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This study evaluated the potential of the aqueous moringa (Moringa oleifera) leaf extract (ME) as a prebiotic and its effect on the growth performance of the whiteleg shrimp, Penaeus vannamei Boone, 1931. The growth trial of probiotics in the broth containing ME showed that Lactobacillus acidophilus growth rate was significantly higher () than that of Lactobacillus plantarum and Lactobacillus reuteri. Furthermore, L. acidophilus exhibited also better growth () than Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio harveyi in the broth containing ME. The minimum inhibitory concentration of ME (MIC) and minimum bactericidal concentration (MBC) were the same against V. alginolyticus, V. parahaemolyticus, and V. harveyi at concentrations of 10, 7.5, and 7.5 mg/ml, respectively. By contrast, ME’s MIC and MBC against L. acidophilus were 50 and >100 mg/ml, respectively. The prebiotic scores of L. acidophilus against V. parahaemolyticus and V. harveyi were significantly higher () than that against V. alginolyticus. The MIC, MBC, and prebiotic scores indicated that ME enhanced the growth of L. acidophilus more than that of Vibrio bacteria. Six diets were formulated for the feeding trial; 3 diets contained ME at 0 (ME0), 2.5 (ME2.5) and 5.0 g/kg (ME5.0), respectively, and the same 3 diets were also prepared with L. acidophilus (ME0 + P, ME2.5 + P, and ME5.0 + P, respectively). After the feeding trial, the number of lactic acid bacteria in the gut was higher () in the ME2.5 + P group than in the control and ME2.5 groups, but no differences were found in the vibrio-like bacterial count among the treatments. The ME2.5 + P group had a higher () final weight, weight gain, and specific growth rate than did the ME or L. acidophilus alone and the control groups. Therefore, ME exhibits a prebiotic function and exerts a symbiotic effect with L. acidophilus, thus increasing the growth performance of P. vannamei.

This study investigated the synergistic effects of oxolinic acid (OA) combined with oxytetracycline (OTC) on white shrimp (Penaeus vannamei). Disk diffusion susceptibility testing was performed to analyze the sensitivity of Vibrio alginolyticus and Vibrio parahaemolyticus to different concentrations of OA and OTC. The results revealed that 50 mg OA/L combined with 50 mg OTC/L exhibited stronger antibacterial effects on V. alginolyticus and V. parahaemolyticus. The results of in vitro tests indicated that cotreatment with OA and OTC significantly reduced superoxide anion production and phenoloxidase activity, but not phagocytic activity. Subsequently, feeding trials were performed to investigate the immunomodulatory effects and bioaccumulation of dietary OA combined with OTC on shrimp. The healthy shrimp (15.13 ± 1.02 g) were divided into four groups: control, 100 mg OA/kg combined with 50 mg OTC/kg, 50 mg OA/kg combined with 100 mg OTC/kg, and 50 mg OA/kg combined with 50 mg OTC/kg. The shrimp were sampled to determine innate immunity parameters and residual OA and OTC levels in the muscle during a 28-day feeding regimen; the shrimp were fed the experimental diet from day 1 to day 5 and a commercial diet from day 6 to day 28. Residual OA levels were considerably higher in the group fed 100 mg OA/kg combined with 50 mg OTC/kg compared with the other groups and peaked on day 4. The residual OA levels of all the groups were below the detection limit after without providing OA. The residual OTC levels of the group fed 50 mg OA/kg combined with 100 mg OTC/kg were considerably higher from day 1 to day 4. The residual OTC levels in all the groups decreased rapidly and could not be detected on day 28. The administration of 50 mg OA/kg combined with 100 mg OTC/kg exerted the least effect on the white shrimp. Moreover, the survival rates of the treatment groups after the V. parahaemolyticus challenge were higher than those of the control group, especially the group fed 50 mg OA/kg combined with 100 mg OTC/kg. This result indicated that the synergistic effects of dietary OA and OTC are safe and effective. Combination therapy is a new method of antibiotic use in aquaculture.

This study investigated the effect of the moringa (Moringa oleifera) leaf extract and Lactobacillus acidophilus individually or combined on growth performance, enzyme activity, intestinal and hepatopancreatic histology, intestinal microbiota, immune response, and resistance against Vibrio alginolyticus and Vibrio parahaemolyticus in whiteleg shrimp (Penaeus vannamei). Six diets were formulated: three diets without L. acidophilus containining 0 (control, ME0), 2.5 (ME2.5), and 5.0 g/kg of moringa (ME5.0) and the same three diets containing L. acidophilus at 1 × 10

This study investigated the effects of two probiotics, namely Lactobacillus paracasei and Bifidobacterium longum, as feed additives on growth performance, nonspecific immunity, immune-related gene expression, and disease resistance against Vibrio parahaemolyticus in Penaeus vannamei. The experimental diets were prepared using L. paracasei and B. longum at concentrations of 10

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.

