoelectric point of (pI) of 5.16. The multiple alignment found that Lvp70S6K was highly homologous with other invertebrates. Lvp70S6K mRNA was detected in all the tested tissues and the Lvp70S6K expression levels was significantly down-regulated and reached the lowest level (0.44-fold, p 0.01) at 1.5 h after low temperature stress. The subcellular localization of Lvp70S6K could be detected in cytoplasm. ROS production was significantly up-regulation (1.19-fold, p 0.01), total hemocyte count (THC) was significantly down-regulation (0.22-fold, p 0.01), apoptosis rate was markedly increased (1.09-fold, p 0.01), apoptosis-related genes of LvPDCD4 (1.61-fold, p 0.01) and LvCyt.C (1.23-fold, p 0.01) were up-regulated, and anti-apoptotic gene of LvBcl-2 (0.69-fold, p 0.01), LvIAP1 (0.68-fold, p 0.01) and LvIAP2 (0.45-fold, p 0.01) were decreased after low temperature stress in hemolymph of Lvp70S6K-silenced shrimp at 1.5 h. Silencing of LvPTEN significantly increased Lvp70S6K, LvPI3K, LvAKT and LvmTOR expression. In summary, these results indicated that Lvp70S6K play a crucial role in oxidative and apoptosis, which was able to negatively regulate by PTEN.

Gills as the respiratory tissue of crustacean encounter various threats in the environment. The immune defense in gills is essential to the health of shrimp. In the present study, we identified a novel gill-abundant crustin, LvCrustin Ⅱ-1, from the shrimp Litopenaeus vannamei. The full-length open reading frame of LvCrustin Ⅱ-1 is 522 bp, which encodes 173 amino acid residues. The amino acid sequence of LvCrustin Ⅱ-1 contained a signal peptide, a glycine rich region, a cysteine rich region and a whey acidic protein (WAP) domain. The expression level of LvCrustin Ⅱ-1 was significantly up-regulated at different time points after Vibrio parahaemolyticus immersion. When LvCrustin Ⅱ-1 was silenced by dsRNA interference, the count of bacteria colonies increased significantly in the hepatopancreas of shrimp after V. parahaemolyticus immersion, which indicated that the infection progress of pathogenic bacteria was accelerated after LvCrustin Ⅱ-1 knockdown. Compared with the microbiota of seawater, the lower proportion of aquatic bacteria and higher proportion of symbiont in the gills microbiota of shrimp indicated the bacterial colonization was modulated by the host. Knockdown of LvCrustin Ⅱ-1 changed the proportion of some potential pathogens and aquatic bacteria, which supported the idea that the new identified crustin in the gills played important roles in modulation of the microbiota community in shrimp. The present data provided new insights into the multiple functions of crustin in the immunity of shrimp.

The white shrimp Litopenaeus vannamei is one of the most commercially important aquatic species. However, intensive farming to meet economic demands has been linked to animal stress and subsequent disease outbreaks. In this study, we explore the immunomodulatory effects of Sargassum horneri hot-water extract (SHE) on the immune parameters of L. vannamei. Hemocytes incubated in vitro with 10 mg/ml of SHE showed the highest response in phenoloxidase (PO) activity and reactive oxygen species production. In the in vivo trials, shrimp were fed diets containing 0 (control), 2.5 (SHE 2.5), 5.0 g (SHE 5.0), and 10 g (SHE10.0) of lyophilized SHE per kg feed for four weeks, after which immune parameters were measured. Group SHE10.0 had the highest total hemocyte count, reactive oxygen species production, and phagocytic rate during the 4-week feeding period, followed by groups SHE5.0 and SHE2.5. However, group SHE5.0 showed a better growth performance among all examined groups after four weeks of rearing. Furthermore, gene expression analysis revealed that L. vannamei fed diets containing 0.5% SHE over 28 days showed significant modulation of 11 immune-related genes, including prophenoloxidase I, prophenoloxidase II, peroxinectin, α2macroglobulin, clotting protein, lysozyme, superoxide dismutase, glutathione peroxidase, penaiedin2-4, and crustin. Combined, these results show that SHE is a functional feed additive that can be applied orally to enhance innate immunity and growth performance of white shrimps.

Two trials were conducted as follows: the first trial was a 90-day experiment to determine the effects of reducing feeding level on shrimp status; the second trial (90 days) is based on the first trial to explore the suitable C/N ratio of biological flocs for shrimp culture in outdoor soil ponds. Results showed that the BFV levels increased gradually and then tended to be stable in the treatment groups. Concentrations of TAN and NO2–N were maintained low level in each treatment pond during experimental period. The final body weight, biology body length and yield of the shrimp in each trial with no significantly different (P > 0.05) while food coefficient and THC of the shrimp in 70% feeding level and C/N12 treatment were slightly lower than in the 100% feeding level and C/N16 treatment respectively (p 0.05). The shrimp in 70% feeding level and C/N12 treatment had the higher T-AOC in both the plasma and the hepatopancreas when compared with 100% feeding level and C/N16 treatment group (p 0.05). The effects of two feeding levels and C/N ratios on the GSH level and the ratio of GSH/GSSG in the plasma and the hepatopancreas of shrimp showed no significant difference (P > 0.05). The results showed that 70% feeding level and C/N12 ratio could provide adequate nutrition for shrimp to maintain a normal physical health status with the presence of bioflocs.

Cobalt (Co) is an important component of vitamin B12, but is toxic to aquatic animals at a high level. In this study, the Pacific white shrimp, Litopenaeus vannamei were exposed to three Co concentrations (0, 100, and 1000 μg/L) for 4 weeks. The survival and condition factor in shrimp exposed to the Co treatments were not different from the control, but the shrimp exposed to 100 μg Co/L gained more weight than in other two groups, and the shrimp exposed to 1000 μg Co/L gained less weight than in other groups. The SOD and GSH-PX activities were higher in shrimp exposed to 100 μg Co/L, but lower in the shrimp exposed to 100 μg Co/L compared with the control, respectively. The MDA contents in the hepatopancreas decreased in the 100 μg Co/L, but increased in the 1000 μg Co/L. The serum lysozyme decreased with ambient cobalt, was lower in the shrimp exposed to 1000 μg Co/L than in other two groups. The expression of C-type lectin 3 was down-regulated by Co concentrations. The Toll and immune deficiency in shrimp exposed to 100 μg Co/L was higher than in other two groups. The mucin-1 was lower in the 1000 μg Co/L group than in other two groups, but mucin-2 and mucin-5AC were higher in the 1000 μg Co/L group than in the control. With increasing Co concentration, Shannon and Simpson indexes of the intestinal microbial communities were decreased. The abundance of pathogenic bacteria (Ruegeria and Vibrio) increased in both Co groups. This study indicates that chronic exposure to waterborne cobalt could affect growth, cause oxidative stress, stimulate the immune response, damage intestinal histology, and reshape intestinal microbiota community L. vannamei.

