Effect of Lactobacillus plantarum isolated from digestive tract of wild shrimp on growth and survival of white shrimp (Litopenaeus vannamei) challenged with Vibrio harveyi

Two hundred and two strains of lactic acid bacteria (LAB) isolated from digestive tracts of cultivated and wild adult shrimp, including Litopenaeus vannamei, Metapenaeus brevicornis and Penaeus merguiensis were selected based on their antibacterial activity against Vibrio harveyi. LAB strain of MRO3.12 exhibiting highest reduction of V. harveyi was identified as Lactobacillus plantarum MRO3.12 based on the nucleotide sequence of its 16S rDNA, which showed 99% (780/786 bp) homology to L. plantarum strain L5 (GenBank accession number DQ 239698.1). Co-cultivation of V. harveyi and L. plantarum MRO3.12 showed complete reduction of V. harveyi at 24 h under aerobic and anaerobic conditions, whereas L. plantarum increased from 5.29 to 9.47 log CFU ml⁻¹. After 6-week feeding trial with L. plantarum supplemented diet, white shrimp (L. vannamei) exhibited significant differences (p < 0.05) in relative growth rate (% RGR), feed conversion ratio (FCR) and survival compared to the control group fed with non-supplemented diet. LAB-fed group showed 98.89% survival, whereas only 68.89% survival was observed in the control group. LAB from the digestive tract of probiotic-fed shrimp showed higher level of 5.0 ± 0.14 log CFU/g than the non-supplemented ones (3.34 ± 0.21 log CFU/g). However, total bacterial and non-fermenting vibrios counts decreased in shrimps fed on L. plantarum. Ten days after infection with V. harveyi (5.3-5.5 log CFU ml⁻¹), significant survival (p < 0.05) of 77% was observed in LAB supplemented shrimp, while only 67% survival was observed in the control.     

Two C-type lectins from shrimp Litopenaeus vannamei that might be involved in immune response against bacteria and virus

C-type lectins play crucial roles in innate immunity to recognize and eliminate pathogens efficiently. In the present study, two C-type lectins from shrimp Litopenaeus vannamei (designated as LvLectin-1 and LvLectin-2) were identified, and their expression patterns, both in tissues and toward pathogen stimulation, were then characterized. The full-length cDNA of LvLectin-1 and LvLectin-2 was 567 and 625 bp, containing an open reading frame (ORF) of 471 and 489 bp, respectively, and deduced amino acid sequences showed high similarity to other members of C-type lectin superfamily. Both two C-type lectins encoded a single carbohydrate-recognition domain (CRD). The motif of Ca²⁺ binding site 2 in CRD, which determined carbohydrate-binding specificity, was QPN (Gln¹²²-Pro¹²³-Asn¹²⁴) in LvLectin-1, but QPD (Gln¹²⁸-Pro¹²⁹-Asp¹³⁰) in LvLectin-2. Two C-type lectins exhibited similar tissue expression pattern, for their mRNA were both constitutively expressed in all tested tissues, including hepatopancreas, muscle, gill, hemocytes, gonad and heart, furthermore they were both mostly expressed in hepatopancreas, though the expression level of LvLectin-2 was much higher than LvLectin-1. The expression level of two C-type lectins mRNA in hemocytes varied greatly after the challenge of Listonella anguillarum or WSSV. After L. anguillarum challenge, the expression of both C-type lectins were significantly (P < 0.01) up-regulated compared with blank group, and LvLectin-1 exhibited higher level than LvLectin-2; while after the stimulation of WSSV, the expression of LvLectin-2 was significantly up-regulated at 6 h (P < 0.01) and 12 h (P < 0.05), but the expression level of LvLectin-1 down-regulated significantly (P < 0.01) to 0.4-fold at 6 and 12 h post-stimulation. The results indicated that the two C-type lectins might be involved in immune response toward pathogen infection, and they might perform different recognition specificity toward bacteria or virus.     