This study was to examine the immunomodulatory effects of oxytetracycline (OTC) on the white shrimp Litopenaeus vannamei (Boone, 1931). Immune parameters were not suppressed by OTC treatment in haemocytes in vitro. A total of 96 healthy shrimp were divided into four groups. Group 1 was the control group. Group 2 received 50 mg OTC/kg body weight/day. Group 3 received 1 g β-glucan/kg feed. Group 4 received 50 mg OTC/kg body weight/day and 1 g/kg β-glucan. OTC residues were more highly accumulated in the hepatopancreas than in muscle tissue during the rearing period, and the addition of β-glucan prolonged the retention time of OTC in the muscle tissue. Additionally, dietary administration of OTC in combination with β-glucan facilitated the excretion of OTC from the hepatopancreas. Shrimp that orally ingested OTC alone had an elevated total haemocyte count, phagocytosis, superoxide anion production and prophenoloxidase activity, and these immunostimulatory effects were further enhanced in shrimp receiving OTC in combination.

The objective of this study was to investigate the effects of Caulerpa racemosa (Agardh, 1873) polysaccharides (CRP) on non-specific immunity of the white shrimp, Litopenaeus vannamei (Boone, 1931). The extracted polysaccharide consists of galactose, glucose, and mannose. Total polyphenolic compound and total flavonoids content in CRP were determined. In vitro CRP treatment did not alter cell viability of haemocytes and significantly activated phenoloxidase (PO) activity, superoxide anion production capacity, phagocytic rate, and index in haemocytes. Immune parameters, such as total haemocyte count, PO activity, phagocytosis, and superoxide anion production, were significantly elevated in CRP-fed shrimp during the rearing period. Malondialdehyde levels in the hepatopancreas of CRP-fed shrimp were lower than in control shrimp from day 7 to day 28. These results show that CRP exhibits antioxidant activity and can activate immune responses of white shrimp in vitro and in vivo. Therefore, CRP can be considered a functional feed additive to improve white shrimp immunity.

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.

BackgroundShrimp aquaculture has suffered huge economic losses over the past decade due to the outbreak of acute hepatopancreatic necrosis disease (AHPND), which is mainly caused by the bacteriaVibrio parahaemolyticus(V. parahaemolyticus) with the virulence pVA1 plasmid, which encodes a secretory photorhabdus insect-related (Pir) toxin composed of PirA and PirB proteins. The Pir toxin mainly attacks the hepatopancreas, a major metabolic organ in shrimp, thereby causing necrosis and loss of function. The pandemic of antibiotic-resistant strains makes the impact worse.MethodsMild pyrolysis of a mixture of polysaccharide dextran 70 and the crosslinker 1,8-diaminooctane at 180 ℃ for 3 h to form carbonized nanogels (DAO/DEX-CNGs) through controlled cross-linking and carbonization. The multifunctional therapeutic CNGs inherit nanogel-like structures and functional groups from their precursor molecules.ResultsDAO/DEX-CNGs manifest broad-spectrum antibacterial activity againstVibrio parahaemolyticusresponsible for AHPND and even multiple drug-resistant strains. The polymer-like structures and functional groups on graphitic-carbon within the CNGs exhibit multiple treatment effects, including disruption of bacterial membranes, elevating bacterial oxidative stress, and neutralization of PirAB toxins. The inhibition ofVibrioin the midgut of infected shrimp, protection of hepatopancreas tissue from Pir toxin, and suppressing overstimulation of the immune system in severeV. parahaemolyticusinfection, revealing that CNGs can effectively guard shrimp fromVibrioinvasion. Moreover, shrimps fed with DAO/DEX-CNGs were carefully examined, such as the expression of the immune-related genes, hepatopancreas biopsy, and intestinal microbiota. Few adverse effects on shrimps were observed.ConclusionOur work proposes brand-new applications of multifunctional carbon-based nanomaterials as efficient anti-Vibrioagents in the aquatic industry that hold great potential as feed additives to reduce antibiotic overuse in aquaculture.Graphical Abstract

