Influence of dietary carbohydrate on the metabolism of juvenile Litopenaeus stylirostris

Cockles Cerastoderma edule were fed two different concentrations ( approximately 0.8 and 2 mm(3) l(-1)) of two diets with different qualities ( approximately 10 and 60% of organic content) which were achieved by mixing different proportions of ashed silt particles with cells of the microalgae Tetraselmis suecica. Clearance, ingestion and absorption rates of organic matter and biochemical components were measured after 3 days (acute response) and 11 days (acclimated response) of exposure to the diets. With low quality diets cockles were found to reject part of the filtered matter ( approximately 25-35%) through pseudofaeces production both in the acute and acclimated responses. In the acute response, absorption rate of organic matter was positively dependent on food quality and quantity, but the physiological response to increasing food concentrations differed with food quality: with low qualities, increasing absorption rate resulted from the simultaneous increase of clearance ( approximately 2 times) and ingestion rate ( approximately 4 times) as well as absorption efficiency of organic matter ( approximately 22%). However, those fed high qualities, were found to compensate increasing food concentration by reducing ( approximately 50%) clearance rate. The resulting moderate increase of ingestion rate ( approximately 1.6 times) was accompanied with a reduction in absorption efficiency ( approximately 20%). Irrespective of food quality and quantity, protein and lipids were absorbed, respectively, with the highest (from 61.7 to 80.0%) and the lowest (from 42.6 to 66.8%) efficiency. Acclimated response was entirely affected by food quality: with low qualities, cockles greatly improved the energetic intake from available ration ( approximately 4 and 2 times, with low and high food concentrations, respectively). Both preingestive and digestive mechanisms were involved in this response: at the preingestive level, clearance rate and preingestive selection efficiency were significantly increased. At the digestive level, cockles were capable of maintaining absorption efficiency of organic matter with rising ingestion rate. On the contrary, acclimation to high quality diets brought about no significant increase in organic absorption rate: with low ration, clearance rate was kept constant, whereas with high ration the increase in clearance and ingestion rate ( approximately 2 times) promoted a compensatory reduction in absorption efficiency. However, the biochemical composition of the absorbed matter was found to be absolutely modified, both at low and high food rations, due to an strong reduction of lipid absorption efficiency. The observed modifications of absorption rate and/or the biochemical composition of the absorbed matter suggests the capability of cockles to adjust the digestive performance.     

Protection of blue shrimp (Litopenaeus stylirostris) against the White Spot Syndrome Virus (WSSV) when injected with shrimp lysozyme

In this study we found that a blue shrimp (Litopenaeus stylirostris) lysozyme gene (Lslzm) was up-regulated in WSSV-infected shrimp, suggesting that lysozyme is involved in the innate response of shrimp to this virus. Shrimp were intramuscularly injected with Lslzm protein to identify how this recombinant protein protects L. stylirostris from WSSV infection and to determine how this protein influences nonspecific cellular and humoral defense mechanisms. Higher survival rates and a lower viral load (compared with controls) were reported for shrimps that were first injected with the Lslzm protein and then infected with WSSV. In addition, the Lslzm expression level and the immunological parameters (including THC, phagocytic activity, respiratory burst activity, phenoloxidase activity and lysozyme activity) were all significantly higher in the WSSV-infected shrimp treated with the Lslzm protein, compared with the controls. These results indicate that lysozyme is effective at blocking WSSV infection in L. stylirostris and that lysozyme modulates the cellular and humoral defense mechanisms after they are suppressed by the WSSV virus.     

Size-dependent haemagglutinating activity in the haemolymph from sub-adult blue shrimp (Penaeus stylirostris stimpson)

• 1.1. The blue shrimp (Penaeus stylirostris) haemolymph is capable of agglutinating the red blood cells of several vertebrates to different titres. However, the haemagglutinin is considered non-specific because it is incapable of differentiating erythrocytes of human blood types A, B and O.
• 2.2. Haemagglutinating activity and serum protein content were determined for male and female blue shrimp ranging in size from 8.5 to 16 cm. Haemagglutinating activity decreased significantly with animal size, while protein content was unaffected.
• 3.3. The above finding is probably related to maturation of the immune system and could explain the higher susceptibility of young shrimp to parasitic and viral diseases.