Effect of the addition of four potential probiotic strains on the survival of pacific white shrimp (Litopenaeus vannamei) following immersion challenge with Vibrio parahaemolyticus

Four bacterial strains isolated from the gastrointestinal tract of adult shrimp Litopenaeus vannamei, Vibrio alginolyticus UTM 102, Bacillus subtilis UTM 126, Roseobacter gallaeciensis SLV03, and Pseudomonas aestumarina SLV22, were evaluated for potential use as probiotics for shrimp. In vitro studies demonstrated antagonism against the shrimp-pathogenic bacterium, Vibrio parahaemolyticus PS-017. Feeding shrimp with diets containing the potential probiotics showed the best feed conversion ratio in comparison with the control groups. After feeding with the potential probiotics for 28 days, challenge by immersion indicated effectiveness at reducing disease caused by V. parahaemolyticus in shrimp.     

Immunostimulation of white shrimp (Litopenaeus vannamei) following dietary administration of Ergosan

Ergosan an algal product containing 1% alginic acid, developed for use in aquaculture and reported to have immunomodulatory activity, was administered orally to intermoult adult white shrimp (Litopenaeus vannamei) for 15 days. Examination of haemolymph proteins using SDS-PAGE did not reveal any obvious differences between control and Ergosan treated shrimp. Similarly, total haemocyte counts were found to be roughly equivalent for both the control and experimental samples. However, differential analysis of haemocyte populations revealed marked changes in terms of the relative levels of hyaline, semi-granular, and particularly granular haemocytes between the two groups. Moreover, enhancement of the in vitro antimicrobial activity of haemolymph towards two shrimp pathogenic Vibrio isolates was recorded for shrimp fed with Ergosan. Finally, shrimp fed with Ergosan showed a significant increase in relative growth when compared with control groups.     

Combined effect of temperature and salinity on the Thermotolerance and osmotic pressure of juvenile white shrimp litopenaeus vannamei (Boone)

Thermotolerance (CTMax) was determined in L. vannamei in three salinities and five acclimation temperatures 20, 23, 26, 29 and 32 °C. In white shrimp, the CTMax was not significantly affected by salinity (P>0.05). A direct relationship was obtained between CTMax and acclimation temperature. The end point of the CTMax in L. vannamei exposed to different combinations of temperature and salinity was defined as the loss of the righting response (LRR). The acclimation response ratio (ARR) for the juveniles of white shrimp ranged from 0.42 to 0.49; values in agreement with other crustaceans from tropical and sub tropical climates. The osmotic pressure of the hemolymph was measured in control organisms and in organisms exposed to CTMax; significant differences were found in organisms maintained in 10 and 40 psu, but there were no significant differences in hemolymph osmotic pressure in those that were acclimated to 26 psu.     

A model of Taura syndrome virus (TSV) epidemics in Litopenaeus vannamei

Taura syndrome virus (TSV) is a highly virulent pathogen of Litopenaeus vannamei, has affected shrimp aquaculture throughout the world, and threatens wild populations. Despite its importance, little work has been done on the pathogen's formal epidemiology. Therefore we developed a compartment model for epidemics of TSV in closed populations of L. vannamei. The model includes five compartments, uninfected susceptible, prepatently infected, acutely infected, chronically infected, and dead infected shrimp. The transmission coefficients, patency coefficient, virulence coefficients, and removal coefficient (disappearance of dead infected shrimp) control the dynamics of the model. We estimated the coefficients in laboratory studies and inserted the estimates in the model to characterize TSV epidemics and to estimate the basic reproduction ratio R(0) and threshold density for TSV epidemics in L. vannamei. Further we examined through computer simulation the effect of varying the coefficients on R(0). Decreases in transmission decrease R(0), decreases in virulence increase R(0), increases in patency do not affect R(0), and increases in recovery most likely increase R(0) but under some conditions might decrease it.     

Isolation,identification, and synthesis of a disulfated sulfakinin from the central nervous system of an arthropods the white shrimp Litopenaeus vannamei

