Resistance Of Shrimp Research Articles

Effect of dietary supplementation with butyrate and probiotic on the survival of Pacific white shrimp after challenge with Vibrio alginolyticus

ABSTRACT This study evaluated the performance, immunology, and survival of the Pacific white shrimp Litopenaeus vannamei to experimental challenge to Vibrio alginolyticus based on the use of the probiotic Lactobacillus plantarum and the combined use of probiotic and butyrate. Four different diets resulted from the addition of additives: butyrate, probiotic, butyrate + probiotic, and control (no additives). The attractiveness of the diets was assessed by the percentage of positive choices and rejections, using a dual-choice Y-maze format aquarium. The shrimps were fed during four weeks and performance parameters, intestinal microbiota, and immunological parameters were all evaluated. Subsequently, the shrimps were challenged with V. alginolyticus and after 48 h, survival and immunological parameters were evaluated. The results showed increased attractiveness and intake, but only with diets supplemented with sodium butyrate. However, other diets were not rejected. No difference in performance or immunological parameters was observed among the different diets. Also, among the treatments, no difference in Vibrio spp., or total heterotrophic bacteria counts, was found in the intestinal tract. However, the lactic acid bacteria count was higher in the intestinal tract of shrimps fed diets supplemented with probiotic. After bacterial challenge, shrimp fed all diets had a greater survival when compared with the control group. Lactobacillus plantarum and sodium butyrate increase the resistance of shrimp to infection with V. alginolyticus, but do so without affecting performance, immunological parameters, or Vibrio spp., and total heterotrophic bacteria counts in the intestinal tract.

Brown seaweeds as feed additive for white-leg shrimp: effects on thermal stress resistance, midgut microbiology, and immunology

This study evaluated the midgut microbiology, hemato-immunological parameters and the resistance to thermal shock of the white-leg shrimp Litopenaeus vannamei fed with diets containing 0.5, 2, and 4% of the brown seaweeds Undaria pinnatifida and Sargassum filipendula. Shrimp were reared in clear seawater for 15 days in seven 800-L tanks with 100 shrimp (5.9 ± 0.2 g), under constant aeration and temperature (29 °C), with 80% daily water exchange. Animals were fed four times per day (6% of biomass) during the experimental period. At day 16, bacterial count of midgut and hemato-immunological parameters were assessed before the thermal shock; during thermal shock, shrimp were transferred from seawater at 29 to 12.5 °C for 1 h, and then back to 29 °C, and kept for 48 h to estimate the cumulative mortality. Animals fed with 4% of U. pinnatifida had lower Vibrio spp. quantity in the midgut and showed an overall improvement of evaluated hemato-immunological parameters; however, only phenoloxidase activity was statistically significant. Those fed with 0.5 and 2% of S. filipendula showed lower cumulative mortality after thermal shock, in comparison with control and those fed with 4% of this species. Increasing levels of U. pinnatifida had negative effects on thermal shock resistance. In conclusion, U. pinnatifida enhances shrimp immunity and specifically inhibit Vibrio spp., while S. filipendula increases shrimp resistance to thermal variation.

Gene set based association analyses for the WSSV resistance of Pacific white shrimp Litopenaeus vannamei

White Spot Syndrome Virus (WSSV) is regarded as a virus with the strongest pathogenicity to shrimp. For the threshold trait such as disease resistance, marker assisted selection (MAS) was considered to be a more effective approach. In the present study, association analyses of single nucleotide polymorphisms (SNPs) located in a set of immune related genes were conducted to identify markers associated with WSSV resistance. SNPs were detected by bioinformatics analysis on RNA sequencing data generated by Illimina sequencing platform and Roche 454 sequencing technology. A total of 681 SNPs located in the exons of immune related genes were selected as candidate SNPs. Among these SNPs, 77 loci were genotyped in WSSV susceptible group and resistant group. Association analysis was performed based on logistic regression method under an additive and dominance model in GenABEL package. As a result, five SNPs showed associations with WSSV resistance at a significant level of 0.05. Besides, SNP-SNP interaction analysis was conducted. The combination of SNP loci in TRAF6, Cu/Zn SOD and nLvALF2 exhibited a significant effect on the WSSV resistance of shrimp. Gene expression analysis revealed that these SNPs might influence the expression of these immune-related genes. This study provides a useful method for performing MAS in shrimp.

