A native strain of Bacillus subtilis increases lipid accumulation and modulates expression of genes related to digestion and amino acid metabolism in Litopenaeus vannamei.

In the field of shrimp aquaculture, the utilization of probiotics represents a promising avenue, due to the well-documented benefits conferred by these microorganisms. In the current study, a Bacillus subtilis strain, referred to as strain E, was isolated from the gastrointestinal tract of the shrimp Litopenaeus vannamei and subsequently identified via molecular methods and phylogeny. The probiotic potential of strain E was characterized, and its application as a feed shrimp additive was evaluated in a 45-day experiment. Several parameters were assessed, including zootechnical performance, muscle tissue proximate composition, hepatopancreas lipid concentration, and the expression of genes associated with digestion, amino acid metabolism, and antioxidant defense mechanisms in various shrimp tissues. Although no significant impact on zootechnical performance was observed, supplementation with strain E led to an increase in lipid concentration within both muscle and hepatopancreas tissues. Furthermore, a marked decrease in the expression of genes linked to digestion and amino acid metabolism was noted. These findings suggest that the addition of the B. subtilis strain E to shrimp feed may enhance nutrient absorption and modulate the expression of genes related to digestion and amino acid metabolism.

Effects of dietary melatonin on antioxidant and immune function of the Pacific white shrimp (Litopenaeus vannamei), as determined by transcriptomic analysis.

Melatonin (MT) is regarded as an antioxidant and immunostimulant that can efficiently scavenge free radicals and activate antioxidant enzymes. The aim of this study was to investigate the effects of dietary MT on the growth performance and immune function of the Pacific white shrimp (Litopenaeus vannamei). Six groups of L. vannamei were supplemented with dietary MT at 0, 22.5, 41.2, 82.7, 165.1, and 329.2 mg/kg levels for 2 months. RNA-Seq analysis was performed to obtain transcriptome data of the control group and the group supplemented with dietary MT at 82.7 mg/kg BW. In total, 1220 DEGs (799 up-regulated and 421 down-regulated) were identified. Pathways and genes related to growth performance and immune function were verified by real-time quantitative polymerase chain reaction. The total hemocyte count, phagocytosis rate, and respiratory burst were significantly increased in the MT (82.7 mg/kg BW) group as compared to the control group. Analysis of antioxidant-related enzymes in the hepatopancreas showed that dietary MT (82.7 mg/kg BW) significantly increased activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, while dietary MT at 41.2 mg/kg BW significantly increased activities of glutathione S-transferase, lysozyme (LZM), and phenoloxidase (PO). At the transcriptional level, dietary MT up-regulated expression levels of genes associated with antioxidant immunity and growth, which included PO, SOD, LZM, GPx, chitin synthase, ecdysone receptor, calcium-calmodulin dependent protein kinase I, and retinoid X receptor. In conclusion, dietary MT may improve the growth performance and immune function of L. vannamei to some extent.

Effect of fed dietary yeast (Rhodotorula paludigena CM33) on shrimp growth, gene expression, intestinal microbial, disease resistance, and meat composition of Litopenaeus vannamei.

Yeast is a health-promoting and bio-therapeutic probiotic that is commonly used in aquaculture. Rhodotorula paludigena CM33 can accumulate amounts of intracellular carotenoids and lipid, which are regarded as nutritionally beneficial compounds in various aspects. The aim of this study was to evaluate the impact of different levels of R. paludigena CM33 (RD) incorporated in a dietary composition at 0% (control), 1% (1% RD), 2% (2% RD), and 5% (5% RD) on the growth of shrimp (Litopenaeus vannamei), their immune-related gene expression, intestinal health, resistance to Vibrio parahaemolyticus (VPAHPND) infection, and meat composition. The results showed significant improvements in the specific growth rate, weight gain, and survival of shrimp fed with 1% RD, 2% RD, and 5% RD, which were higher than the control group after 4 weeks of administration. The administration of 5% RD group resulted in a decrease in cumulative mortality upon VPAHPND challenge when compared to the control group. Furthermore, the expression levels of immune-responsive genes, including proPO system (prophenoloxidase-2: PO2), antioxidant enzyme (superoxide dismutase: SOD, glutathione peroxidase: GPX, and catalase: CAT), JAK/STAT pathway (signal transducer and activator of transcription: STAT, gamma interferon inducible lysosomal thiol reductase: GILT), IMD pathway (inhibitor of nuclear factor kappa-B kinase subunit beta and epsilon: IKKb and IKKe), and Toll pathway (Lysozyme) genes, were up-regulated in the 5% RD group. In the context of microbiota, microbiome analysis revealed that the main phyla in shrimp intestines were Proteobacteria, Firmicutes, Bacteroidota, Campilobacterota, Actinobacteriota, and Verrucomicrobiota. At the genus level, Vibrio was found to be reduced in the 5% RD group, whereas the abundance of potentially beneficial bacteria Bifidobacterium was increased. The 5% RD group showed a significant increase in the levels of crude protein and crude lipid, both of which are essential nutritious components. Our results show the capability of R. paludigena CM33 as a probiotic supplement in shrimp feed in improving growth, antimicrobial responses against VPAHPND, and meat quality by increasing protein and lipid content in shrimp.