Copper can be accumulated in water through excessive sewage discharge or residual algaecide to generate toxic effect to aquatic animals. In this study, the juvenile of Pacific white shrimp, Litopenaeus vannamei was exposed to 0 (control), 0.05, 0.1, 0.2, 0.5 or 1 mg Cu2+ L−1 for 30 days. Growth, immune function, anti-oxidative status and gut microbiota were evaluated. Weight gain and specific growth rate of L. vannamei were significantly decreased with the increase of ambient Cu2+. Enlarged lumen and ruptured cells were found in the hepatopancreas of shrimp in the 0.5 or 1 mg Cu2+ L−1 treatment. Total hemocyte counts of shrimp in 0.5 or 1 mg Cu2+ L−1 were significantly lower than in the control. The hemocyanin concentration was also significantly increased in 0.2 or 0.5 mg Cu2+ L−1. Lysozyme contents were reduced in shrimp when Cu2+ exceeded 0.2 mg L−1. Meanwhile, activities of superoxide dismutase and glutathione peroxidase were increased in the hepatopancreas and the activity of Na+-K+ ATPase was decreased in the gills with increasing Cu2+. The mRNA expressions of immune deficiency, toll-like receptor and caspase-3 were all significantly higher in the hepatopancreas in 0.05 mg Cu2+ L−1 than in the control. For the diversity of intestinal microbes, Bacteroidetes significantly decreased in 1 mg Cu2+ L−1 at the phylum level. KEGG pathway analysis demonstrates that 1 mg L−1 Cu2+ can significantly alter metabolism, cellular processes and environmental information processing. This study indicates that the concentration of 1 mg L−1 Cu can negatively impact growth, hemolymph immunity, anti-oxidative capacity and gut microbiota composition of L. vannamei.

Although information about invertebrate lysozymes is scarce, these enzymes have been described as components of the innate immune system, functioning as antibacterial proteins. Here we describe the first thermodynamic and structural study of a new C-type lysozyme from a Pacific white shrimp Litopenaeus vannamei (LvL), which has shown high activity against both Gram (+) and Gram (−) bacteria including Vibrio sp. that is one of the most severe pathogens in penaeid shrimp aquaculture. Compared with hen egg-white lysozyme, its sequence harbors a seven-residue insertion from amino acid 97 to 103, and a nine-residue extension at the C-terminus only found in penaeid crustaceans, making this enzyme one of the longest lysozyme reported to date. LvL was crystallized in the presence and absence of chitotriose. The former crystallized as a monomer in space group P61 and the latter in P212121 with two monomers in the asymmetric unit. Since the enzyme crystallized at a pH where lysozyme activity is deficient, the ligand could not be observed in the P61 structure; therefore, we performed a docking simulation with chitotriose to compare with the hen egg lysozyme crystallized in the presence of the ligand. Remarkably, additional amino acids in LvL caused an increase in the length of α-helix H4 (residues 97–103) that is directly related to ligand recognition. The Ka for chitotriose (4.1 × 105 M−1), as determined by Isothermal Titration Calorimetry, was one order of magnitude higher than those for lysozymes from hen and duck eggs. Our results revealed new interactions of chitiotriose with residues in helix H4.

Litopenaeus vannamei, as an important marine aquaculture species, has attracted more and more attentions in past several years. More recently people got its genome fine mapping, which unveiled a gene treasure. In this study, we have identified a novel trypsin-like protein which came from previous WSSV-infected shrimp plasma iTRAQ data. This protein is a 39 kDa protein with 363 amino acids. It contains a conserved trypsin-domain and could be strongly induced with WSSV infection. Interestingly, knockdown of this protein made shrimps vulnerable to WSSV infection. Further exploration unveiled that this fragility was probably due to the fact that knockdown of this protein could cause shrimp hemocytes apoptosis, which indicated that this protein played key roles in preventing shrimp hemocytes from apoptosis. To further explore how LvTLAP protected shrimp hemocytes from apoptosis, GST pull down assay was applied to screen LvTLAP interacting protein in shrimp plasma. L. vannamei growth and transformation-dependent-like protein (LvGTD-like protein) was identified as a LvTLAP interacting protein, which played proapoptotic roles in cells. Thus, a possible explanation for LvTLAP anti-apoptosis activity was that this protein could block LvGTD-like protein proapoptotic activity to protect shrimp hemocytes from death. In general, our study has uncovered a novel WSSV responsive shrimp plasma protein, which played key roles in shrimp hemocytes anti-apoptosis and shrimp against WSSV infection.

The purpose of this study was to profile the mechanisms of action of probiotic, Bacillus subtilis E20 in activating the immunity of white shrimp, Litopenaeus vannamei. Two groups of shrimp were studied. One group was fed a control diet without probiotic supplementation and the other was fed a probiotic-containing diet at a level of 109 cfu kg diet−1. After the 8-week feeding regimen, the metabolite composition in the hepatopancreas of shrimp were investigated using 1H nuclear magnetic resonance (1H NMR) based metabolomic analysis. Results from the 1H NMR analysis revealed that 16 hepatopancreatic metabolites were matched and identified among groups, of which 2 metabolites, creatinine and glutamine were significantly higher in probiotic group than in the control group. This result was confirmed by the reverse-phase high-performance liquid chromatography (RP-HPLC) and spectrophotometric analysis. Transcriptome analysis indicated the expressions of 10 genes associated with antioxidant enzymes, pattern recognition proteins and antimicrobial molecules, more active expression in the shrimp fed a diet supplemented with probiotic as compared to that of shrimp in control. In addition, the expressions of 4 genes involved with hexosamine biosynthesis pathway (HBP) and UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase for protein O-glycosylation were also higher in hepatopancreas of probiotic-treated shrimp than in shrimp fed a control diet. Western blot and enzyme-linked immunosorbent assay showed that heat shock factor 1, heat shock protein 70, and protein O-glycosylation in hepatopancreas were higher in probiotic group than the control group. These findings suggest that probiotic, B. subtilis E20 promotes the digestibility of glutamine in the diet, and that the increased glutamine in shrimp can be used as fuel for immune cells or may be used to regulate immune molecule expressions and protein O-glycosylation via the HBP to increase protein O-glycosylation, thereby improving the health of shrimp.