Two myotropic peptides displaying tyrosyl sulfation have been isolated from an extract of central nervous systems (brain, suboesophageal ganglion, thoracic ganglia, and ventral nerve cord) of the white shrimp Litopenaeus vannamei. Both peptides were identified by mass spectrometry and belong to the sulfakinin family of neuropeptides, which are characterized by the C-terminal hexapeptide Y(SO(3)H)GHMRF-NH(2) preceded by two acidic amino acid residues. Pev-SK 1 (AGGSGGVGGEY(SO(3)H)DDY(SO(3)H)GH(L/I) RF-NH(2)) has two sulfated tyrosyl residues and a unique (L/I) for M substitution in the C-terminal sequence. Pev-SK 2 (pQFDEY(SO(3)H)GHMRF-NH(2)) fully complies with the typical sulfakinin core sequence and is blocked by a pyroglutamyl residue. Synthetic analogs (sulfated and unsulfated) were synthesized and the tyrosyl sulfations were confirmed by myotropic activity studies and co-elution with the native fractions. Pev-SK 1 is the first disulfated neuropeptide elucidated in the phylum of the arthropoda, with the only other reported disulfated neuropeptide, called cionin, found in a protochordate. The similarities in amino acid sequence and posttranslational modifications of the crustacean sulfakinins and protochordate cionin provide further evidence for the hypothesis stating that gastrin/CCK, cionin, and sulfakinins originate from a common ancestral gastrin/CCK-like peptide.     

Effect of copper sulfate on the immune response and susceptibility to Vibrio alginolyticus in the white shrimp Litopenaeus vannamei

White shrimp Litopenaeus vannamei (Boone) held in 35 per thousand seawater were challenged with Vibrio alginolyticus at a dose of 3 x 10(5) colony-forming units (cfu) shrimp(-1), and then placed in water containing concentrations of Cu2+ at 0 (control), 1, 5, 10 and 20 mg l(-1). Mortality of shrimp in 5, 10 and 20 mg l(-1) Cu2+ was significantly higher than those in 1 mg l(-1) Cu2+ and the control solution after 24-96 h. In another experiment, L. vannamei which had been exposed to control, 1, 5, 10 and 20 mg l(-1) Cu2+ for 24, 48 and 96 h were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity and clearance efficiency to V. alginolyticus. Copper concentrations at 1 mg l(-1) or greater for 24h resulted in decreased THC, phenoloxidase activity, phagocytic activity and clearance efficiency, whereas copper concentration at 20 mg l(-1) caused significant increase in respiratory burst of L. vannamei. In conclusion, concentration of Cu2+ at 1 mg l(-1) or greater increased the susceptibility of L. vannamei to V. alginolyticus infection by a depression in immune ability. The release of superoxide anion by L. vannamei exposed to 20 mg l(-1) Cu2+ was considered to be cytotoxic to the host.     

Role of invariant tyrosines in a crustacean mu-class glutathione S-transferase from shrimp Litopenaeus vannamei: site-directed mutagenesis of Y7 and Y116

Y6 and Y115 are key amino acids involved in enzyme-substrate interactions in mu-class glutathione S-transferase (GST). They provide electrophilic assistance and stabilize substrates through their hydroxyl groups. Two site-directed mutants (Y7F and Y116F) and the wild-type shrimp GSTs were expressed in Escherichia coli, and the steady-state kinetic parameters were determined using CDNB as the second substrate. The mutants were modeled based on a crystal structure of a mu-class GST to obtain further insights about the changes at the active site. The Y116F mutant had an increase in kcat contrary to Y7F compared to the wild type. Molecular modeling showed that the shrimp GST has a H108 residue that may contribute to compensate and lead to a less deleterious change when conserved tyrosine residues are mutated. This work indicates that shrimp GST is a useful model to understand the catalysis mechanisms in this critical enzyme.     

Characteristics of NADH-dependent lipid peroxidation in sarcoplasmic reticulum of white shrimp,Litopenaeus vannamei,and freshwater prawn,Macrobrachium rosenbergii

The iron-catalyzed NADH-dependent lipid peroxidation system in sarcoplasmic reticulum (SR) of cultured white shrimp, Litopenaeus vannamei and freshwater prawn, Macrobrachium rosenbergii was characterized. Production of thiobarbituric acid reactive substances was used to measure the activity of lipid peroxidation. In both species, the system preferred NADH to NADPH as the reducing agent. Lipid peroxidation activities of SR from both species increased when reaction temperatures increased from 6 to 26 °C. At 66 °C, the reaction was no longer NADH-dependent. Acidic pH amplified the lipid peroxidation activity. Sarcoplasmic reticular lipid peroxidation activity in white shrimp was always greater than in freshwater prawn. Fatty acid composition of SR lipids could be a major factor for this outcome. The proportion of n–3 highly unsaturated fatty acids, such as C20:5 and C22:6, in sarcoplasmic reticular lipids of white shrimp was twice of that in freshwater prawn. The results of this study provide important tools required for anti-oxidative nutrient study at sub-cellular level.     