The upregulation of immune responses in tyrosine hydroxylase (TH) silenced Litopenaeus vannamei

Catecholamines (CAs) play a crucial role in maintaining physiological and immune homeostasis in invertebrates and vertebrates under stressful conditions. Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in CA synthesis. To develop an effective CA-related immunological defense system against stress and pathogen infection, various criteria, were evaluated in TH double-stranded (ds) RNA-injected white shrimp, Litopenaeus vannamei. Specifically, the relative transcript quantification of TH, dopamine β-hydroxylase (DBH), crustacean hyperglycemic hormone (CHH), and other immune-related genes; TH activity in the haemolymph; and the estimation of l-dihydroxyphenylalanine (l-DOPA), glucose, and lactate levels in the haemolymph were examined. TH depletion revealed a significant increase in the total haemocyte count; granular cells; semigranular cells; respiratory bursts (RBs, release of superoxide anion); superoxide dismutase (SOD) activity; phagocytic activity and clearance efficiency; and the expression of lipopolysaccharide and β-1,3-glucan-binding protein and peroxinectin, SOD, crustin, and lysozyme genes. In addition, the reduction of TH gene expression and activity was accompanied by a decline of phenoloxidase (PO) activity per granulocyte, lower glucose and lactate levels, and significantly low expression of DBH and CHH genes. However, the number of hyaline cells, activity of PO, RBs per haemocyte, and expression of POI and POII genes were not significantly different in the LvTH-silenced shrimp. Notably, the survival ratio of LvTH-silenced shrimp was significantly higher than that of shrimp injected with diethyl pyrocarbonate–water and nontargeting dsRNA when challenged with Vibrio alginolyticus. Therefore, the depletion of TH can enhance disease resistance in shrimp by upregulating specific immune parameters but downregulating the levels of carbohydrate metabolites.

The effects of sediment redox potential and stocking density on Pacific white shrimp Litopenaeus vannamei production performance and white spot syndrome virus resistance

Redox potential represents the intensity of anaerobic condition in the pond sediment, which may affect the dominant microbial transformations of substances, the toxins production, mineral solubility, as well as the water quality in the sediment–water interface inhabited by the shrimp. This study evaluates the effect of sediment redox potential in conjunction with stocking density on shrimp production performance, immune response and resistance against white spot syndrome virus (WSSV) infection. A completely randomized two factors experimental design was applied with three different sediment redox potential, i.e. −65, −108 and −06 mV, and two shrimp densities, i.e. low (60 shrimp m−2) and high (120 shrimp m−2). Shrimp juveniles with an initial mean body weight of 5.32 ± 0.22 g were maintained in semi-outdoor fibre tanks (270 L in capacity) for 35 days of experimental periods. At the bottom of each tank, 5-cm deep soil substrate with different redox potential was added according to the treatments. The survival and biomass production were significantly reduced at −206 mV sediment redox potential, regardless of stocking density. Highly negative sediment redox potential (−206 mV) and higher stocking density significantly reduced total haemocyte counts and phenoloxydase activity, and shrimp resistance to WSSV infection. We recommend to maintain the redox potential of pond sediment at a level of more than −206 mV.

Effects of dietary taurine, carnitine and cholesterol supplementation on growth performance and immunological status ofLitopenaeus vannameiunder cold exposure