Effect of Clostridium butyricum on intestinal microbiota and resistance to Vibrio alginolyticus of Penaeus vannamei.

In order to evaluate the effect of Clostridium butyricum (C. butyricum) feeding on intestinal microorganisms and protection against infection by Vibrio alginolyticus (V. alginolyticus) in Penaeus vannamei (P. vannamei). We set up two groups, CG30 (fed normal feed) and CB30 (fed feed supplemented with C. butyricum), for the 30d C. butyricum feeding test, and four groups, CG (CG30 group injected with PBS), CB (CB30 group injected with PBS), VACG (CG30 group injected with V. alginolyticus), and VACB (CB30 group injected with V. alginolyticus), for the 24 h infection test. The protective effect of C. butyricum against acute V. alginolyticus infection in P. vannamei was explained in terms of survival, histopathology, changes in enzyme activity, transcriptome analysis, and immune-related genes. We found that feeding C. butyricum significantly altered intestinal microbial populations' abundance and significantly reduced Vibrio spp. In the V. alginolyticus stress test, C. butyricum improved the survival rate and alleviated pathological changes in hepatopancreatic tissues, alleviated the reduction of superoxide dismutase (SOD) and phenoloxidase (PO) activity caused by infection, and increased the lysozyme content in P. vannamei. VACB group compared with the VACG group, 1730 up-regulated differentially expressed genes (DEGs) and 2029 down-regulated DEGs were screened. Quantitative real-time PCR (qRT-PCR) showed that dietary supplementation with C. butyricum suppressed the upregulation of alkaline phosphatase (AKP) transcription factors and the downregulation of prophenoloxidase (proPO), alpha-2-macroglobulin (A2M), and anti-lipopolysaccharide factor (ALF) induced by V. alginolyticus infection. In conclusion, feed supplementation with C. butyricum changed P. vannamei's population ratio of intestinal microorganisms. Moreover, C. butyricum has the potential to act as an inhibitor of V. alginolyticus infection and enhance the resistance of P. vannamei to V. alginolyticus infection.

Identification and Expression Analysis of Dsx and Its Positive Transcriptional Regulation of IAG in Black Tiger Shrimp (Penaeus monodon).

Doublesex (Dsx) is a polymorphic transcription factor of the DMRTs family, which is involved in male sex trait development and controls sexual dimorphism at different developmental stages in arthropods. However, the transcriptional regulation of the Dsx gene is largely unknown in decapods. In this study, we reported the cDNA sequence of PmDsx in Penaeus monodon, which encodes a 257 amino acid polypeptide. It shared many similarities with Dsx homologs and has a close relationship in the phylogeny of different species. We demonstrated that the expression of the male sex differentiation gene Dsx was predominantly expressed in the P. monodon testis, and that PmDsx dsRNA injection significantly decreased the expression of the insulin-like androgenic gland hormone (IAG) and male sex-determining gene while increasing the expression of the female sex-determining gene. We also identified a 5'-flanking region of PmIAG that had two potential cis-regulatory elements (CREs) for the PmDsx transcription. Further, the dual-luciferase reporter analysis and truncated mutagenesis revealed that PmDsx overexpression significantly promoted the transcriptional activity of the PmIAG promoter via a specific CRE. These results suggest that PmDsx is engaged in male reproductive development and positively regulates the transcription of the PmIAG by specifically binding upstream of the promoter of the PmIAG. It provides a theoretical basis for exploring the sexual regulation pathway and evolutionary dynamics of Dmrt family genes in P. monodon.