     

The effects of temperature and salinity on the swimming ability of whiteleg shrimp, Litopenaeus vannamei

Swimming endurance of whiteleg shrimp, Litopenaeus vannamei exposed to various temperatures (15, 20, and 25 degrees C) and salinities (15, 32, and 40 per thousand) was determined in a swimming channel against one of five flow velocities (5.41, 6.78, 8.21, 10.11, and 11.47 cm s(-1)) for up to 9000 s. No shrimp swam the full 9000 s throughout the experiment. The swimming endurance decreased as swimming speed was increased at any of the temperatures and salinities tested and was significantly affected by temperature and salinity (P<0.05). The power model (nu x t(b) = a) showed a better fit to the relationship between swimming endurance (t, in s) and swimming speed (nu, in cm s(-1)) at any of the temperatures and salinities tested. The swimming ability index (SAI), defined as SAI = integral(0)(9000) vdt x 10(-4) (cm), was found to be temperature- and salinity-dependent in L. vannamei. The optimum temperature and salinity and corresponding maximum SAI were Topt = 21.3 degrees C and SAI(max21.3) = 7.37 cm; Sopt = 27.6 per thousand and SAI(max27.6) = 7.47 cm, respectively. The range of temperatures and salinities within which SAI is >90% of the maximum was estimated between 17.6 and 24.9 degrees C and between 18.5 and 36.7 per thousand, respectively. The results suggest that the power model fits well to the observed endurance estimates and the SAI is a good index to quantitatively describe the overall swimming ability of L. vannamei. Furthermore, temperature and salinity can limit the swimming performance of L. vannamei.     

Possible toxic effects of the marine blue-green alga, Spirulina subsalsa, on the blue shrimp, Penaeus stylirostris.

Blooms of a marine species of blue-green algae identified as Spirulina subsalsa (Cyanophyta, Oscillatoriacae) were found to be related to a particular disease syndrome in raceway-reared blue shrimp, Penaeus stylirostris. The disease was characterized by necrosis of the lining epithelium of the midgut, dorsal cecum, and hindgut gland, and a consequent hemocytic enteritis. Bacterial infections due predominately to Vibrio alginolyticus were common in affected shrimp and presumed to be a secondary condition resulting from necrosis of the gut epithelium. These bacterial infections were expressed as local abscesses near or on the gut or as fulminating septicemias.     

Correlation between detection of a plasmid and high-level virulence of Vibrio nigripulchritudo, a pathogen of the shrimp Litopenaeus stylirostris

Vibrio nigripulchritudo, the etiological agent of Litopenaeus stylirostris summer syndrome, is responsible for mass mortalities of shrimp in New Caledonia. Epidemiological studies led to the suggestion that this disease is caused by an emergent group of pathogenic strains. Genomic subtractive hybridization was carried out between two isolates exhibiting low and high virulence. Our subtraction library was constituted of 521 specific fragments; 55 of these were detected in all virulent isolates from our collection (n = 32), and 13 were detected only in the isolates demonstrating the highest pathogenicity (n = 19), suggesting that they could be used as genetic markers for high virulence capacity. Interestingly, 10 of these markers are carried by a replicon of 11.2 kbp that contains sequences highly similar to those of a plasmid detected in Vibrio shilonii, a coral pathogen. The detection of this plasmid was correlated with the highest pathogenicity status of the isolates from our collection. The origin and consequence of this plasmid acquisition are discussed.     

Molecular epidemiology of Vibrio nigripulchritudo, a pathogen of cultured penaeid shrimp (Litopenaeus stylirostris) in New Caledonia

A collection of 57 isolates of Vibrio nigripulchritudo from either diseased or healthy shrimp and from shrimp farms environment was studied in order to gain a better understanding of the epidemiology of this pathogen, notably isolated from two distinct shrimp disease complexes. Molecular typing using two different techniques, arbitrarily primed PCR (AP-PCR) and multi-locus sequence typing (MLST), studied together with experimental pathology data allowed a relevant epidemiological insight into this possibly emerging pathogen. Additionally, results obtained with the two molecular typing techniques were congruent and allowed discriminating the strains associated with the "Summer Syndrome" from strains isolated from other contexts, especially the other shrimp vibriosis "Syndrome 93". These results highlight that the "Summer Syndrome" is most probably caused by an emergent clonal pathogen that therefore deserves surveillance and that AP-PCR can satisfactorily be used for that purpose.     