Trivalent arsenic (As (III)) contamination in the marine environment can produce adverse effects in crustaceans. The present study investigated the chronic toxicity of As (III) in white shrimp (Penaeus vannamei) by analyzing the tissue bioaccumulation and non-specific immune responses. Shrimps were exposed to 0 (control), 50, 500, and 2500 μg/L of As (III) for 21 days. The results showed that the hepatopancreas was the main tissue of arsenic accumulation in white shrimp. The cumulative concentration of total arsenic and inorganic arsenic but not arsenobetaine was positively correlated with the exposure concentration. In vitro As (III) treatment (0-2500 μg/L) with haemocytes isolated from healthy shrimp did not cause the cytotoxicity, but this arsenic treatments inhibited the phagocytic rate and O

Simple Summary In this study, we found that phycoerythrin from Colaconema sp. can differentially stimulate the immune response of whiteleg shrimp in vitro and in vivo and could potentially be used as an immunomodulator in shrimp culture. Abstract We investigated whether phycoerythrin (PE), a pigment sourced from marine algae, could act as an immunomodulatory agent in whiteleg shrimp (Litopenaeus vannamei). To this end, PE was extracted and purified from a PE-rich macroalgae, Colaconema sp. Our in vitro analysis demonstrated that PE enhanced prophenoloxidase and phagocytosis activity but inhibited the production of reactive oxygen species in hemocytes. Additionally, the PE signal could be detected using an in vivo imaging system after its injection into the ventral sinus of the cephalothorax of whiteleg shrimp. The expression profiles of fourteen immune-related genes were monitored in hemocytes from whiteleg shrimp injected with 0.30 μg of PE per gram of body weight, and crustin, lysozyme, penaiedin 4, and anti-lipopolysaccharide factor showed up-regulated post-stimulation. The induction of immune genes and enhancement of innate immune parameters by PE may explain the higher survival rates for shrimp that received different doses of PE prior to being challenged with Vibrio parahaemolyticus or white spot syndrome virus compared to controls. Combined, these results show that PE from Colaconema sp. can differentially stimulate the immune response of whiteleg shrimp in vitro and in vivo and could potentially be used as an immunomodulator in shrimp culture.

This study investigated the effects of guava leaf extract (GLE) on immune responses, growth performance, and resistance to Vibrio parahaemolyticus in white shrimp (Penaeus vannamei). To examine the effect of GLE on the immune response of white shrimps, they were treated with various concentrations of GLE on hemocyte (in vitro) and were orally administered (in vivo) feed containing various concentrations of 0, 1, 5, and 10 g kg−1 GLE (control, GLE1, GLE5, and GLE10, respectively) for 28 days. Furthermore, their growth performance was evaluated for 56 days. In a separate experiment, the shrimps were challenged with V. parahaemolyticus injection after 7 days of culture. In vitro experiments indicated that GLE is nontoxic and can activate immune response. In vivo experiments revealed that the GLE5 led to the highest total hemocyte count, phenoloxidase activity, phagocytic activity, and superoxide anion production and the highest upregulation of lipopolysaccharide, β-1,3-glucan-binding protein, peroxinectin, lysozyme, crustin, penaeidin 2, penaeidin 3, clotting protein, superoxide dismutase, and glutathione peroxidase. Moreover, better growth performance was observed in the GLE groups, with GLE5 exhibiting the highest specific growth rate, weight gain, and feed conversion rate. In addition, GLE5 enhanced resistance to V. parahaemolyticus, with a survival rate of 72.27%. In conclusion, GLE was found to be effective in enhancing nonspecific immune response and growth performance and in reducing V. parahaemolyticus infection in white shrimp.