Effects of dietary chenodeoxycholic acid supplementation in a low fishmeal diet on growth performance, lipid metabolism, autophagy and intestinal health of Pacific white shrimp, Litopenaeus vannamei.

An 8-week feeding trial was conducted to evaluate the effects of chenodeoxycholic acid (CDCA) on growth performance, body composition, lipid metabolism, and intestinal health of juvenile white shrimp, Litopenaeus vannamei fed a low fishmeal diet. Four practical diets were formulated: HFM (25% fishmeal), LFM (15% fishmeal), LB1 (LFM + 0.04% CDCA), LB2 (LFM + 0.08% CDCA). Each diet was assigned to four tanks with forty shrimp (initial weight 0.33 ± 0.03 g) per tank. The results indicated that the growth performance of shrimp were similar between the four groups; the crude lipid content of shrimp fed the LB2 diet was significantly lower than those fed the HFM diet (P < 0.05). The lipase activity content in hepatopancreatic were significantly higher in the two CDCA supplemented groups than that in LFM group; the contents of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol in hemolymph were significantly lower in LFM group, LB1 group and LB2 group than that in HFM group (P < 0.05). The shrimp fed LB1 diet was significantly decreased the intestinal expression levels of tube than those fed in HFM diet; the intestinal gene expression of imd and toll were significantly lower in LB2 group than those in HFM group (P < 0.05). The results of hepatopancreas gene expression suggest that shrimp fed the LFM diet showed significantly upregulated expression levels of sterol regulatory element-binding protein (srebp), acetyl-CoA carboxylase (acc), and carnitine palmitoyltransferase 1 (cpt-1) than those fed the HFM diet; shrimp fed the LB1 diet showed significantly upregulated expression levels of srebp, acc, and AMP-activated protein kinase (ampk) than those fed the HFM diet; shrimp fed the LB2 diet had higher expression levels of srebp, acc, and cpt-1 than those fed the HFM diet (P < 0.05). In the hepatopancreas, the shrimp fed the LFM diet shown significantly up-regulated the expression levels of beclin1 compared to those fed HFM diet; the expression levels of autophagy-related protein13 (atg3), autophagy-related protein 12 (atg12) of in shrimp fed the LB1 diet were significantly higher than those fed the HFM diet; and the expression levels of autophagy-related protein13 (atg13), beclin1, atg3, atg12, autophagy-related protein 9 (atg9) of shrimp fed LB2 diet were significantly higher than those fed the HFM diet (P < 0.05). The atg3 in intestine of shrimp fed the LB2 diet were significantly higher than those fed the HFM diet (P < 0.05). Intestinal mucous fold were damaged, hepatic tubules were disorganized and B cells appeared to be swollen in LFM group. The fold height and width of shrimp fed the diets supplemented with CDCA increased significantly than those fed the LFM diet (P < 0.05), the hepatic tubules were neatly arranged, and R cells increased. In conclusion, supplementary CDCA in a low fishmeal diet promoted lipid metabolism, enhanced autophagy of shrimp, also improved the health of the intestine and hepatopancreas.

Transcriptomic and Histological Analysis of the Greentail Prawn (Metapenaeus bennettae) Following Light Crude Oil Exposure.

Oil spills pose a significant threat to marine biodiversity. Crude oil can partition into sediments where it may be persistent, placing benthic species such as decapods at particular risk of exposure. Transcriptomic and histological tools are often used to investigate the effects of hydrocarbon exposure on marine organisms following oil spill events, allowing for the identification of metabolic pathways impacted by oil exposure. However, there is limited information available for decapod crustaceans, many of which carry significant economic value. In the present study, we assess the sublethal impacts of crude oil exposure in the commercially important Australian greentail prawn (Metapenaeus bennettae) using transcriptomic and histological analyses. Prawns exposed to light, unweathered crude oil "spiked" sediments for 90 h were transferred to clean sediments for a further 72 h to assess recovery. Chemical analyses indicated that polycyclic aromatic hydrocarbons increased by approximately 65% and 91% in prawn muscle following 24 and 90 h of exposure, respectively, and significantly decreased during 24- and 72-h recovery periods. Transcriptomic responses followed an exposure and recovery pattern with innate immunity and nutrient metabolism transcripts significantly lowered in abundance after 24 h of exposure and were higher in abundance after 72 h of recovery. In addition, transcription/translation, cellular responses, and DNA repair pathways were significantly impacted after 24 h of exposure and recovered after 72 h of recovery. However, histological alterations such as tubule atrophy indicated an increase in severity after 24 and 72 h of recovery. The present study provides new insights into the sublethal impacts of crude oil exposure in greentail prawns and identifies molecular pathways altered by exposure. We expect these findings to inform future management associated with oil extraction activity and spills. Environ Toxicol Chem 2022;41:2162-2180. © 2022 John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Immune gene expression and antioxidant response to vitamin E enriched diets for males Litopenaeus vannamei breeder (Boone, 1931).