Modulation of the innate immune system in white shrimp Litopenaeus vannamei following long-term low salinity exposure

Immune parameters, haemocyte lifespan, and gene expressions of lipopolysaccharide and β-glucan-binding protein (LGBP), peroxinectin (PX), integrin β, and α2-macroglobulin (α2-M) were examined in white shrimp Litopenaeus vannamei juveniles (0.48 ± 0.05 g) which had been reared at different salinity levels of 2.5‰, 5‰, 15‰, 25‰, and 35‰ for 24 weeks. All shrimp survived during the first 6 weeks. The survival rate of shrimp reared at 2.5‰ and 5‰ was much lower (30%) than that of shrimp reared at 15‰, 25‰, and 35‰ (76%~86%) after 24 weeks. Shrimp reared at 25% grew faster. Shrimp reared at 2.5‰ and 5‰ showed lower hyaline cells (HCs), granular cells (GCs), phenoloxidase activity (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, and lysozyme activity, but showed a longer haemocyte lifespan, and higher expressions of LGBP, PX, integrin β, and α2-M. In another experiment, shrimp which had been reared at different salinity levels for 24 weeks were challenged with Vibrio alginolyticus (6 × 10(6) cfu shrimp(-1)), and WSSV (10(3) copies shrimp(-1)) and then released to their respective seawater. At 96-144 h, cumulative mortalities of shrimp reared at 2.5‰ and 5‰ were significantly higher than those of shrimp reared at 15‰, 25‰, and 35‰. It was concluded that following long-term exposure to 2.5‰ and 5‰ seawater, white shrimp juveniles exhibited decreased resistance against a pathogen due to reductions in immune parameters. Increases in the haemocyte lifespan and gene expressions of LGBP, integrin β, PX, and α2-M indicated that shrimp had the ability to expend extra energy to modulate the innate immune system to prevent further perturbations at low salinity levels.     

Grooming behavior and morphology of the caridean shrimp Pandalus danae Stimpson (Decapoda: Natantia: Pandalidae)

Grooming or cleaning is a frequent behaviorial activity of the shrimp Pandalus danae . Setal brushes on thoracic limbs scrape and rub other appendages and general surfaces of the exoskeleton. Chelate limbs nip and pick at edges of crevices in the cephalothoracic region. Scanning electron microscopy reveals that the setae composing the grooming brushes are equipped with characteristic tooth- and scale-like setules which serve as the actual rasping devices. Antennules, antennae and pereopods are frequently preened by the third maxillipeds and first pereopods, while large areas of the exoskeleton are cleaned by the third pair of walking legs and the chelate limbs.
Shrimp with the general cleaning limbs ablated develop significantly greater infestations of the epizoic suctorian Ephelota than animals allowed to groom. Olfactory hairs on the antennules of shrimp deprived of the third maxillipeds become fouled with diatoms and debris while those on controls do not. Grooming behavior clearly prevents a build-up of settling organisms between molts and repeated cleaning of sensory sites is essential in maintaining contact with the environment.     

RAPD markers as predictors of infectious hypodermal and hematopoietic necrosis virus (IHHNV) resistance in shrimp (Litopenaeus stylirostris).

Random amplified polymorphic DNA (RAPD) fingerprints of two shrimp populations (Litopenaeus stylirostris) were compared to find genetic marker(s) that may be associated with infectious hypodermal and hematopoietic necrosis virus (IHHNV) resistance or susceptibility. Of the 100 10-mer random primers and 100 intersimple-sequence repeat (ISSR) primers screened, five provided markers specific to the Super Shrimp population and three provided markers specific to the wild caught population. The two populations were further characterized for relative viral load (reported as cycle threshold, CT) using real-time quantitative PCR with primers specific to the IHHNV genome. The beta-actin gene was amplified to serve as a control for normalization of the IHHNV viral load. The mean viral load was significantly lower (C(T) = 34.58; equivalent to 3.3 x 10(1) copies of IHHNV genome/ng DNA) in Super Shrimp than in the wild caught population (CT = 23.49; equivalent to 4.2 x 10(4) copies/ng DNA; P < 0.001; CT values are inversely related to viral load). A preliminary prediction model was created with Classification and Regression Tree (CART) software (Salford Systems, San Diego, Calif.), where the resultant decision tree uses the presence or absence of seven RAPD markers as predictors of the relative viral load.     