The aim of this study was to investigate effects of dietary feed additives on growth and immunological status of Litopenaeus vannamei under cold exposure. Four experimental diets with 0.2% taurine, 0.01% carnitine, 0.1% cholesterol supplementation and control were fed to L. vannamei (IBW 0.86 ± 0.03 g) for 30 days at 30 ± 1°C before water temperature was dropped to 16°C with a rate of −4°C h−1. Growth performance parameters including weight gain rate (WGR), feed conversion ratio (FCR) and survival rate before the cold exposure, as well as immunological parameters related to haemocytes including total haemocyte count (THC), percentages of hyaline cells (SC%), granular cells and semigranular cells (SC + GC%), non-specific esterase (NSE) activity and apoptotic cell ratio before and after the cold exposure were determined. The results showed that: (1) WGR, FCE and survival rate were not significantly different among groups before the cold exposure. Most of immunological parameters did not differ either, except the apoptotic cell ratio of carnitine and cholesterol groups was significantly lower than that of control and taurine groups (P < 0.05). (2) After the cold exposure, THC was decreased by 48.1% (P < 0.05), 54.1% (P < 0.05), 30.1% and 2.2%; NSE activity was decreased by 31.7% (P < 0.05), 21.4%, 7.0% and 3.5% in control, taurine, carnitine and cholesterol respectively. (3) Conversely, ROS production was increased by 23.8% (P < 0.05), 15.1%, 7.2% and 13.0%; apoptotic cell ratio was increased by 10.4%, 12.8%, 6.4% and 7.2% in control, taurine, carnitine and cholesterol respectively. (4) An increase in HC%, decrease in SC + GC% in all groups was observed after cold exposure, and was significant in cholesterol group. This study showed that cholesterol, carnitine and taurine affected immunological status of L. vannamei under cold exposure to different degrees, possibly by modulating some immunological parameters, as discussed. Present study provides theoretical data for nutritional strategies to enhance cold resistance of shrimp.

Ingestion of food pellets containing Escherichia coli overexpressing the heat-shock protein DnaK protects Penaeus vannamei (Boone) against Vibrio harveyi (Baumann) infection.

Feeding aquatic animals with bacterial encapsulated heat-shock proteins (Hsps) is potentially a new method to combat vibriosis, an important disease affecting aquatic animals used in aquaculture. Food pellets comprised of shrimp and containing Escherichia coli overexpressing either DnaK-DnaJ-GrpE, the prokaryotic equivalents of Hsp70-Hsp40-Hsp20, or only DnaK were fed to juveniles of the white leg shrimp Penaeus vannamei, and protection against pathogenic Vibrio harveyi was determined. Maintaining pellets at different temperatures for varying lengths of time reduced the number of live adhering E. coli, as did contact with sea water, demonstrating that storage and immersion adversely affected bacterial survival and attachment to pellets. Feeding P. vannamei with E. coli did not compromise their survival, indicating that the bacteria were not pathogenic to shrimp. Feeding P. vannamei with pellets containing bacteria overproducing DnaK (approximately 60 cells g(-1) pellets) boosted P. vannamei survival twofold against V. harveyi, suggesting that DnaK plays a role in Vibrio tolerance. Pellets containing DnaK were effective in providing protection to P. vannamei for up to 2 weeks before loss of viability and that DnaK encapsulated by these bacteria enhanced shrimp resistance against Vibrio infection.

Cloning and characterization of a novel hemocyanin variant LvHMCV4 from shrimp Litopenaeus vannamei

Recently, we found 3 variants of hemocyanin subunit with higher molecular weight in shrimp Litopenaeus vannamei (Named as LvHMCV1-3). In this study, a novel L. vannamei hemocyanin variant (Named as LvHMCV4) was further cloned and characterized. Bioinformatic analysis predicted that LvHMCV4 contains one open reading frame of 2137 bp and encodes a polypeptide of 678 amino acids. It shares 84-99% cDNA sequences identity to that of the classical form of L. vannamei hemocyanin (LvHMC, AJ250830.1) and LvHMCV1-3. LvHMCV4 possesses a conserved structure characteristic of the hemocyanin family and can be clustered into one branch along with other arthropod hemocyanins in a phylogenetic tree. Further, the full-length DNA of LvHMCV4 contains 2660 bp and two introns, which are located at the 80-538 bp and 2063-2227 bp regions, respectively. In addition, the mRNA transcript of LvHMCV4 was expressed highly in the hepatopancreas, lymphoid, brain and hemocytes, and weakly in the heart, intestine and gill, while no expression was found in the muscle, stomach and gut. Infection by Escherichia coli K12, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio fluvialis, Streptococcus pyogenes or Staphylococcus aureus up-regulated significantly LvHMCV4 mRNA expression in the hepatopancreas. Furthermore, the recombinant protein of LvHMCV4 (rLvHMCV4) was prepared, which showed agglutination activities against six pathogenic bacteria at concentrations ranging from 15.6 to 125 μg/ml. When co-injected with V. parahaemolyticus in L.vannamei, rLvHMCV4 significantly increased the survival rate after 48 h injection. Together, these studies suggested that hemocyanin variant, LvHMCV4, might be involved in shrimp resistance to pathogenic infection.