The is aim of this study was to evaluate if vitamin E supplementation to diet could improve the spermatic quantity and quality in males of Litopenaeus vannamei. A basal diet with 171.5 mg/kg (included in commercial vitamin premix) and experimental diets with 3 concentrations of vitamin E: 620 (diet A), 1069 (diet B) and 1517 (diet C) mg/kg were tested by 30 days. 500 L tanks were set up for each treatment, and seven males were set in each tank. Circulant cholesterol, triglyceride (TG), glucose, total protein was measured. Sperm quantity and quality were classified as normal, abnormal, or dead. In reproductive tract was measured the activity and differential expression lipid soluble antioxidant system (LSAS) and of immune genes system. The higher sperm quantity was obtained in A diet and decreased in shrimp fed B and C diets (p < 0.05). Males fed diets B and C presented a higher activity and relative expression of lipid system oy suggesting oxidative stress. A significant increment of prophenol oxidase (proFo) and α-2-macroglobulin (α-2 M) transcripts were observed in males fed C diet (p < 0.05). All these variables were present in males with low sperm amount and low hemocyanin gene expression (p < 0.05). Therefore, diet A supplemented with 620 mg/kg vitamin E could benefit males L. vannamei broodstock, taking a count the sperm quantity and all the lipid and immune genes response.

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

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

Molecular characterization and expression profiling of transformer 2 and fruitless-like homologs in the black tiger shrimp, Penaeus monodon.

Transformer 2 (tra 2) and fruitless (fru) genes have been proven to play a key role in sex determination pathways in many Arthropods, including insects and crustaceans. In this study, a paralog of P. monodon tra 2 (Pmtra 2), P. monodon ovarian associated transformer 2 (PmOvtra 2) and 2 isoforms of P. monodon fruitless-like gene (Pmfru-1 and Pmfru-2) were identified and characterized. The full cDNA sequence of PmOvtra 2 consisted of 1,774 bp with the longest open reading frame (ORF) of 744 bp encoding for 247 amino acids. The PmOvtra 2 exhibited a predicted RNA-recognition motif (RRM) domain and two arginine-serine (RS) regions, suggesting its function in RNA splicing. The full cDNA sequence of Pmfru-1 consisted of 1,306 bp with 1,182 bp ORF encoding for 393 amino acids, whereas the full cDNA sequence of Pmfru-2 consisted of 1,858 bp with 1,437 bp ORF encoding 478 amino acids. The deduced amino acid sequences of Pmfru-1 and Pmfru-2 exhibited highly conserved domains of Fru proteins, including Broad-complex, Tramtrack and Bric-a-brac (BTB), and zinc finger (ZF) domains. In addition, Pmfru-1 and Pmfru-2 were suggestively originated from the same single genomic locus by genomic sequence analysis. Specifically, Pmfru pre-mRNA was alternatively spliced for Pmfru-1 and Pmfru-2 to include mutually exclusive exon 7 and exon 6, respectively. Temporal and spatial expression of PmOvtra 2, Pmfru-1, and Pmfru-2 were also investigated by qPCR. The results showed that all were expressed in early developmental stages with undifferentiated gonads starting from nauplius until postlarvae. The expression of PmOvtra 2 started at nauplius stage and gradually increased from mysis to postlarvae (PL) 1. However, the expression of Pmfru-1 was low at the nauplii stage and slightly increased from protozoea to PL5, whereas the expression of Pmfru-2 maintained a low level from nauplius to mysis and then gradually increased at the PL stages. Expressions of PmOvtra 2, Pmfru-1, and Pmfru-2 were detected in various tissues including nervous tissue, gill, heart, hepatopancreas, gut, and gonads. Interestingly, the sexually dimorphic expression of PmOvtra 2, Pmfru-1, and Pmfru-2 was demonstrated in fully developed gonads in which the ovary showed significantly higher expressions than the testis. The great difference in the expression pattern of PmOvtra 2, Pmfru-1, and Pmfru-2 in the ovary and testis suggested their roles in the female sex determination in P. monodon.