Probiotics in the intestinal tract of juvenile whiteleg shrimp Litopenaeus vannamei: modulation of the bacterial community

Molecular analysis of the 16S rDNA of the intestinal microbiota of whiteleg shrimp Litopenaeus vannamei was examined to investigate the effect of a Bacillus mix (Bacillus endophyticus YC3-b, Bacillus endophyticus C2-2, Bacillus tequilensisYC5-2) and the commercial probiotic (Alibio^®) on intestinal bacterial communities and resistance to Vibrio infection. PCR and single strain conformation polymorphism (SSCP) analyses were then performed on DNA extracted directly from guts. Injection of shrimp with V. parahaemolyticus at 2.5 × 10⁵ CFU g^?1 per shrimp followed 168 h after inoculation with Bacillus mix or the Alibio probiotic or the positive control. Diversity analyses showed that the bacterial community resulting from the Bacillus mix had the highest diversity and evenness and the bacterial community of the control had the lowest diversity. The bacterial community treated with probiotics mainly consisted of α- and γ-proteobacteria, fusobacteria, sphingobacteria, and flavobacteria, while the control mainly consisted of α-proteobacteria and flavobacteria. Differences were grouped using principal component analyses of PCR-SSCP of the microbiota, according to the time of inoculation. In Vibrio parahaemolyticus-infected shrimp, the Bacillus mix (~33 %) induced a significant increase in survival compared to Alibio (~21 %) and the control (~9 %). We conclude that administration of the Bacillus mix induced modulation of the intestinal microbiota of L. vannamei and increased its resistance to V. parahaemolyticus.     

Effect of methyl parathion on the susceptibility of shrimp Litopenaeus vannamei to experimental vibriosis

Following increasing calls for environmental safety over the past 2 decades, persistent pesticides are being replaced by more rapidly degradable products. However, even these pesticides can affect non-target species, and may be associated with slow growth and increased susceptibility to viral and bacterial infections. In this study, juvenile white shrimp Litopenaeus vannamei (also named Penaeus vannamei) were challenged by intramuscular injection with Vibrio parahaemolyticus after 4 d prior exposure to methyl parathion in feed pellets at 0.080 microg g(-1). The bacterial injection control group consisted of shrimp fed pellets containing the methyl parathion-carrier solvent acetonitrile. Three additional control groups comprised 2 sterile saline-injection groups fed pellets containing methyl parathion or acetonitrile prior to injection, and 1 uninjected group fed normal pellets. Cumulative mortalities were recorded on the 4th and 8th days, and the presence of histological lesions was recorded on the 8th day. Cumulative mortalities were significantly higher in the group exposed to methyl parathion and bacteria on Day 8. Histological lesions, typical of vibriosis, were significantly associated with the injection of V. parahaemolyticus. The study provides strong experimental evidence that prior exposure to methyl parathion can increase the severity of Vibrio infections.     

Effect of probiotic Pediococcus acidilactici on antioxidant defences and oxidative stress of Litopenaeus stylirostris under Vibrio nigripulchritudo challenge

Antioxidant defences and induced oxidative stress tissue damage of the blue shrimp Litopenaeus stylirostris, under challenge with Vibrio nigripulchritudo, were investigated for a 72-h period. For this purpose, L. stylirostris were first infected by immersion with pathogenic V. nigripulchritudo strain SFn1 and then antioxidant defences: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx), Total antioxidant status (TAS), glutathiones and induced tissue damage (MDA and carbonyl proteins) were determined in the digestive gland at 0, 12, 24, 48 and 72 h post-infection (h.p.i.). In the meantime, TAS was also measured in the blood. Infection level of the shrimps during the challenge was followed by determining V. nigripulchritudo prevalence and load in the haemolymph of the shrimps. Changes in all these parameters during the 72-h.p.i. period were recorded for control shrimps and shrimps previously fed for one month with probiotic Pediococcus acidilactici MA18/5M at 10⁷ CFU g^?1 of feed.Our results showed that immersion with V. nigripulchritudo led to maximal infection level in the haemolymph at 24 h.p.i. preceding the mortality peak recorded at 48 h.p.i. Significant decreases in the antioxidant defences were detected from 24 h.p.i. and beyond that time infection leaded to increases in oxidative stress level and tissue damage. Compared to control group, shrimps fed the probiotic diet showed lower infection (20% instead of 45% at 24 h.p.i. in the control group) and mortality (25% instead of 41.7% in the control group) levels. Moreover, infected shrimp fed the probiotic compared to uninfected control shrimps exhibited very similar antioxidant status and oxidative stress level. Compared to the infected control group, shrimps fed the probiotic sustained higher antioxidant defences and lower oxidative stress level.This study shows that bacterial infection leads to oxidative stress in L. stylirostris and highlighted a beneficial effect of P. acidilactici, suggesting both a competitive exclusion effect leading to a reduction of the infection level and/or an enhancement of the antioxidant status of the shrimps.     