The effect of different acclimation temperatures on the prophenoloxidase system and other defence parameters in Litopenaeus vannamei

Water temperature changes (higher and lower than 24 degrees C) were shown to have a significant effect on dopamine (DA) concentration, haemocyte count and the proPO system in the white shrimp Litopenaeus vannamei. No significant difference in any of the parameters was observed in the control group. DA concentration in haemolymph in the experimental groups increased to a peak value at 0.5 days; meanwhile serine protease (SP) activity and proteinase inhibitor (PI) activity decreased. Total haemocyte count (THC), differential haemocyte count (DHC) and PO activity were lowest at 1 day. All defence parameters became stable after 1-3 days, while the total haemocyte and large granular cell count stabilized after 6 days. After these stabilized, there was no significant difference in DA concentration and PI activity between the control and experimental groups, as was the case for the THC, DHC, PO and SP activities of shrimp held at higher temperatures. However these latter four parameters in the lower temperature groups were distinctly lower than the control group. alpha(2)-Macroglobulin activity in the experimental groups increased to a peak value after 1 day compared with the control and then stabilized after 6 days when the activity levels in higher temperature groups were higher than the control, while the lower temperature groups had no significant difference from the control. It was therefore concluded that water temperature changes modulated the immune system of L. vannamei.     

Immune responses of Litopenaeus vannamei to thermal stress: a comparative study of shrimp in freshwater and seawater conditions

The effects of hypothermal (22–16?°C) and hyperthermal (22–28?°C) stress on the immune system responses of the shrimp Litopenaeus vannamei cultured in either freshwater or seawater were measured and compared. The following immune system indicators were measured for comparison: total hemocyte count (THC), activity of phenoloxidase (PO), nitric oxide synthase (NOS), superoxidase (SOD), and malondialdehyde (MDA) content. Thermal stress significantly decreased THC in both freshwater and seawater shrimp within 6–12?h (P?0.05). After hypothermal stress, all shrimp had a significantly lower THC level than their prechallenge levels (P?0.05). Under both types of thermal stress, the activity of PO, NOS, and SOD first increased and then decreased. After 48?h of thermal stress, shrimp PO and NOS activity decreased in both freshwater and seawater. After 48?h of thermal stress, the reduction in the SOD activity in the hemolymph of freshwater shrimp was greater than that in seawater shrimp. During exposure to stress, the MDA content in freshwater shrimp was significantly higher than in seawater shrimp, which demonstrated that lipids in freshwater shrimp were more susceptible to peroxidation than those in seawater shrimp, particularly at low temperatures. Large temperature fluctuations, particularly sudden cooling, should be avoided when rearing L. vannamei because of high rates of lipid peroxidation and decreased immunity. These effects are more marked in freshwater than in seawater.     

The use of flow cytometry in the evaluation of cell viability of cryopreserved sperm of the marine shrimp (Litopenaeus vannamei)

Although the cryopreservation of penaeid prawn sperm or embryos has definite applications in the aquaculture industry, there is no protocol routinely used for this procedure. One of the main problems relies on the limitations for the determination of sperm cell viability. In this study, we evaluated the toxicity and cryoprotectant effect of four agents, at three different concentrations, in sperm suspension, spermatic mass, and complete spermatophore of the marine shrimp Litopenaeus vannamei. Cells were frozen by fast and slow cooling rates. After thawing, they were analyzed by optical microscopy and flow cytometry, which was also utilized to determine spermatic viability by DNA staining with propidium iodine. Considering viability by morphotype analysis, the best result was obtained when the spermatic mass was frozen by slow cooling rate in the presence of methanol (61.6%). There was a positive correlation between morphotype analysis and flow cytometry, although the percentage of viable cells was always lower when determined by the later. These results show that flow cytometry is a valuable tool to evaluate sperm cell viability in decapod species and it is more sensitive technique than optical microscopy.