A 56-day feeding trial was done to investigate the interactive effects of cholesterol (CHO) and phospholipids (PL) on the growth performance, immune response, expression of immune-related genes, and resistance against Vibrio alginolyticus of freshwater cultured white shrimp (Litopenaeus vannamei). A 3 × 3 experimental design was conducted with nine experimental diets containing three levels of CHO (0, 0.2%, and 0.4%) and three levels of PL (0, 2%, and 4%). The results indicated that the growth performance significantly (P 0.05) increased with the increase in dietary CHO levels. Interactive effects between dietary CHO and PL on the growth parameters were not observed. Superoxide dismutase (SOD) and lysozyme activities were also significantly affected by dietary CHO levels. Furthermore, the interaction between these two additives was only detected in SOD activity. Shrimp fed experimental diet with CHO and PL supplementation showed better tolerance against Vibrio alginolyticus compared to the control, interactive effects (P 0.05) were also detected on these two factors. The expression of immune deficiency (IMD) and lysozyme mRNA was up-regulated in shrimp fed diets with CHO and PL. The expression level of Toll-like receptor mRNA directly reflected the dietary CHO levels, which was not affected by dietary PL. The interaction between dietary CHO and PL was shown as the significant factor (P 0.05) both in the expression of IMD and lysozyme mRNA, which indicated that different dietary levels of CHO and PL could strongly affect expression levels of some immune-relevant genes of the juvenile freshwater cultured L. vannamei.

Surface display can expose foreign antigenic protein on the surface of the vaccine vector, which is promising choice to elicit better immune responses. In this study, we apply this strategy to develop an immunoactivator by using a live attenuated Vibrio harveyi as an antigenic protein carrier with surface displayed VP28, a major envelope protein of white spot syndrome virus (WSSV), for two major pathogens of Litopenaeus vannamei. As a result, the immunoactivator showed self-limited growth and attenuation of virulence in shrimp via different inoculation routes either with single-repetitive dose or high dose. Moreover, either intramuscular injection or oral administration of the immunoactivator did not affect growth of shrimp body weight or cause pathologic changes. Additionally, the rapid immunoprotection was induced by the immunoactivator after administration for one week with highly relative percent survival (RPS) more than 90% against both V. harveyi and WSSV. Until 4 weeks post administration, the immunoactivator still possessed efficient immune effect with no less than 60% RPS for both pathogens. Totally, the attenuated V. harveyi surface displaying VP28 could be a potential immunoactivator for WSSV and vibriosis control in L. vannamei.

Akirin, which are members of the NF-κB signaling pathway, play critical roles in regulating the expression of antimicrobial peptides. In the present study, the Akirin gene from Penaeus monodon was identified from a transcriptome database and designated as PmAkirin. The complete sequence of the PmAkirin cDNA was 1508 bp, encoding a protein of 213 amino acids, and it showed 99% amino acid identity to the Litopenaeus vannamei Akirin. Two predicted nuclear localization signals (NLSs) were found, and the amino acid sequence alignments showed that PmAkirin was highly conserved at the N-terminus and C-terminus. PmAkirin expression was found to be the highest in the hemolymph, followed by the heart, gill, stomach, hepatopancreas, intestine, and muscle. When challenged with Vibrio parahaemolyticus infection, the PmAkirin mRNA and three antimicrobial peptides (AMPs: PmALF2, PmALF3, and PmCrus4) were upregulated. However, another five AMPs (PmALF6, PmCrus1, PmPEN3a, PmPEN3b, and PmPEN5) were downregulated by V. parahaemolyticus infection. Silencing PmAkirin by dsRNA significantly decreased the expression of the eight AMPs, which lead to an increase in the blood concentration of V. parahaemolyticus and higher mortality in the shrimp. In contrast, the overexpression of PmAkirin significantly increased the expression of the eight AMPs, which led to a reduction in the blood concentration of V. parahaemolyticus and promoted the survival of the shrimp. Taken together, we concluded that PmAkirin plays an important role in regulating the expression of AMPs in black tiger shrimp to defend against V. parahaemolyticus infection.

As a crucial neuroendocrine-immune factor, dopamine (DA) could regulate the immune system of Litopenaeus vannamei. To understand the immune mechanisms and regulatory pathways of DA in L. vannamei, the transcriptome analysis of hemocytes of L. vannamei with injection of DA (10−6 mol/shrimp) at 3 and 12 h were performed in this study. Moreover, quantitative real-time PCR (qPCR) method was applied to validate the accuracy of transcriptome sequencing and analyze the expression pattern of candidate differentially expressed genes (DEGs) at different time points (0, 3, 6, 12, and 24 h) after DA injection. The results showed that a total of 51382 unigenes with a N50 length of 2341 bp were generated. And 1397 and 457 DEGs were obtained by comparative transcriptome at 3 and 12h respectively. Moreover, the results of functional annotation and enriched pathway showed that the DEGs were involved in phagosome (ko04145), lysosome (ko04142), Endocytosis (ko04144), and NOD-like receptor signaling pathway (ko04621). Besides, the Pearson's correlation coefficient (R) between transcriptome sequencing and qPCR was 0.845, which confirmed the reliability of the transcriptome sequencing results and the accuracy of assembly. Furthermore, the expression pattern of 15 candidate DEGs, containing 9 up-regulated and 6 down-regulated DEGs at 3 h, indicated the regulation of DA in physiological functions especially in the immune system. Therefore, these results revealed that DA induced the expressions of membrane receptors or proteins, activated intracellular signaling pathways, regulated cellular and humoral immune systems, controlled antioxidation and apoptosis, and was involved in the regulation of neuroendocrine system. These findings are helpful to promote the understanding on the effects of biogenic amines on physiological functions and regulatory networks of crustacean, and offer a substantial material and foundation for researching the immune response of crustacean.

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.

Hypoxia is a common stressor for aquaculture species. The Pacific white shrimp Litopenaeus vannamei survives low dissolved oxygen (DO) conditions by adjusting its energy metabolism. In vertebrates, the transcription factor p53 regulates glucose metabolism under stress through diverse target genes like the Tp53-induced glycolysis and apoptotic regulator (TIGAR), a protein similar to fructose-2,6-bisphosphatase that has a pro-survival role in cells participating in the defense against oxidative damage. Until now, TIGAR has been not reported in any invertebrate species, including crustaceans. In this work, we report the molecular cloning of the white shrimp TIGAR. The cDNA sequence is 765 bp encoding a 254 amino acid protein. Bioinformatics analyses predicted that although the overall sequence identities of L. vannamei TIGAR and vertebrate proteins are not very high (33.61%-35.34%), they have a remarkable predicted structural similarity with full conservation of catalytic residues, secondary and three-dimensional structures. Gene expression analysis by RT-qPCR revealed that the mRNA abundance of TIGAR in white shrimp is tissue-specific under normal oxygen conditions, with higher expression in gills than hepatopancreas and muscle. Also, gene expression in gills and hepatopancreas is modified by environmental hypoxia, suggesting that TIGAR participates in the cellular tolerance of L. vannamei to this stressor.