Cellular and molecular immune response and production performance of Indian white shrimp Penaeus indicus (H. Milne-Edwards, 1837), reared in a biofloc-based system with different protein levels of feed.

The present study focuses on the immunity and growth of Penaeus indicus fed with varying protein levels (25%, 30%, and 35%) in a biofloc based rearing system. A 120 days growth trial was carried out using juvenile Penaeus indicus (0.71 ± 0.01) with dietary protein level, 25% (LP), 30% (MP), and 35% (HP), and a control diet-fed with 35% acted as control group resulting in 4 treatments each with four replicates and were randomly assigned 16 tank units (7500 L each). A combination of different carbon sources (molasses, wheat flour, and rice bran in 2:1:1 ratio), yeast and a probiotic (Bacillus sp.) consortium were used for the development of biofloc. At the end of the trial, the growth parameters of shrimps viz., initial weight, feed conversion ratio (FCR), and daily growth coefficient (DGC) were computed. The results indicated that shrimp fed with medium (30%) protein (MP) diet recorded significantly (P < 0.05) improved growth performance compared to high protein fed group (35%) and low protein (25%) fed group (LP) in a biofloc system and control group (35%). The immunological parameters such as hemagglutination activity (HA) assay, serum protein, lysozyme, phenol oxidase (PO), and inhibition of superoxide dismutase (SOD) activity were observed in serum, plasma, and hemocyte lysate supernatant (HLS). The HA activity, PO activity in plasma was found to be higher in high protein fed animals, whereas medium protein resulted in enhanced PO activity in serum. Similarly, lysozyme and SOD were inhibited well in high protein fed animals compared to the low protein fed group. The vital immune genes's mRNA profiling showed a potential rise in the expressional pattern in MP and HP treatments compared to LP and control. BGBP (beta-1,3-glucan binding protein) and hemocyanin mRNA transcript levels were highly upregulated in the HP (5 fold) and moderately expressed in MP (2 fold) and LP (1-2 fold). The transcripts of peroxinectin, antimicrobial peptides like crustin showed significant upregulation in HP followed by in MP and LP and control. Likewise, other immune genes, such as SOD, prophenoloxidase (proPO), showed a similar trend in a marginal way, indicating immunomodulation in the biofloc groups. This study suggested that biofloc with high protein (35%) supplementation can substantially enhance the immune response of shrimps, although medium protein level (30%) is optimum for improving the survival, growth, and in turn economic return in Indian white shrimp.

Dietary Supplementation of Brown Seaweed and/or Nucleotides Improved Shrimp Performance, Health Status and Cold-Tolerant Gene Expression of Juvenile Whiteleg Shrimp during the Winter Season.

This study was aimed to evaluate the efficiency of Sargassumpolycystum and nucleotides- supplemented diets to improve immune response and cold-tolerance of juvenile Litopenaeus vannamei. Four treatments were evaluated: T1, the control, shrimp received only a basal diet; T2, a basal diet with 500 ppm nucleotides; T3, a basal diet with 500 ppm S.polycystum powdered; T4, a basal diet with 500 ppm nucleotides and 500 ppm S.polycystum powdered. Shrimp were fed experimental diets for 56 days. Results revealed shrimp fed T4 diet exhibited the best significant improvement in water quality, survival, growth, and feed utilization indices followed by T2, and T3, while T1 showed the worst values. Additionally, nonspecific immune responses (phagocytosis (%), lysozyme, phenoloxidase, super oxide dismutase (SOD) activity, total nitric oxide) were improved with 1.7-3.2-fold in T4 higher than T1. Histomorphology of hepatopancreas in T4 showed the most increased activation of the hepatic glandular duct system compared with the other treatments. Moreover, nucleotides/seaweed-supplemented diets upregulated relative expression of cMnSOD, Penaeidin4, and heat shock protein70 (HSP70) genes, while translationally controlled tumor protein (TCTP) was downregulated. In conclusion, the synergistic effects of both S. polycystum and nucleotides have many advantages as a growth promoter, immunostimulant, antimicrobial, and cold-tolerant stimulant to L. vannamei.