Effects of low salinity on juvenile pink shrimp Farfantepenaeus paulensis (Perez-Farfante 1967, Crustacea)

It is of paramount importance to study salinity tolerance of commercially important crustaceans, such as the pink shrimp Farfantepenaeus paulensis to determine possible mortality causes in the wild and in aquaculture in oligohaline waters. The aim of this study was to determine the lethal salinity concentration (LC50) for juvenile pink shrimp F. paulensis and measure its oxygen consumption and ammonia excretion at different salinity levels. Shrimp of two length classes (49.4?±?4.3 and 78.5?±?5.5?mm) were placed in 10-L containers and exposed to salinity levels of 35, 30, 25, 20, 15, 10 and 5. The experiments were tripled, with seven shrimp in each container. The average lethal concentration (LC50s) for an exposure of 24?h was 13.33 (11.26–15.78) and 10.26 (8.60–12.64), respectively, for the two classes of juveniles. For an exposure of 48?h, LC50s were 12.71 (10.68–15.12) for the larger animals and 9.20 (7.34–11.52) for the smaller ones. There was an inverse relationship between salinity and rates of oxygen consumption and ammonia excretion. The average reduction in specific oxygen consumption in salinities 20, 25 and 30 showed a decrease in metabolic rate of 63, 80 and 82%, respectively, in relation to salinity level 0. The same occurred for the averages of ammonia excretion at salinity levels of 15, 20, 25, 30 and 35, which represented low metabolic rates of 57, 61, 70, 71 and 74% respectably in relation to salinity level 0.     

Acute toxicity of carbon dioxide to juvenile marine shrimp Litopenaeus vannamei (Boone 1931)

Elevated concentrations of dissolved carbon dioxide (CO₂) and reduced pH levels are observed during the culture and transportation of aquatic organisms. Studies on the toxicity effects of CO₂ in penaeid shrimp are scarce when compared to the amount of research in fish. The objective of the present study was to determine the lethal concentration and safety levels of CO₂ for juvenile white shrimp Litopenaeus vannamei. Juveniles (1.76 ± 0.36 g) were exposed for 96 h to one of six concentrations of dissolved CO₂ (14.5, 23.8, 59.0, 88.0, 115.0, and 175.0 mg/L) or a control condition (without the addition of CO₂), and their survival was monitored for 96 h. The LC₅₀ values with 95% confidence limits at 24, 48, 72, and 96 h were 130.05 (104.2–162.1), 77.2 (73.8–80.02), 69.65 (65.47–74.32), and 59.12 (53.08–66.07) mg/L of CO₂, respectively. The calculated safety level was 5.9 mg/L of CO₂, and the highest concentration that did not induce significantly higher mortality than that observed in controls (NOEC) was 23.8 mg/L of CO₂. We recommend that CO₂ levels should be kept below the safety level obtained in this study.     

Histopathology of aflatoxicosis in the marine shrimp Penaeus stylirostris and P. vannamei

The acute and subacute toxicity of aflatoxin B₁ to the marine shrimp Penaeus stylirostris and P. vannamei (Order: Decapoda, Class: Crustacea) was investigated. Experimental shrimp were exposed to a range of concentrations of the toxin directly by intramascular injection (from 2 to 160 μg aflatoxin B₁/g body weight), or by multiple per os dosing with the feed (from 53 to 300 μg aflatoxin B₁/g feed) for up to 25 days. The histopathogenesis of aflatoxicosis in the aflatoxin-exposed animals was followed and found to be time and dose dependent in the hepatopancreas, mandibular organ, and in the hematopoietic organs. Less significant and/or inconsistent lesions were also observed in other organs and tissues, but a time-dose dependency was not noted. The principal lesions of aflatoxicosis in penaeid shrimp occur in the hepatopancreas and the mandibular organ. In the former organ, subacute and acute aflatoxicosis is expressed as necrosis of the hepatopancreatic tubule epithelium that proceeds from the proximal (older) portion of the tubules, in the center of the organ, to the peripheral (younger) tubule tips. A marked intertubular hemocytic inflammation followed by encapsulation and fibrosis of affected tubules follows in subacute aflatoxicosis, but is not as developed as in acute aflatoxicosis. The mandibular organ in aflatoxicosis displays a necrosis of the peripheral epithelial cells of the cords within the gland that progresses proximally to the central vein. Only a slight hemocytic inflammation accompanies the degenerative changes in this latter organ.