To understand the homeostasis mechanism of crustacean hepatopancreas to cold stress, iTRAQ proteomics based on the genome database of Litopenaeus vannamei (L. vannamei) was applied to investigate proteins changes and variety of the hepatopancreas during cold stress stage in this study. A total of 4062 distinct proteins were identified, 137 differentially expressed proteins (DEPs) including 62 differentially up-regulated proteins (DUPs) and 75 differentially down-regulated proteins (DDPs) were identified in G1 (18 °C) compared with CK (28 °C), 359 DEPs including 131 DUPs and 228 DDPs were identified in G2 (13 °C for 24 h) compared with CK. Based on bioinformatics analysis, the cold tolerance of L. vannamei might be related to energy metabolism such as amino acid, carbohydrate, lipid, and oxidative phosphorylation. Moreover, shrimp immunity was declined during cold stress stage. However, L. vannamei could cope with cold stress by enhancing the production of ATP and UFA. Notably, arginine kinase, heat shock proteins, and histones may act as positive regulators in L. vannamei under cold stress. Ten randomly selected proteins were used for validation using qRT-PCR and the expressions on the transcription level for most of the genes were similar to the results of iTRAQ. These results indicated that L. vannamei can maintain the organism homeostasis by a series of orderly regulatory process during cold stress. Furthermore, the results can provide guidance for shrimp farming.

Our previous work showed that Lactobacillus pentosus HC-2 has high antibacterial and adhesion activity, and as a probiotic could improve the nutrients and immunomodulatory effects in the Litopenaeus vannamei farming. In order to further investigate the influence of HC-2 surface protein on its probiotic effects, the immune and digestion related genes expression and enzymes activities, the colonization numbers of HC-2, and the histologic characteristics were analysis in shrimp hepatopancreas after feeding either the intact surface proteins, the probiotic treated with lithium chloride (LiCl) to remove noncovalently bound surface proteins or no probiotic for four weeks. The results showed that the immune genes expression of lysozyme, proPO, LGBP, Penaeidins-3α, crustin and C-type lectin, the immune enzymes activities of superoxide dismutase, catalase and Alkaline phosphatase, and the digestion enzymes of Trypsin, Lipase and α-Amylase were significantly higher in hepatopancreas of shrimp fed with intact HC-2 than that in shrimp fed with base diet or striped surface proteins HC-2 post feeding and challenge. In addition, the shrimp fed with intact HC-2 leads to the bacteria cells adhesion to hepatopancreas was significantly higher than that in shrimp fed with no surface proteins HC-2. Furthermore, the tissue damages of hepatopancreas caused by pathogenic vibrio were obviously observed in shrimp fed with base diet or no surface proteins HC-2, but no signs of damages were found in shrimp fed with intact HC-2. These results demonstrate that surface proteins are important components for HC-2 to execute probiotic effect that improve hepatopancreas immune response and nutrition digestion to protect shrimp against pathogen damage.

The survival and immune responses of Litopenaeus vannamei were evaluated during white spot syndrome virus (WSSV) or Vibrio parahaemolyticus single and concurrent infections. The mortality, WSSV load, activities of 4 immune enzymes: acid phosphatase (ACP), alkaline phosphatase (AKP), peroxidase (POD) and superoxide dismutase (SOD), and the transcription of Evolutionarily Conserved Signaling Intermediate in Toll pathways of L.vannamei (LvECSIT) were quantified at 0, 3, 6, 12, 24, 48, 72 and 96 h post-infection (pi). The results showed: (i) the cumulative mortality of the co-infection group (WSSV and V. Parahaemolyticus 83%) was significantly lower than the WSSV infection group (97%) (P < 0.05) at 96 hpi; (ii) copies of WSSV in the co-infection group were significantly lower than that of the single infection group from 24 to 96 hpi (P < 0.05); (iii) ACP, AKP,POD and SOD activity in the gills of the co-infection group was higher than that of the WSSV group at12, 48 and 96 hpi (P < 0.05).The expression of LvECSIT mRNA in the co-infection group was significantly higher than in the WSSV infection group from 12 to 72 hpi (P < 0.05).The results indicate that proliferation of WSSV is inhibited by V.parahaemolyticus infection. In addition, infection with WSSV alone causes a significant reduction in some immune responses of shrimp than co-infection with WSSV and V.parahaemolyticus occurs at 26 °C. Third, LvECSIT, an essential member of TLR signaling pathway might play a crucial role in shrimp defense against WSSV – Vibrio co-infection.

White spot syndrome, which is caused by white spot syndrome virus (WSSV), is a highly contagious disease of penaeid shrimp. However, there is currently incomplete understanding of the infection mechanism and pathogenesis of WSSV. In this study, a novel gene of a previously uncharacterized WSSV response protein (LvWRP) in Litopenaeus vannamei was identified and characterized. The LvWRP gene has an open reading frame (ORF) of 879 bp encoding a putative protein of 292 amino acids. Sequence analysis revealed that LvWRP shared 24.9% identity with an uncharacterized protein of Penaeus monodon nudivirus. Real-time qPCR analysis showed that LvWRP was ubiquitously expressed in shrimp tissues, with transcript levels induced in hemocytes upon immune challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and WSSV. In addition, RNA interference-mediated knockdown of LvWRP followed by WSSV challenge revealed significant decrease in the transcript levels of WSSV IE1 and VP28 genes coupled with a reduction in WSSV copies in shrimp hemocytes. Moreover, depletion of LvWRP followed by WSSV challenge significantly increased the transcript levels of Vago4 and Vago5 as well as increased the phosphorylation of STAT, while hemocytes apoptosis in terms of caspase 3/7 activity was decreased. These results suggest that LvWRP is important for WSSV replication in shrimp, and therefore one of the vital host factors in WSSV infection.

Comparative transcriptome analysis via high throughput sequencing was applied to gain knowledge on the immune response in Litopenaeus vannamei reared in biofloc technology systems (BFT). Two types of carbon sources, namely, traditional carbon sources (molasses) and biodegradable polymers [hydroxybutyric acid-co-3-hydroxyvaleric acid (PHBV)] were used in BFT systems. Clear water systems without the addition of carbon sources were treated as the control. Water quality assays showed that the average concentrations of several stress factors, including nitrite, nitrate and TSS, were the highest in molasses-based BFT systems. After sequencing and comparing the transcriptome profiles of the L. vannamei hepatopancreas, 743 and 201 genes were significantly differentially expressed in molasses- and PHBV-based BFT systems, respectively. GO enrichment analysis, which was performed using the differentially expressed genes, revealed seven significantly over-represented GO terms in molasses-based BFT systems, including catabolic process, hydrolase activity, cellular localization, organic substance metabolic process, cellular metabolic process, establishment of localization and response to stress. The captured key genes were mainly involved in the pathways including cellular stress response, immune response and pathogen recognition. However, no GO terms were significantly over-represented in PHBV-based BFT systems compared with control. This study indicates that shrimp are subject to stress in BFT systems when molasses serves as the carbon source. Thus, PHBV may be a better alternative.