Pandanus tectorius fruit extract promotes Hsp70 accumulation, immune-related genes expression and Vibrio parahaemolyticus tolerance in the white-leg shrimp Penaeus vannamei.

Plants and herbal extracts are indispensable for controlling the spread of disease-causing bacteria, including those that infect aquatic organisms used in aquaculture. The use of plant or herbal extract is expected to be safe for aquatic animals and less harmful to the environment, as opposed to conventional therapeutic alternatives such as antibiotics that promote the occurrence of potential antibiotic-resistant bacteria when used improperly. The efficacy of Pandanus tectorius fruit extract in the regulation of Hsp70 expression, pro-phenoloxidase (ProPO), peroxinectin, penaeidin, crustin and transglutaminase, all immune peptides essential for Vibrio tolerance in white leg shrimp, Penaeus vannamei, was investigated in this study, which included the determination of the safety levels of the extract. Tolerance of shrimp against Vibrio parahaemolyticus, a pathogenic bacteria that causes Acute Hepatopancreas Necrosis Disease (AHPND), was assessed on the basis of median lethal dose challenge survival (LD50 = 106 cells/ml). Mortality was not observed 24 h after exposure of 0.5-6 g/L of the fruit extract, indicating that P. tectorius was not toxic to shrimp at these concentrations. A 24-h incubation of 2-6 g/L of the fruit extract increased shrimp tolerance to V. parahaemolyticus, with survival doubled when the maximum dose tested in this study was used. Concomitant with a rise in survival was the increase in immune-related proteins, with Hsp70, ProPO, peroxinectin, penaeidin, crustin and transglutaminase increased 10, 11, 11, 0.4, 8 and 13-fold respectively. Histological examination of the hepatopancreas and muscle tissues of Vibrio-infected shrimp primed with P. tectorius extract revealed reduced signs of histopathological degeneration, possibly due to the accumulation of Hsp70, a molecular chaperone crucial to cellular protein folding, tissue repair and immune response of living organisms, including Penaeid shrimp.

The effect of dietary supplementation of Astragalus membranaceus and Bupleurum chinense on the growth performance, immune-related enzyme activities and genes expression in white shrimp, Litopenaeus vannamei.

A 56-day feeding trial was conducted to investigate the effects of dietary supplementation of Astragalus membranaceus or/and Bupleurum chinense on the growth performance, immune enzymes, and related gene expression of Pacific white shrimp (Litopenaeus vanammei). Six experimental diets were formulated and supplemented with two levels (0.25% and 0.5%) of each herb and their combination. At the end of the trial, the specific growth rate and feed conversion ratio of shrimp were significantly (P < 0.05) improved by herbal diets. Besides, the activities of immune-related enzymes such as superoxide dismutase (SOD), alkaline phosphatase (AKP), and lysozyme in serum and hepatopancreas were significantly (P < 0.05) elevated in shrimp fed A. membranaceus or/and B. chinense. The high expression levels of immune deficiency (IMD), lysozyme, and Toll-like receptor mRNA directly or indirectly reflected the activation effect of innate immune in shrimp by dietary A. membranaceus or/and B. chinense. However, no significant difference (P > 0.05) among the herbal incorporated treatments was detected on the growth performance and immune response. In conclusion, the results suggest that A. membranaceus and B. chinense could be used as a beneficial feed additives and alternatives to antibiotics for white shrimp aquaculture.

Development of a specific isothermal nucleic acid amplification for the rapid and sensitive detection of shrimp allergens in processed food.