Glucosamine: fructose-6-phosphate amidotransferase in the white shrimp Litopenaeus vannamei: characterization and regulation under alkaline and cadmium stress.

Heavy metal residues and chemical contaminators considered as relevant sources of aquatic environmental pollutants have a generally immunosuppressive effect on aquatic organisms, depressing metabolic activities and immune response. Glutamine: fructose-6-phosphate aminotransferase (GFAT, EC2.6.1.16) is the first, and rate-limiting, enzyme in the hexosamine biosynthetic pathway, and is involved in the regulation of chitin biosynthesis and glycosylation of proteins. We have isolated and characterized GFAT from the white shrimp Litopenaeus vannamei. Amino acid sequence similarity of the Lv-GFAT (L.vannamei-GFAT) was highest to GFATs isolated from insects and mammals (83 % similarity to that of Haemaphysalis longicornis). The open-reading frame of the Lv-GFAT codes for a protein of 41.6 kDa with a calculated isoelectric point of 5.03. RT-PCR assays showed that endogenous Lv-GFAT mRNA is most strongly expressed in the intestine. Further analysis of Lv-GFAT gene expression in hepatopancreas by quantitative real-time PCR demonstrated that Lv-GFAT transcript levels increased when the shrimp were exposed to alkaline pH (9.3) and cadmium stress, but the time when its mRNA expression level peaked differed under these stresses. We also first expressed the recombinant protein of GFAT from shrimps in Escherichia coli. Western blot analyses confirmed that the Lv-GFAT protein was strongly expressed in the hepatopancreas after exposure to the LC-Cd stress. These results suggest that Lv-GFAT expression is stimulated by alkaline pH and cadmium stress and that it may play important roles in resistance of shrimp to environmental stresses.

Farm-raised tiger shrimp, Penaeus monodon, fed commercial feeds with added organic acids showed enhanced nutrient utilization, immune response and resistance to Vibrio harveyi challenge

The threat of disease in the aquaculture industry, coupled with greater restrictions or ban on antibiotic use, has increased interest in evaluating antibiotic alternatives. A promising alternative is organic acids, which have been used for decades in the livestock feed industry as an antimicrobial and growth promoter. However, very limited information exists on their applications to the shrimp aquaculture industry. The aim of the current study was to examine the potential beneficial effects of a novel microencapsulated organic acid blend (OAB) to the production of tiger shrimp, Penaeus monodon, in earthen ponds at a commercial farm when fed commercial feeds without (Diet A) or with (Diet B) organic acid supplementation at 2% OAB throughout the grow-out period. Farm-raised shrimp were randomly sampled and transferred to the laboratory to examine any effects on nutrient utilization, resistance of shrimp to Vibrio harveyi and associated hepatopancreatic histopathology and phenoloxidase (PO) activity. Results showed that after 22weeks of culture in ponds, shrimp growth was similar between treatments. Lower nitrite-N and nitrate-N concentrations in the pond water indicated potential improved protein utilization from shrimp fed Diet B. This was supported by data from the digestibility trial, demonstrating crude protein, but also dry matter, ash and phosphorous utilization was significantly enhanced (P<0.05). Total viable bacteria and presumptive Vibrio spp. counts were lower at the end of the grow-out period in the pond water of shrimp fed Diet B. Shrimp fed Diet B showed significantly higher survival to V. harveyi challenge, likely due to enhanced PO activity and less hepatopancreatic damage. Total viable bacterial and Vibrio counts in the hepatopancreas of shrimp fed Diet B were significantly lower compared to Diet A. This study provides the first reported data on the use of dietary organic acids in a commercial shrimp farm setting. The enhancement to nutrient utilization may reduce feeding costs and improve water quality while the higher resistance of shrimp to pathogenic bacteria such as Vibrio spp. may provide shrimp farmers with an effective method to mitigate disease outbreaks in the global shrimp aquaculture industry.