Hemocyanin is primarily a respiratory copper-containing glycoprotein present in the hemolymph of mollusks and arthropods. Recently, hemocyanin has attracted huge research interest due to its multifunctionality and polymorphism. Most previous immune-related studies on shrimp hemocyanin have focused on the C-terminal. Moreover, we previously reported that the C-terminal domain of Litopenaeus vannamei hemocyanin possesses single nucleotide polymorphisms (SNPs), but little is known about the molecular diversity of the N-terminal domain. In the current study, diversity within the N-terminal domain of L. vannamei hemocyanin (LvHMC-N) was explored using bioinformatics and molecular biology techniques as well as immune challenge. Twenty-five LvHMC-N variants were identified using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing, with multiple sequence alignment showing that the 25 variants shared 87%–99 % sequence homology with LvHMC (AJ250830.1). In different shrimp individuals and different shrimp tissues (i.e., hemocytes, stomach, muscle and hepatopancreas), the LvHMC-N variants were expressed differently. Pathogen challenge could modulate the molecular diversity of LvHMC-N, as three LvHMC-Nr variants (LvHMC-Nr1, LvHMC-Nr2 and LvHMC-Nr3) were identified by sequencing following Vibrio parahaemolyticus challenge. Most importantly, recombinant proteins of these three variants (rLvHMC-Nr1, rLvHMC-Nr2 and rLvHMC- Nr3) had relatively high in vitro agglutinative activities against V. parahaemolyticus, Vibrio alginolyticus and Streptoccocus iniae. Our present data indicates that the N-terminus of L. vannamei hemocyanin also possess molecular diversity, which seems to be associated with immune resistance to pathogenic infections.

An 8-week trial was conducted to evaluate the effect of dietary β-glucan supplement (0, 0.01%, 0.02%, or 0.04%) on growth and health of Pacific white shrimp Litopenaeus vannamei at low salinity of 3 practical salinity unit (psu). The L. vannamei fed 0.02% and 0.04% β-glucan gained more weight and showed higher activities of protease, amylase, superoxide dismutase, and glutathione peroxidase in the intestine than in the control (0% β-glucan). The L. vannamei fed 0.04% β-glucan had a higher condition factor than those fed the control diet. Amylase activity in the hepatopancreas of L. vannamei fed 0.02% β-glucan was higher than those fed the control diet. Dietary β-glucan supplement increased the mRNA expressions of Toll-like receptor, myostatin, immune deficiency or heat shock protein 70, but decreased the mRNA expressions of tumor necrosis factor-α and C-type lectin 3 in both hepatopancreas and intestine. The response of intestine microbiota in L. vannamei fed 0.04% β-glucan was further compared to the control. The 0.04% β-glucan supplement reduced richness and diversity of the intestinal microbial community as indicated by the low values of Chao1 estimator, ACE estimator, Simpson index and Shannon diversity index. Abundances of Bacillus, Chitinibacter, Geobacillus and Vibrio in the intestine increased, while Flavobacterium, Microbacterium and Mycobacterium decreased significantly in L. vannamei fed 0.04% β-glucan compared to the control. This study indicates that dietary β-glucan supplement at 0.02%-0.04% can significantly improve digestibility, antioxidant capacity and immunity in L. vannamei, and thus improve growth performance and survival at low salinity. These beneficial effects of β-glucan probably are related to the dominance of probiotics over potential pathogens in the intestine.

This study evaluated the effects of replacing fish meal (FM) with defatted silkworm pupae meal (SPM) on growth, diet digestibility, antioxidant capacity, hepatopancreas histology and molting process in Pacific white shrimp (Litopenaeus vannamei). A basal diet was formulated to contain 24% FM, then 25, 50, 75 or 100% of FM was substituted with SPM referred to as FM, SPM25, SPM50, SPM75 and SPM100 diets, respectively. Quadruplicate groups (36 shrimp per replicate) of shrimp (0.2 ± 0.02 g) were fed the test diets to apparent satiation three times daily for eight weeks. No significant difference (P > .05) in growth performance could be found among FM and SPM groups. Apparent digestibility coefficients of dry matter, gross energy and phosphorous were significantly improved by SPM inclusion. Serum antioxidant parameters including total antioxidant capacity and malondialdehyde concentration were beneficially influenced by SMP inclusion. Higher number of B-cells and lower number of E-cells were detected in hepatopancreatic tubules of SPM100 group compared to the other groups. Moreover, total replacement of FM triggered separation of basal membrane and greater interstitial spaces between the hepatopancreatic tubules. Molting time significantly decreased when FM was entirely replaced with SPM. Also, chitin deacetylase activity was enhanced in SPM100 group, but β-N-Acetyl- d -glucosaminidase activity, and expression of ecdysteroid receptor and retinoid X receptor remained unchanged. To conclude, the entire replacement of FM with SPM did not influence shrimp growth, and its beneficial impacts were found on diet digestibility, antioxidant capacity and molting time. However, the substitution level is recommended to be restricted to 75% as total replacement led to shrinkage of hepatopancreatic cells.

Aflatoxin B1 (AFB1) is a mycotoxin mainly produced by Aspergillus flavus and Aspergillus parasiticus contaminating food, feed ingredients and products of animal origin. In mammals, this toxin causes widespread organ-specific damage; it is immunotoxicity and could promote hepatotoxicity, alter intestinal functions and so on. In this study, we conducted transcriptome and histomorphology analyses of hepatopancreas and intestinal in Litopenaeus vannamei (L. vannamei) challenged with AFB1. Totally 12,014 and 1387 differentially expression genes (DEGs) were identified in the hepatopancreas and intestine, respectively. In hepatopancreas, a total of 1995 DEGs were mainly annotated and grouped into 18 processes or pathways related to animal immune system. With respect to intestine, a total of 152 DEGs were mainly annotated to 7 processes or pathways related to animal immune system. Meanwhile, we determined the relative mRNA expression of several crucial representative immune genes including Toll, immune deficiency (IMD), prophenoloxidase (proPO), Rab and glutathione S-transferase (GST) in the hepatopancreas and intestines of shrimp at 3-, 6-, 12-, 18-, 24- and 30-d after challenged by AFB1. Exposure to AFB1 increased mortality, decrease weight gain rate, severely destroyed the histomorphology of hepatopancreas and intestine, and resulted in the damaged of immune system of shrimp. The present data reveals the different roles between hepatopancreas and intestine of L. vannamei in immune response to AFB1 challenge, and provides insight into the molecular basis of the relationship between hepatopancreas and intestinal immunity during either homeostasis or inflammation.