Food allergens that cause anaphylactic reactions have become an important health problem worldwide. Among them, shrimp is a popular seafood in many cuisines. The best way to avoid allergic reactions is to mitigate the intake of food allergens. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of shrimp DNA. Using LAMP primers, the identification of shrimp DNA by the LAMP assay was specific and rapid (within 30 min). It exhibited no cross-reaction with the DNA of other Crustacea, including crabs and lobster, and at least 0.01% shrimp DNA existed in the test sample. Additionally, the sensitivity of LAMP for detecting shrimp DNA was 100-fold greater than that of conventional PCR. LAMP for the detection of shrimp DNA was reproducible regardless of whether the genomic DNA was extracted from boiled, steamed or roasted shrimp samples. In summary, the LAMP assay established herein not only could be potentially used for diagnosing shrimp DNA but could also be applicable for identifying shrimp allergens in commercial food products in marketplaces.

Interactive effects of dietary cholesterol and phospholipids on the growth performance, expression of immune-related genes and resistance against Vibrio alginolyticus in white shrimp (Litopenaeus vannamei).

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.

The intracellular signaling pathway of octopamine upregulating immune resistance functions in Penaeus monodon.

Octopamine (OA), a biogenic monoamine, is known to mediate several immune responses. This study analyzed the effects of OA on immunological regulation in the tiger shrimp Penaeus monodon. The immune parameters including total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, superoxide dismutase activity, and phagocytic activity and clearance efficiency in response to the pathogen, Photobacterium damselae, were determined when shrimp were individually injected with saline or OA at 100 or 1000 pmol shrimp-1. In addition, the intracellular second messengers in haemocyte such as Ca2+ and adenosine 3',5'-cyclic monophosphate (cAMP) were examined in shrimp receiving saline or OA at 1 or 10 nmol shrimp-1. Results showed that all of the immune parameters significantly increased at 2-4 h in OA-injected shrimp except hyaline cells in 100 pmol shrimp-1-injected shrimp at 4 h, but phenoloxidase activity per granulocyte significantly decreased at 2-4 h. However, these had returned to saline control levels after receiving OA for 8 h except differential haemocyte count and phenoloxidase activity per granulocyte for 16 h. An injection of OA also significantly increased the survival rate of shrimp challenged with Pho. damselae. Shrimp receiving OA at 1 and 10 nmol shrimp-1 significantly increased the intracellular Ca2+ concentration ([Ca2+]i) at 30-60 min and 30 min, and cAMP concentration [cAMP]i) at 5-15 min and 15 min, respectively. However, [Ca2+]i at 50-60 min, and [cAMP]i at 30-60 min returned to saline control when the shrimp received OA at 10 nmol shrimp-1, and at 1 and 10 nmol shrimp-1, respectively. These results suggest that OA administration by injection at ≤1000 pmol shrimp-1 mediates transient upregulation of immunity together with the increased resistance of P. monodon to Pho. damselae, which are modulated through intracellular Ca2+ and cAMP second messenger pathways.

Alginate from Sargassum siliquosum Simultaneously Stimulates Innate Immunity, Upregulates Immune Genes, and Enhances Resistance of Pacific White Shrimp (Litopenaeus vannamei) Against White Spot Syndrome Virus (WSSV).

Although alginate is known as an immunostimulant in shrimp, the comprehensive and simultaneous study on its activity to resolve the relationship of the hematological parameters, upregulation of immune-related gene expression, and resistance to pathogen has not been found in shrimp. We performed experiments to evaluate the effect and mechanism of alginate from S. siliquosum on Pacific white shrimp immune system. Hematological parameters were examined after oral administration of Na alginate in the shrimp. White spot syndrome virus (WSSV) was injected to the shrimp at 14 days, and its copy number was examined quantitatively (qRT-PCR). Immune-related gene expression was evaluated by qRT-PCR. Alginate increased some hematological immune parameters of shrimp. Before WSSV infection, expression levels of Toll and lectin genes were upregulated. The lectin gene were upregulated post infection, and the Toll gene in all the treatments were downregulated, except the shrimps fed with alginate at 6.0 g kg-1 at 48 h post infection (hpi). The shrimps fed with alginate at 6.0 g kg-1 were the most resistant and gave the least WSSV copy number at 48 hpi. Resistance of shrimps fed the alginate-supplemented diets against WSSV was significantly higher compared to that of the control treatment with 56% and 10% of survival rates, respectively. Oral administration of alginate did not affect the growth and total protein plasma. At 120 h post challenge, alginate treatment at 6.0 g kg-1 exhibited the highest survival rate. It is concluded that oral administration of alginate enhanced the innate immunity by upregulating immune-related gene expression. Consequently, the enhancement of the shrimp innate immunity improves the resistance against WSSV infection.