Expression of innate immunity genes in kuruma shrimp Marsupenaeus japonicus after in vivo stimulation with garlic extract (allicin)

In recent times, attention has focused on immunostimulants and plant products which could have beneficial effects in disease control. At present, the application of immunostimulants has been considered a more effective approach to health management in aquaculture through the enhancement of immune capability and disease resistance in shrimp. Garlic possesses bactericidal property against bacteria and can inhibit the growth of protozoa as well as gregarine infection in cultured shrimp. However, its effect on viral disease infection has not been well studied and requires further investigation. Garlic can also stimulate shrimp haemocytes as determined by cellular immune responses (phagocytic activity, superoxide anion production and phenoloxidase activity) suggesting that the immune-stimulatory properties of garlic will be useful for improvement of shrimp health. Here, we determined the expression of the immune-related genes Penaeidin, Crustin, Lysozyme, Toll-like, and tumour necrosis factor in kuruma shrimp, upon stimulation with allicin extract. The expression of these factors was measured for the first time and was found to be elevated in intestine and lymphoid organ after stimulation for 3, 12, 24 and 48 h. We conclude that garlic can be used in shrimp culture as an alternative to antibiotics or chemotherapeutic agents; however, further research is needed under field conditions.

Effects of dietary chlorogenic acid on growth performance, antioxidant capacity of white shrimp Litopenaeus vannamei under normal condition and combined stress of low-salinity and nitrite

An eight-week feeding trial followed by an acute combined stress test of low-salinity and nitrite were performed to evaluate effects of chlorogenic acid (CGA) on growth performance and antioxidant capacity of white shrimp Litopenaeus vannamei. Shrimp were randomly allocated in 12 tanks (30 shrimp per tank) and triplicate tanks were fed with a control diet or diets containing different levels of CGA (100, 200 and 400 mg kg−1 feed) as treatment groups. Growth performance including weight gain (WG), biomass gain (BG), feed conversion ratio (FCR), and feed intake were determined after feeding for 56 days. Antioxidant capacity were evaluated by determining the activity of total antioxidant status (TAS), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) as well as the gene expression of GSH-Px and CAT in the hepatopancreas of shrimp at the end of feeding trial and again at the end of the combined stress test. The results indicated that supplemention of CGA had no significant effects on the growth performance and the activities of TAS, SOD, GSH-Px and CAT in hepatopancreas of shrimp cultured under normal conditions for 56 days. However, compared with the control group, CGA (200, 400 mg kg−1 feed) significantly improved the resistance of L. vannamei against the combined stress of low-salinity and nitrite, as indicated by the significant (P < 0.05) higher survival, higher activities of TAS, GSH-Px and CAT, as well as higher transcript levels of GPx and CAT gene in shrimp treated with CGA in the combined tress test. Our findings suggested that CGA possessed dual-modulatory effects on antioxidant capacity of L. vannamei and could be a potential feed additive that can enhance shrimp resistance against environmental stresses. The recommended application dosage is 200 mg kg−1 and further studies are needed to clarify the action model of CGA efficiency.

Immune response and disease resistance of shrimp fed biofloc grown on different carbon sources