DCOIT (4,5-dichloro-2-n-octyl-4-isothiazolin-3-one) is the main component of SeaNine-211, a new antifouling agent that replaces tributyltin to prevent the growth of undesirable organisms on ships. There have been some studies on the toxicity of DCOIT, but the mechanism of DCOIT’s toxicity to crustaceans still requires elucidation. This study examined the chronic toxicity (4 weeks) of 0, 3, 15, and 30 μg/L DCOIT to the Pacific white shrimp (Litopenaeus vannamei) from the aspects of growth and physiological and histological changes in the hepatopancreas and gills. A transcriptomic analysis was performed on the hepatopancreas to reveal the underlying mechanism of DCOIT in shrimp. The exposure to 30 μg/L DCOIT significantly reduced the survival and weight gain of L. vannamei. High Na+/K+-ATPase activity and melanin deposition were found in the gills after 4 weeks of 15 μg/L or 30 μg/L DCOIT exposure. The highest concentration of DCOIT (30 μg/L) induced changes in hepatopancreatic morphology and metabolism, including high anaerobic respiration and the accumulation of triglycerides. Compared with the exposure to 3 μg/L DCOIT, shrimp exposed to 15 μg/L DCOIT showed more differentially expressed genes (DEGs) than those in the control, and these DEGs were involved in biological processes such as starch and sucrose metabolism and choline metabolism in cancer. The findings of this study indicate that L. vannamei is sensitive to the antifouling agent DCOIT and that DCOIT can induce altered gene expression at a concentration of 15 μg/L and can interfere with shrimp metabolism, growth and survival at 30 μg/L.

Pacific white shrimp, Litopenaeus vannamei (9.38 ± 0.17 cm, 10.08 ± 0.35 g), with different ammonia-N tolerances were exposed to NH3 (1.61 mg/L) for 192 h, and the levels of key enzymes and biochemical substances involved in energy metabolism were compared to assess the role of the regulation of energy metabolism on the shrimp's adaptation to ammonia-N stress. Higher ammonia-N tolerance in the shrimp (Tolerance group) was achieved through nutritional fortification, whereas shrimp that were not nutritionally fortified comprised the Control group. The mortality rates in the Control and Tolerance groups at the end of the period of ammonia-N stress exposure were 64.44% and 40.00%, respectively. Within 1 h of exposure to ammonia-N stress, the glucose concentration in both groups declined rapidly, and no significant difference was detected between the two groups. In general, the triglyceride and cholesterol concentrations in the Control group were higher than those in the Tolerance group, and accumulations and/or fluctuations in these metabolites to varying degrees were observed. The Tolerance group presented higher phosphofructokinase (PFK) and pyruvate kinase (PK) activity compared with the Control group from 1 to 48 h of exposure to ammonia-N stress, whereas the opposite result was observed from 96 to 192 h. Similarly, during exposure to ammonia-N stress, the Tolerance group showed higher and lower lactate dehydrogenase (LDH) activity than the Control group from 1 to 24 h and from 48 to 92 h, respectively. In addition, compared with the Control group, the shrimp in the Tolerance group exhibited higher succinate dehydrogenase (SDH) activity, especially from 48 to 192 h of exposure to ammonia-N stress. The results of this study suggest that anaerobic carbohydrate (in the early stage) and aerobic metabolism (in the late stage) plays an important role in the shrimp's response to ammonia-N stress. In addition, maintenance of the normal operation of lipid metabolism is equally important for improving the tolerance of L. vannamei to ammonia-N stress.

A 48-day feeding trial and an acute ammonia-N stress test were performed to investigate the effects on the growth performance, energy status, antioxidant status, and ammonia-N tolerance of Litopenaeus vannamei (5.0 ± 0.2 cm, 1.6 ± 0.4 g) when commercial feed was supplemented with freeze-dried powder of Ampithoe sp. (FDPA) on an interval and a continuous schedule. Shrimp fed with commercial feed were defined as the control group. For the interval feeding group (INF group), commercial feed supplemented with FDPA was provided once every 4 days, while commercial feed was used on the remaining days. The continuous feeding group (COF group) received commercial feed with added FDPA every day. The shrimp in the INF group and COF group showed better growth performance than did the control shrimp, and the survival rate and average yield in the COF group were significantly higher than those in the control group. During the culture period, the concentrations of glucose, triglycerides and total cholesterol in the hemolymph of shrimp were generally higher in the INF and COF groups than in the control group, and the concentrations in the COF group were generally higher than those in the INF group. Compared with the control group, shrimp in the INF and COF groups had elevated glutathione peroxidase (GSH-Px) activity, total antioxidant capacity (T-AOC), and expression levels of ATPase-α and PFK in the hemolymph or hepatopancreas, along with reduced malondialdehyde (MDA) concentrations and expressions of AMPK-α and FAS in the hepatopancreas. At 48 to 192 h during the ammonia-N stress test, the concentrations of urea-N and ammonia-N in the hemolymph generally remained lower in the INF group and COF groups than in the control group. At the end of the ammonia-N stress test, the shrimp mortality rates in the INF and COF groups were 28.1% and 40.6%, respectively, which were lower than the rate of 75.0% observed in the control group. In conclusion, dietary supplementation with FDPA had a positive influence on shrimp growth performance, energy status, antioxidant status and ammonia-N tolerance, and continuous feeding appeared to be more effective than interval feeding at improving antioxidant capacity.

The enzymatic oxidation process that causes black spots on shrimp, named melanosis, leads to spoilage and economic losses. Therefore, there is an urgent need for strategies to reduce melan...

The extraction of polyphenoloxidase (PPO) from Litopenaeus vannamei was studied at different temperatures, pH and ionic strength conditions. Optimum extracts at pH = 7.2 were treated by hiph pressure carbon dioxide (HPCD) technology at 20 MPa and in a temperature range from 35 to 50 °C. Inactivation kinetics for PPO extracts were determined and fitted to the Weibull model. The effect of HPCD on PPO activity was also evaluated in shrimps in vivo, finding that PPO could be easier inactivated than in the crude PPO extract. Color of Litopenaeus vannamei was visually followed right after HPCD treatment and thermal treatment at the same temperature and during storage at 4 °C during a week, showing good results when shrimps were treated with HPCD in the absence of water., Spanish Government (MINECO) and the European Regional Development Fund for financial support of project CTQ2015-64396-R