This study investigates the effects of three medicinal herbal extracts, namely Bidens pilosa (BPE), Lonicera japonica (LJE), and Cyathula officinalis (COE), on nonspecific immune parameters of cobia (Rachycentron canadum) in vitro and in vivo. During in vitro tests, BPE treatment increased reactive oxygen species (ROS) production in a dose-dependent manner in primary head kidney leukocytes. Similarly, ROS production rates were enhanced by LJE (50 and 100 mg/ml) and COE (100 mg/ml). This suggests that these three herbal extracts possess immunostimulating properties. We then conducted two feeding trials to examine the effects of these three herbal extracts on growth and innate immune parameters of cobia, and sought an optimal dietary supplementation proportion required for activating the non-specific immune responses. In the first trial, we supplemented the diet with 1, 5, or 10% of the individual extracts. After a ten-week feeding trial, no negative impacts on weight gain, feed conversion rate, and survival rate were observed in fish offered experimental diets. Further, ROS production, phagocytic capacity of the head kidney leukocytes, and serum lysozyme activity were enhanced by differing degrees in fish fed the herbal extracts compared to fish in the control group. A similar albumin/globulin ratio was seen between each experimental group and the control group regardless of the type and dose of herbal extract used, indicating these medicinal herbal extracts are safe for cobia. We then performed a 30-day feeding trial with lower extract concentrations (1, 3, and 5% of the diet) to identify dose responses in cobia at various time points so that we could establish a cost-effective manner of administering the three extracts for cobia. All BPE fed fish had higher ROS production compared to the control group, while phagocytosis rate and index were simultaneously raised in only the BPE30 group (3% BPE). Immune parameters such as ROS production, phagocytic rate, and serum lysozyme activity were triggered when fish received 30 g LJE per kg of feed. However, ROS production only increased in the LJE10 group (1% LJE) on day 30 and was not enhanced in the LJE50 group (5% LJE). Additionally, although the phagocytic rate and phagocytic index were induced in the LJE50 group, serum lysozyme activity was not elevated in this group (LJE50) at any time point examined. ROS production was greatly improved in all COE fed groups, but only the COE30 group (3% COE) showed prolonged enhanced phagocytic rate over the 30-day feeding trial.

White spot syndrome virus (WSSV) is the causative agent of white spot syndrome (WSS), a disease that has led to severe mortality rates in cultured shrimp all over the world. The WSSV is a large, ellipsoid, enveloped double-stranded DNA virus with a wide host range among crustaceans. Currently, the main antiviral method is to block the receptor of the host cell membrane using recombinant viral proteins or virus antiserum. In addition to interference with the ligand-receptor binding, disrupting the structure of the virus envelope may also be a means to combat the viral infection. Carbon quantum dots (CQDs) are carbonaceous nanoparticles that have many advantageous characteristics, including small size, low cytotoxicity, cheap, and ease of production and modification. Polyamine-modified CQDs (polyamine CQDs) with strong antibacterial ability have been identified, previously. In this study, polyamine CQDs are shown to attach to the WSSV envelope and inhibit the virus infection, with a dose-dependent effect. The results also show that polyamine CQDs can upregulate several immune genes in shrimp and reduce the mortality upon WSSV infection. This is first study to identify that polyamine CQDs could against the virus. These results, indeed, provide a direction to develop effective antiviral strategies or therapeutic methods using polyamine CQDs in aquaculture.

Bidens alba and Plectranthus amboinicus are common medicinal plants. This study investigated the effects of a diet containing B. alba extract (BAE) and P. amboinicus extract (PAE) on the immune responses, growth, and their resistance to Vibrio alginolyticus in white leg shrimp (Penaeus vannamei). An in vitro experiment revealed that the BAE and PAE are nontoxic to white leg shrimp haemocytes, and the extracts increased phagocytic activity, superoxide anion (O2−) production, and phenoloxidase (PO) activity. In in vivo trials, shrimp were fed diets containing 10, 20, or 40 g of BAE (B1, B2 and B4) or PAE (P1, P2 and P4 group) per kg of regular feed for 28 days, and immune parameters were measured. All BAE and PAE groups exhibited enhanced nonspecific immune responses including increased total haemocyte count, phagocytic activity, O2− production rate, and PO activity. Shrimp fed the BAE or PAE diet for 7 days exhibited increased expression of Lysozyme (Lyz), Penaeidin 2 (Pen2), Penaeidin 3 (Pen3), and Penaeidin 4 (Pen4). After a V. alginolyticus challenge, the treated shrimp exhibited higher survival rates than exhibited by those fed the control diet. The growth performance results of 56 feeding trials revealed that shrimp fed BAE or PAE at 20 g/kg (B2 or P2 group) gained significantly more weight and has a significantly higher specific growth rate than did those on the control diet. Thus, these results suggest that administration of 20 g of BAE or PAE per kilogram of feed can enhance immunity, growth, and resistance to V. alginolyticus in white leg shrimp.