Involvement of a novel protein kinase C (nPKC) in immunocompetence in hemocytes of white shrimp, Litopenaeus vannamei.

Complementary (c)DNA encoding novel protein kinase C (PKC) messenger (m)RNA of the white shrimp Litopenaeus vannamei, consisted of 2454-bp cDNA containing an open reading frame (ORF) of 2232 bp, belonging to the novel (n)PKC family of proteins characterized by their containing two phorbol ester/diacylglycerol-binding domains (C1 domain), a C2 domain, and a catalytic domain of the serine/threonine kinase, designated LvnPKC. A comparison of amino acid sequences showed that LvnPKC was closely related to arthropod nPKC. LvnPKC cDNA was detected in all tested tissues with a real-time PCR including the hepatopancreas, gills, muscles, subcuticular epithelium, abdominal nerve, thoracic nerve, brain, the stomach, heart, and especially in hemocytes and the intestines. Moreover, significantly upregulated LvnPKC expression was only observed in the eyestalk, brain, and hepatopancreas of shrimp transferred from 28 °C to 18 °C for 30 min. Induction of LvnPKC expression in hemocytes of L. vannamei injected with Vibrio alginolyticus at 105 cfu shrimp-1 was detected earlier than in those injected with 103 cfu shrimp-1. Shrimp received LvnPKC-dsRNA for 1 days specifically depleted the expression of LvnPKC mRNA in hemocytes compared those of diethylpyrocarbonate water treatment. After that, significantly decreased expressions of lipopolysaccharide - and ß-1,3-glucan-binding protein, prophenoloxidase-activating enzyme, peroxinectin, prophenoloxidase I, and prophenoloxidase II in the prophenoloxidase-activating system; lysozyme and cytosolic manganese superoxide dismutase and mitochondrial manganese superoxide dismutase in the antioxidant system were observed. We therefore concluded that LvnPKC is involved in immune defense of L. vannamei exposed to hypothermal stress or infected with V. alginolyticus.

Effects of dietary dosage forms of copper supplementation on growth, antioxidant capacity, innate immunity enzyme activities and gene expressions for juvenile Litopenaeus vannamei.

The experiment was conducted to evaluate the effects of different dietary dosage forms of copper supplementation on growth performance, hematological characteristics, antioxidant capacity, immune responses and gene expressions related to innate immune of juvenile Pacific white shrimp Litopenaeus vannamei. Three isonitrogenous and isolipidic diets were formulated to contain three dosage forms of copper: copper sulfate (Diet I-Cu), copper sulfate + copper amino acid complex (1: 1, Diet M-Cu) and copper amino acid complex from Availa®Cu100 (Diet Availa-Cu), respectively. 360 Pacific white shrimp juveniles (initial weight 1.86 ±â€¯0.03 g) were randomly allocated in 12 tanks corresponding to quadruplicate tanks of the three dietary treatments, and the 8-week feeding trail was conducted. The results indicated that percent weight gain (PWG) and specific growth rate (SGR) in shrimp fed M-Cu diet were significantly higher than that fed I-Cu diet. Survival, feed efficiency (FE), protein efficiency ratio (PER) of shrimp were not significantly different between all treatment groups. High contents of total protein (TP) and glucose (GLU) were found in shrimp fed the diet containing M-Cu, whereas contents of cholesterol (CHOL) and triacylglycerol (TAG) in shrimp fed M-Cu diet were significantly lower than that in I-Cu diet group. In hemolymph, shrimp fed M-Cu diet had high activities of phenoloxidase (PO), alkaline phosphatase (ALP) and acid phosphatase (ACP). While, Cu/Zn superoxide dismutase (Cu/Zn SOD), ceruloplasmin (CP) and lysozyme (LZM) in hemolymph were not significantly affected by different dietary dosage forms of copper. High activities of Cu/Zn SOD, ALP, ACP and LZM in hepatopancreas were observed in shrimp fed M-Cu diet. Shrimp fed diet supplemented with Availa-Cu showed a significantly higher gene expression levels of Cu/Zn sod, alp, acp and lzm in hepatopancreas than that fed I-Cu diet. This study indicated that copper amino acid complex was more effective than copper sulfate to improve growth performance and enhance antioxidant ability and innate immune system.