Biofloc systems are microbial mature environments that are potentially less conducive disease outbreaks. We hypothesized that the way in which biofloc microbial communities are managed determines the level of disease protection. To investigate such hypothesis, Litopenaeus vannamei post-larvae were cultured for 21 days in biofloc environments created by different water management procedures. Five different types of bioflocs were created: autotrophic bioflocs without probiotics, autotrophic bioflocs with probiotics, heterotrophic bioflocs without probiotics, heterotrophic bioflocs with probiotics, and a flow-through system as a control. Heterotrophic bioflocs were obtained by daily addition of carbon (glucose) at an estimated C/N ratio of 18 throughout the experiment. For autotrophic bioflocs this input of carbon was applied only to start up the system and upon appearance of bioflocs (TSS > 100 mg L−1) and a drop in total ammonium nitrogen concentration below 0.05 mg L−1, carbon dosing was stopped. Bioflocs cultured with addition of probiotics received a 0.5 ppm dose every 48 hours. After 21-d culture period, a 96 h challenge test was performed with a Vibrio parahaemolyticus strain known to cause AHPND. For each biofloc type, this challenge was performed in three different approaches: 1- Shrimp were taken out of their biofloc tanks and challenged by applying new seawater; 2-Shrimp from biofloc tanks were challenged in their respective biofloc suspensions; and 3- Non-experimental shrimp, randomly selected from a recirculation (RAS) system were challenged in the types of biofloc suspensions. Mortality was high when shrimp were challenged in new seawater, independent of treatment. When challenged in their respective biofloc suspensions shrimp survival was the highest in heterotrophic bioflocs with and without probiotic supplementation and the autotrophic bioflocs with probiotics, whereas shrimp survival in autotrophic bioflocs without probiotics was 50%. These results were similar when non-experimental shrimp originating from a RAS system were challenged in these biofloc suspensions. Taken together, results suggest that bioflocs as such can decrease the impact of a Vibrio parahaemolyticus challenge and that this protection depends on the operational parameters of the biofloc system. Moreover, probiotics can be used to complement the protective effect of bioflocs. This information reinforces the importance of microbial community management as a tool to reduce the risk of disease and establish highly biosecure systems.

In Biofloc Technology (BFT) Systems, there are changes in microbial communities during a shrimp culture cycle; usually, heterotrophic bacteria are replaced by a photoautotrophic or chemoautotrophic community, depending on the management employed. When the water is exposed to sunlight, it can abruptly change from a heterotrophic system (dominated by bacteria and protozoa) to a predominantly photoautotrophic system, with a dominance of microalgae. However, there is little information on the characterization and quantification of the microbial community in the culture of the Pacific white shrimp Litopenaeus vannamei in a BFT system with light restriction, especially the contribution of these microrganisms to the zootechnical performance of shrimp. The aim of this study was to evaluate the microbial community in a rearing environment with light restriction. The study was conducted in 12 tanks (800 L) with a storage density of 500 shrimp m−3 and an initial shrimp weight of 0.053 g. The experiment was designed with three treatments and four replicates distributed into the following groups: i) natural photoperiod - 12 h light/12 h dark (NP); ii) 24 h light (24hLI); and iii) 24 h dark (24hDA). Significant differences were found in water quality parameters (lower values of ammonia and nitrite in the 24hDA treatment), and higher values of CO2, light (Ix) and chlorophyll were observed for the NP and 24hLI treatments than for the 24hDA treatment (p

The effect of acute ammonia challenge on survival, immune response and antioxidant status of Litopenaeus vannamei pretreated with diets containing different inositol levels was investigated. Shrimp (initial mean weight 0.40 ± 0.00 g) were randomly allocated in 18 tanks (30 shrimp per tank) and triplicate tanks were fed with a control diet without myo-inositol (MI) supplementation (242.6 mg inositol kg−1 diet) or diets containing diverse levels of inositol (368.8, 459.7, 673.1, 993.8 and 1674.4 mg kg−1 diet) as treatment groups for 8-week. Randomly selected 10 shrimp per tank (final mean weight approximately 11.1–13.8g) were exposed to ammonia stress (total ammonia-nitrogen, 60.21 mg L−1) for 24 h after feeding trial. The results showed that after exposed to ammonia stress, survival rates of MI-supplemented groups were enhanced by 31–77% when compared with the control group. MI supplementation increased activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in plasma, and reduced its activities in hepatopancreas. It also enhanced activities of total antioxidant capacity (T-AOC), glutathione S-transferase (GST) and glutathione peroxidase (GPX) and content of reduced glutathione (GSH), and lowered malondialdehyde (MDA) and protein carbonyl (PC) content in plasma or hepatopancreas. In addition, mRNA expression levels of ferritin (FT), arginine kinase (AK), thioredoxin (Trx), heat shock protein 70 (Hsp70), catalase (CAT) and peroxiredoxin (Prx) were significantly differentially regulated in hepatopancreas owing to MI supplementation. Therefore, it suggested that L. vannamei pretreated with higher dietary inositol content may have better ammonia stress tolerance and antioxidant status after ammonia stress, and the optimum levels ranged from 459.7 to 993.8 mg inositol kg−1 when total ammonia-nitrogen concentration was 60.21 mg L−1.

A 58-day feeding trial was conducted to evaluate the effects of dietary myo-inositol (MI) supplementation on growth performance, haematological parameters, hepatopancreas histopathology and antioxidant status of Litopenaeus vannamei fed with oxidized fish oil (OFO). Control diet contained fresh fish oil (FFO) without MI supplementation. The other four diets contained two oxidation levels of OFO (peroxide value: 133.2 and 268.7 meq kg−1) with or without 200 mg MI kg−1 diets (MI0+L, MI0+H, MI200 + L and MI200 + H). Results showed that OFO-supplemented groups (without MI supplementation) showed better growth performance and lower whole-body inositol content when opposed to control group. MI supplementation significantly improved whole-body inositol content in high-oxidized fish oil (HOFO) groups, and also reduced whole-body lipid in low-oxidized fish oil (LOFO) groups. Moreover, Supplementation of OFO and MI markedly hit the fatty acid profile of muscle. HOFO caused severe histopathological changes in hepatopancreas of shrimp, which slightly alleviated by MI supplementation. MI supplementation also grew the total protein (TP) content and alkaline phosphatase (AKP) activity and decreased the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of serum in OFO-supplemented groups. Ingestion of OFO increased levels of lipid peroxidation and protein oxidation in serum or hepatopancreas, which partly ameliorated by MI supplementation. Activities of antioxidant enzymes exhibited different expression patterns because of OFO and MI. In addition, HOFO markedly increased mRNA expression levels of antioxidant genes including ferritin (FT), thioredoxin (Trx), GPX, glutathione S-transferase (GST) and catalase (CAT) and decreased peroxiredoxin (Prx) expression, in which expression of GPX and Prx were increased owing to MI supplementation. Therefore, it suggested that dietary OFO stimulated growth performance, but also induced oxidative stress and caused impairment to hepatopancreas in L. vannamei. The negative impact brought about by OFO was partially mitigated by dietary MI supplementation.