The objective of this study was to document the immunological effects of growing shrimp in biofloc systems. The experiment consisted of four types of biofloc systems in which bioflocs were produced by daily supplementation of four different carbon sources, i.e. molasses, tapioca, tapioca-by-product, and rice bran, at an estimated C/N ratio of 15 and a control system without any organic carbon addition. Each biofloc system was stocked with Pacific white shrimp (Litopenaeus vannamei) juveniles that were reared for 49 days. The use of tapioca-by-product resulted in a higher survival (93%) of the shrimp as compared to the other carbon sources and the control. The highest yield and protein assimilation was observed when tapioca was used as the carbon source. After 49 days, phenoloxidase (PO) activity of the shrimp grown in all biofloc systems was higher than that of the shrimp from the control system. Following a challenge test by injection with infectious myonecrosis virus (IMNV), the levels of PO and respiratory burst (RB) activity in the shrimp of all biofloc treatments were higher than that of the challenged shrimp from the control treatment. An increased immunity was also suggested by the survival of the challenged shrimp from the experimental biofloc groups that was significantly higher as compared to the challenged shrimp from the control treatment, regardless of the organic carbon source used to grow the bioflocs. Overall, this study demonstrated that the application of biofloc technology may contribute to the robustness of cultured shrimp by immunostimulation and that this effect is independent of the type of carbon source used to grow the flocs.

A new anti-lipopolysaccharide factor (ALF) gene with its SNP polymorphisms related to WSSV-resistance of Litopenaeus vannamei

Anti-lipopolysaccharide factors (ALFs) of crustacean play an important role against bacteria or virus infection. In this study, the cDNA sequence and genomic sequence of one new isoform of ALF designated as nLvALF1 were reported. The open reading frame (ORF) of nLvALF1 consisted of 369 bp encoding 123 amino acids and the genomic structure of nLvALF1 comprised four introns and three exons. The predicted pI of the deduced protein was 8.82 and the molecular weight (MW) was 13.72 KDa. The deduced amino acid sequence of nLvALF1 contained a typical functional domain of ALF: LPS-binding domain. Phylogenetic analysis showed that nLvALF1 had the closest relationship with FcALF1 from Fenneropenaeus chinensis. The nLvALF1 was specifically expressed in lymphoid organ (Oka) of shrimp. Its transcriptional level was significantly up-regulated after white spot syndrome virus (WSSV) challenge, suggesting that nLvALF1 might participate in defense against WSSV in Litopenaeus vannamei. In order to search potential genetic markers associated with WSSV-resistance, we scanned the polymorphisms of the genomic fragment with 397 bp where the LPS-binding domain encoding sequence located and 18 SNPs were found. The distribution frequency of these SNPs was analyzed in WSSV susceptible shrimp and resistant shrimp separately. Significant differences existed in allelic frequencies at loci g.1361-T > C, g.1370-T > C, g.1419-T > A between the WSSV-resistant group and the WSSV-susceptible/susceptible group (P < 0.05). The specific haplotype CT consisted of g.1415-C > A and g.1419-T > A was associated with susceptibility to WSSV (P < 0.05). These findings provide theoretical support for selection of WSSV-resistant varieties of L. vannamei.

Effects of dietary administration of Shewanella haliotis D4, Bacillus cereus D7 and Aeromonas bivalvium D15, single or combined, on the growth, innate immunity and disease resistance of shrimp, Litopenaeus vannamei

The effects of single or combined use of dietary Shewanella haliotis D4, Bacillus cereus D7 and Aeromonas bivalvium D15, isolated from the gut of shrimp, were assessed on the growth, innate immunity and disease resistance of Pacific white shrimp (Litopenaeus vannamei). Shrimp was fed 28 days by five experimental diets — i) control diet with no probiotics, ii) three probiotic diets, i.e. feed supplemented with 107 cell g− 1 of D4 (D4 diet), D7 (D7 diet) and D15 (D15 diet), and iii) mixed diet comprising of strains D4, D7 and D15 at a ratio of 2:1:1, dosed at 107 cell g− 1. Results showed that growth of shrimp, i.e. final weight, weight gain, percent weight gain and specific growth rate, was improved after 28-days of feeding regime with probiotics compared to the control. Respond of several non-specific immune parameters of groups fed probiotics was also enhanced; i.e. diet D4 and D15 led to significant enhancement of both respiratory burst and superoxide dismutase activities than that of control. Moreover, the respiratory burst activity was also enhanced by mixed diet at the end of 28 days. All probiotic groups were able to increase acid phosphatase activity of shrimp between days 7 and 21. Moreover, the expression of prophenoloxidase and < beta>-1, 3-glucan-binding protein was significantly up regulated in shrimp fed D4 and D15 diets compared to control group, but penaeidin 3 expression up regulated significantly only with mixed diet after 28 days. The cumulative mortality of shrimp, experimentally challenged with Vibrio harveyi, was reduced in groups fed probiotics, i.e. D4 = 36.67%, D7 = 41.67%, D15 = 13.33% and mixed = 11.67%, respectively, than that of controls (43.33%) after 10 days of post-infection. Overall, the results of this study indicated the potential of using probiotics to improve growth, immunity and disease resistance of white shrimp.