This study was conducted to examine the combinatory effects of β-glucan and oxytetracycline (OTC) on hybrid giant tiger groupers (Epinephelus fuscoguttatus × Epinephelus lanceolatus). In vitro tests, OTC significantly reduced superoxide anion production and phagocytic activity in primary head kidney leukocytes. However, this suppressive effect was alleviated by co-treatment with β-glucan. Subsequently, feeding trials were performed to investigate the potential immunomodulatory effects of dietary β-glucan alone or in combination with OTC on groupers. A total of 210 healthy groupers (368.00 ± 51.03 g) were divided into six groups. Group 1 was the control group, group 2 (BG) received 5 g β-glucan per kg feed weight, groups 3–5 received 5 g/kg β-glucan in combination with 10, 30, or 50 mg OTC/kg fish weight/day (groups M1, M2, and M3, respectively), and group 6 (O) received 50 mg OTC/kg fish weight/day. Fish were sampled to determine the innate immunity parameters and residual OTC levels in the muscle tissue during a 28-day feeding regimen. Residual OTC levels were considerably higher in groups M3 and O compared with the other groups, and peaked on day 14. This was followed by a slight decrease on day 28, despite a continuous supply of OTC. Notably, fish fed with OTC alone had significantly decreased phagocytic rates and superoxide anion production observed in head kidney leukocytes, as well as poorer protection against Vibrio alginolyticus infection. These immunosuppressive effects were not observed in the fish fed with β-glucan in combination with a lower dose of OTC (group M2). Thus, these data suggest that the combination of dietary β-glucan and OTC exerts synergistic immunostimulating effects that protect groupers from bacterial infection.

White spot syndrome virus (WSSV) is a large enveloped DNA virus, and it causes a serious disease that has led to severe mortalities of cultured shrimps in many countries. To determine the mechanism of virus entry into the cell and to establish an antiviral strategy, the cell receptor for virus entry and receptor binding protein should be identified. A shrimp cell surface protein, glucose transporter1 (Glut1), was found to interact with WSSV in previous study. In this study, this Glut1 was confirmed to have the ability of transporting glucose, and this gene can also be found in other shrimp species. The interaction between Glut1 and some other WSSV envelope proteins in the infectome structure was verified by far western blot and His pull down assay. In vitro and in vivo neutralization using recombinant partial Glut1 revealed that the large extracellular portion of Glut1 could delay WSSV infection. Also, shrimps which were knocked-down Glut1 gene by treated with dsRNA before WSSV challenge showed decreased mortality. These results indeed provide a direction to develop efficient antiviral strategies or therapeutic methods by using Glut1.

Two feeding trials were carried out with Penaeus monodon larvae in a small-scale system at a density of 100 individuals l -1 from zoea I to postlarva V in order to determine the effects of dietary protein quality (white fish meal, krill meal, and soybean meal) on survival, digestive enzyme activities, and protein digestibility in vitro. Zocae fed diet C, containing 41.5% krill meal, and 20% soybean meal, had a higher survival than those fed diet A, containing 45% fish meal and 26.5% krill meal, and diet B, containing 15% fish meal, 26.5% krill meal, and 20% soybean meal. In mysid larvae, however, diet A gave the highest survival. In order to set up the feeding regime for P. monodon larvae, diets A, B, and C were used alternatively to feed zoeae and mysids. Zoeac fed diet C and mysids fed diet A had the highest survival. Using whole zoeae, mysids, and postlarvae tissue as the reference protein, there were no significant differences in essential amino acid index (EAAI) of the three experimental diets containing different protein sources. The digestive enzyme extracted from zoeac had a higher in vitro protein digestibility than those extracted from mysids. The activity of total protease was low during the nauplius stage and peaked at zoea II and mysis II. Also, the activity of α-amylase was low during the nauplius stage and peaked at zoea III and mysis II.

The members of the inhibitor of apoptosis protein (IAP) family are involved in the regulation of diverse cellular processes, including apoptosis, signal transduction and mitosis. Here, we report the cloning and characterization of three IAP genes from Pacific white shrimp Litopenaeus vannamei: LvIAP1, LvIAP2 and LvSurvivin. LvIAP1, the orthologue of Penaeus monodon IAP (PmIAP), consists of three BIR domains and one RING domain; LvIAP2 consists of two BIR domains and LvSurvivin has only one BIR domain. Expression profiling by absolute quantitative real-time RT-PCR revealed that of the three IAP genes, LvIAP1 had the highest expression levels in almost all examined tissues and LvSurvivin had the lowest expression levels. Furthermore, among the examined tissues, the lymphoid organs most strongly expressed all three genes. When LvIAP1 expression was silenced by injection of its corresponding dsRNA, the shrimp died within 48 h after injection, whereas injection of the other two dsRNAs did not cause shrimp death. In LvIAP1-silenced shrimp, the number of circulating haemocytes decreased dramatically because of extensive apoptosis. This suggested that LvIAP1 is central to the regulation of shrimp haemocyte apoptosis. 2012 Elsevier Ltd. All rights reserved.