Increasing evidence has revealed a tight association between host diseases and dysbiosis in its gut bacterial communities. However, it remains uncertain whether this interplay also holds true for gut eukaryotes. To fill this knowledge gap, using Illumina sequencing the eukaryotic 18S rRNA genes, we explored the gut eukaryotic signatures of Pacific white shrimp (Litopenaeus vannamei) suffered from white feces syndrome (WFS). The gut eukaryotic communities in WFS shrimp differed significantly (P

High concentrations of total suspended solids (TSS) need to be controlled, as they can affect shrimp production due to the excess of particles in the water column. Water renewal and clarification are alternatives used to reduce TSS. In order to determine the better method of TSS control, we carried out a study using water renewals and clarification on a commercial scale with nine ponds (600 m2 each) in an intensive biofloc system. A total of 87 shrimp m−2 were stocked in each unit divided into three treatments: R (water renewal), C1 (one clarifier) and C2 (two clarifiers in series). Each treatment had three replicates, and the experiment lasted 105 days. There were no significant differences (p > 0.05) in the parameters of water quality and zootechnical performance. Significant differences (p

Fouling species display characteristics that make them suitable options for bioremediation of aquaculture effluents. We studied the potential use of two fouling species (Amphibalanus amphitrite and Saccostrea cucullata) in remediation of shrimp (Litopenaeus vannamei) farm wastewaters. Their abilities, individually and in combination, in reducing nitrogen, phosphorus, and phytoplankton (chlorophyll a) from effluents at three densities and three retention times (1, 3, 6 h) were investigated. The results showed that all treatments, specifically oysters, could effectively reduce nutrients and plankton concentrations. Oysters reduced total nitrogen, total phosphorus and chlorophyll a to 60.7%, 55.6%, and 7.6% of the initial contents at the high density (i.e. 0.80 oyster L−1) after 6 h. Barnacles and the combined use of both species, however, showed remarkably lower efficiencies. Our results demonstrated that oysters could potentially be used to improve water quality of shrimp farm effluents especially in the areas where oysters can be easily collected or cultured.

The aim of this study was to investigate if the inclusion of tilapia silage (TS) in diets for Litopenaeus vannamei juveniles and the rearing systems (clear-water and biofloc) could affect the shrimp quality when stored on ice for 15 days post-harvest. The experiment was performed in two experimental systems: biofloc (BF) and clear-water (CW) systems at a shrimp stocking density of 63 shrimp m−2. The treatments were based on the percentage of silage inclusion (control or 0, 1.5, 3.0, 4.5 and 6.0% of inclusion) in each system, totalizing ten treatments and four replicates. Survival was above 80% in all treatments regardless of system or diet. Shrimp final weight and specific growth rate (7.17 g and 2.01% day−1 – BF; 6.35 g and 1.82% day−1 – CW) were statistically influenced by the system but not by the diet. Shrimp quality index and shelf life were not affected by the inclusion of TS in L. vannamei diets rather than rearing system with better results for BF. The results suggest that the substitution of fish meal by fish silage in L. vannamei diet in the presence of biofloc conditions increase the sustainability of intensive shrimp culture and the overall shrimp quality and shelf life.

This study aimed at identifying and quantifying greenhouse gas fluxes (CH4, CO2, and N2O) in Litopenaeus vannamei shrimp nurseries submitted to different culturing conditions. The experiment was carried out with a completely randomized design, with two treatments and four simultaneous replicates, totaling eight experimental units. Two management systems were tested. The first one (M1) used a stocking density of 92 shrimp/m2 and fertilizer maintenance through the application of calcium nitrate and molasses. The second one (M2) used a stocking density of 14 shrimp/m2 without fertilizer maintenance. Feeding in both treatments consisted in supplying ration through the volley method. The results showed that there were variations in the pattern of gas emission in both treatments and in the concentrations of the evaluated gases. The recorded mean values of total gas flux were −314.87 mg/m2/day of CH4, -3773.51 mg/m2/day of CO2, and 2.47 mg/m2/day of N2O in M1; and 653.89 mg/m2/day of CH4, 497.52 mg/m2/day of CO2, and 25.59 mg/m2/day of N2O in M2. The results obtained in this study suggest that environmental and management conditions interfere with the cultivation system, which acts as either a source or drainage of gases. These emissions from shrimp farming are potentially critical, mainly due to N2O emissions, when compared to emissions from other production systems. Conversely, the cultivation of L. vannamei, particularly when carried out with the use of organic fertilizers such as molasses, presented a potential absorption of gases such as CH4 and CO2. The greatest fluxes of gases occurred at the beginning of the cultivation due to the initial fertilization. In addition, the contribution of molasses probably favored denitrification and increased natural productivity, which may have contributed to a lower emission of these gases compared to emissions of other systems where molasses was not used.

Biofloc technology involves manipulation of the C/N ratio to convert toxic nitrogenous wastes into useful microbial protein and helps improve the water quality in a closed system. Poly-β-hydroxybutyric (PHB) has been demonstrated to have positive effects on aquatic animals in an aquaculture system. A 40-day lab scale trial was carried out to investigate the potential of PHB as an additional carbohydrate for a shrimp Litopenaeus vannamei bioflocs nursery in brackish water. Three methods of carbohydrate supplementation were investigated. The first method was the addition of glucose daily at a percentage of 75% of the feed (GLU-group). The second method was the addition of PHB plus glucose; when the total ammonia nitrogen (TAN) level above 2 mg L−1 was observed, glucose was added, 6 g C to 1 g TAN (PHB + GLU-group). The third method was the addition of PHB only (PHB-group). During the 40-d trial, most of the ratios of dissolved organic carbon to total ammonium nitrogen (DOC/TAN) were maintained at 20 in the three groups. The average concentration of TAN in the PHB + GLU-group (0.23 ± 0.19 mg L−1) was significantly lower than those of the GLU-group (0.28 ± 0.26 mg L−1) and the PHB-group (0.30 ± 0.33 mg L−1) (P .05). The survival rate and final weight in the PHB + GLU group were significantly higher than those of the other groups (P

This study describes the amoeba (order Dactylopodida) infection in Pacific white shrimp Litopenaeus vannamei cultured in an anonymous shrimp hatchery, located in North America. The diseased shrimp grossly exhibited reduced appetite, lethargy, respiratory distress, eroded carapace, and blackened gill. Histopathology examination revealed a massive amoeba infection (mostly in trophozoite form) mainly in the gill, but also other organs, such as the antennal gland, lymphoid organ, appendages, and nerve cord of the studied shrimp. The irregular-shaped trophozoites, with the prominent centrally located nucleolus and the basophilic parasome, suggested that this parasite is related to the Paramoeba sp. (order Dactylopodida). Analysis of the small subunit rRNA sequence showed 100% sequence similarity with Paramoeba spp. (or Neoparamoeba spp.) from several aquatic crustaceans and echinoderm species, such as sea urchins, crabs, and lobsters. The sequence also matched with other amoeba species, such as Korotnevella sp. and Pseudoparamoeba sp. (order Dactylopodida). Diagnostic in situ hybridization and PCR assays were developed as the initial screening methods for the detection of the amoebic infection in cultured shrimp. This report is the first to describe the amoeba (Paramoeba-like sp.) infection in penaeid shrimp.