The second type of transglutaminase regulates immune and stress responses in white shrimp, Litopenaeus vannamei

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, and phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when white shrimp, Litopenaeus vannamei, (7.5 ± 0.5 g) were individually injected with diethyl pyrocarbonate-water (DEPC-H2O) or different dsRNA at 3 days of injection. In addition, haemolymph glucose and lactate, and haemocytes crustacean hyperglycemic hormone (CHH), transglutaminase I (TGI), transglutaminase II (TGII) and clottable protein (CP) mRNA expression were determined for the shrimp that received DEPC-H2O and different dsRNA after 3 days, and then transferred to 22 and 28 °C from 28 °C. Results showed that respiratory burst, phagocytic activity and clearance efficiency significantly decreased, but hyaline cells significantly increased in the shrimp received LvTGII dsRNA after 3 days. In hypothermal stress studies, LvTGI and CHH were significantly up-regulated in LvTGII-depleted shrimp following exposure to 28 and 22 °C, and haemolymph glucose and lactate were significantly enhanced in LvTGII-depleted shrimp. The injection of LvTGII dsRNA also significantly increased the mortality of L. vannamei challenged with the pathogen V. alginolyticus. These results suggest that LvTGII is an important component on the immune resistance of shrimp, and is involved in the regulation of some immune parameters and carbohydrate metabolites, as well as has a complementary effect with LvTGI in immunological and physiological response of shrimp.

RNA-Seq analysis reveals genes associated with resistance to Taura syndrome virus (TSV) in the Pacific white shrimp Litopenaeus vannamei

Outbreak of Taura syndrome virus (TSV) is one of the major pathogens of the Pacific white shrimp Litopenaeus vannamei. Although selective breeding for improvement of TSV resistance in L. vannamei has been successfully developed and has led to a great benefit to the shrimp farming industry worldwide. The molecular mechanisms underlying the viral resistance in shrimp remain largely unknown. In the present study, we conducted the first transcriptomic profiling of host responses in hemolymph and hemocytes in order to identify the differentially expressed genes associated with resistance to TSV in L. vannamei. High-throughput RNA-Seq was employed, obtaining 193.6 and 171.2 million high-quality Illumina reads from TSV-resistant and susceptible L. vannamei lines respectively. A total of 61,937 contigs were generated with an average length of 546.26bp. BLASTX-based gene annotation (E-value<10−5) allowed the identification of 12,398 unique proteins against the NCBI non-redundant NR database. In addition, comparison of digital gene expression between resistant and susceptible strains revealed 1374 significantly differentially expressed contigs (representing 697 unigenes). Gene pathway analysis of the differentially expressed gene set highlighted several putative genes involved in the immune response activity including (1) pathogen/antigen recognition including immune regulator, adhesive protein and signal transducer; (2) coagulation; (3) proPO pathway cascade; (4) antioxidation; and (5) protease. The expression patterns of 22 differentially expressed genes involving immune response were validated by quantitative real-time RT-PCR (average correlation coefficients 0.94, p-value<0.001). Our results provide valuable information on gene functions associated with resistance to TSV in L. vannamei.

Dieta suplementada com prebiótico, probiótico e simbiótico no cultivo de camarões marinhos

O objetivo deste trabalho foi avaliar o uso de suplemento prebiótico, probiótico e simbiótico, na dieta de camarões marinhos (Litopenaeus vannamei) e seus efeitos sobre o crescimento, a microbiota intestinal, a resposta imune e a resistência ao desafio experimental com Vibrio alginolyticus. Foram utilizados quatro tratamentos: prebiótico inulina; probiótico Lactobacillus plantarum; simbiótico Lactobacillus plantarum + inulina; e controle. Os camarões foram distribuídos em 16 tanques de dez mil litros de água, povoados com 200 camarões cada, cultivados por seis semanas. Avaliaram-se a microbiologia do trato intestinal dos camarões e a reposta imune, antes e após o desafio com V. alginolyticus. A concentração de Vibrio spp. no trato digestório foi menor em camarões alimentados com dieta suplementada com prebiótico, probiótico e simbiótico, enquanto a concentração de bactérias acidoláticas foi superior somente nos camarões alimentados com probiótico e simbiótico. O título aglutinante do soro contra V. alginolyticus aumentou no grupo probiótico e simbiótico, antes da infecção, e foi maior em todos os tratamentos após infecção com V. alginolyticus, em comparação ao controle. Não foi observada diferença entre os tratamentos quanto aos demais parâmetros avaliados. As dietas probióticas, prebióticas e simbióticas alteram a microbiota intestinal e aumentam o título aglutinante do soro contra V. alginolyticus; contudo, não alteram a resistência ao desafio nem o crescimento dos camarões.

Identification of a novel alternative splicing variant of hemocyanin from shrimp Litopenaeus vannamei

Recent evidences suggest that invertebrates express families of immune molecules with high levels of sequence diversity. Hemocyanin is an important non-specific immune molecule present in the hemolymph of both mollusks and arthropods. In the present study, we characterized a novel alternative splicing variant of hemocyanin (cHE1) from Litopenaeus vannamei that produced mRNA transcript of 2579bp in length. The isoform contained two additional sequences of 296 and 267bp in the 5′- and 3′-terminus respectively, in comparison to that of wild type hemocyanin (cHE). Sequence of cHE1 shows 100% identity to that of hemocyanin genomic DNA (HE, which does not form an open reading frame), suggesting that cHE1 might be an alternative splicing variant due to intron retention. Moreover, cHE1 could be detected by RT-PCR from five tissues (heart, gill, stomach, intestine and brain), and from shrimps at stages from nauplius to mysis larva. Further, cHE1 mRNA transcripts were significantly increased in hearts after 12h of infection with Vibrio parahemolyticus or poly I: C, while no significant difference in the transcript levels of hepatopancreas cHE was detected in the pathogen-treated shrimps during the period. In summary, these studies suggested a novel splicing variant of hemocyanin in shrimp, which might be involved in shrimp resistance to pathogenic infection.

Pond shutdown as a strategy for preventing outbreaks of white spot disease in shrimp farms in Mexico

AbstractPond shutdown is a strategy used to eliminate residual pathogens. For 2005–2011, we analysed the relationship between the prevalence of white spot disease (WSD) and the length of pond shutdown (LPS) and discussed its effectiveness for the prevention of WSD in Mexico. The LPS increased significantly from 2.1 months in 2005 to 3.1 months in 2011. The prevalence of WSD diminished significantly from 2005 to 2009, but no evidence of diminution was found when 2010 and 2011 were included in the time series. A χ2‐test for trend indicated that the prevalence of WSD diminished significantly as LPS increased in 2005 through to 2009 and 2005 through to 2011 (P &lt; 0.05). When analysed on an annual basis, the prevalence decreased significantly in relation with LPS in 2006 (P = 0.048), 2007 (P = 0.002), 2008 (P = 0.0001) and 2009 (P = 0.001); no evidence for decreasing the prevalence occurred in 2005 (P = 0.1), 2010 (P = 0.17) or 2011 (P = 0.52). The χ2‐test adjusted for confounding indicated that decreases in prevalence in 2005 to 2009 could be attributed to increases of LPS rather than confounding variables such as increased resistance of shrimp, diminished virulence and improved cultivation practices. We discuss the possible relationship between the warm water temperatures in the Gulf of California and increases in the prevalence of WSD. We conclude that LPS has been an adequate management strategy for preventing WSD, supporting recent compulsory measures taken by the Mexican government to improve biosecurity in shrimp farms.