Effect of salinity and body weight on ecophysiological performance of the Pacific white shrimp (Litopenaeus vannamei)

Ecophysiological responses of Litopenaeus vannamei were evaluated as functions of environmental salinity and animal size. Growth rate, routine metabolic rate, limiting oxygen concentration, and marginal metabolic scope were determined for L. vannamei acclimated to, and tested at, salinities of 2, 10, and 28 ppt, all at 28 °C. Routine metabolic rate (RMR), estimated as oxygen-consumption rate per unit body weight for fasted, routinely-active shrimp, was independent of salinity but decreased with increasing shrimp weight. Limiting oxygen concentration for routine metabolism (LOCr) decreased with increased shrimp weight for the 10 and 28 ppt treatments, but not for the 2 ppt treatment. Marginal metabolic scope (MMS = RMR/LOCr) also decreased with increasing shrimp weight and was independent of salinity. Growth rate was significantly less at 2 ppt than at either 10 or 28 ppt, which gave similar growth rates.     

Purification and characterization of the clotting protein from the white shrimp Penaeus vannamei

The protein responsible for clot formation was isolated from plasma of the white shrimp Penaeus vannamei by affinity chromatography in a heparin–agarose column. The protein, named clotting protein (CP), was found to be a lipoglycoprotein, composed of two 210-kDa subunits covalently bound by disulfide bridges. CP formed large polymers when incubated with hemocyte lysate. Dansylcadaverine can be incorporated into CP by a hemocyte lysate or guinea pig transglutaminase mediated reaction. The amino acid composition and the amino terminal sequence were determined and compared with the clotting protein of the crayfish and the spiny lobster.     

Metabolic responses of the white shrimp, Penaeus vannamei, to ambient ammonia

Juvenile shrimp were individually exposed during 24 h to 0.007 (control), 0.36, 1.07, and 2.14 mmol/l total ammonia-N at 28 degrees C and 39 ppt salinity. After 22 h of ammonia-N exposure, oxygen consumption was measured for 2 h, and then hemolymph, hepatopancreas, and muscle tissues were sampled. Oxygen consumption, and levels of lactate and glycogen in the hepatopancreas increased significantly at the highest ammonia-N concentration. Concentration of oxyhemocyanin, acylglycerol, and cholesterol in hemolymph, and lactate in muscle decreased significantly in the group exposed to the highest ammonia levels. The changes observed in hemolymph and tissue metabolic fuels suggest a reduced use of carbohydrate through anaerobic metabolism and an increase in the use of lipids to satisfy the metabolic demand.     

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.     

Thermal preference,tolerance and oxygen consumption of adult white shrimp Litopenaeus vannamei (Boone) exposed to different acclimation temperatures

Final temperature preferendum of white shrimp adults were determined with acute and gravitation methods. The final preferendum was similar, independent of method (26.2–25.6 °C). A direct relationship was determined between the critical thermal maxima values and the acclimation temperatures (P<0.05). The end point of Critical Thermal Maxima (CTMax) for adults was defined as the loss of righting response (LRR). The acclimation response ratio (ARR) for adults of white shrimp had an interval of 0.36–0.76, values that agreed with others obtained for crustaceans from tropical and subtropical climates. The oxygen consumption rates increased significantly (P<0.05) from 39.6 up to 90.0 mg O₂ kg⁻¹ h⁻¹ wet weight (w.w.) as the acclimation temperature increased from 20 to 32 °C. The range of temperature coefficient (Q₁₀) of the white shrimp between 23 and 26 °C was the lower 1.60. The results obtained in this work are discussed in relation to the species importance in the reproductive scope and maintenance of breeders.     

Identification of a type of human IgG-like protein in shrimp Penaeus vannamei by mass spectrometry

Recently, infectious diseases have delayed the growth of shrimp aquaculture. Interest has been focused on immune molecules and defense mechanisms to reduce these diseases in shrimp aquaculture. In invertebrates, various immunoglobulin superfamily (IgSF) molecules have been characterized in body tissues and fluids, which play a significant role in innate defense. In the current study, we found that a protein in shrimp serum, referred as an IgG-like protein, could be reacted with goat anti-human IgG, specifically. The IgG-like protein was purified from the serum of shrimp Penaeus vannamei by affinity chromatography using CNBr-activated sepharose 4B. The purified protein was subsequently analysed using one-dimensional sodium sulphate-polyacrylamide gel electrophoresis (1-DE), two-dimensional sodium sulphate-polyacrylamide gel electrophoresis (2-DE) and immunoblotting. Furthermore, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry methods were used for peptide mass fingerprint (PMF) and sequencing of the protein, respectively. Sequence information and PMF queried against the NCBI database confirmed the identity of the protein as hemocyanin. Conserved domain search showed that there was an Ig-like conserved domain of 252 amino acid residues in the C-terminus of arthropoda hemocyanins. In addition, four and one conserved regions were found between hemocyanin and human Ig heavy chain and Ig kappa chain, respectively. These results indicate that in addition to copper-binding domains hemocyanin has an Ig-like conserved domain, which would confer some new functions to multifunctional respiratory pigment of crustaceans.     

Penaeus monodon chitin-binding protein (PmCBP) is involved in white spot syndrome virus (WSSV) infection

White spot syndrome virus (WSSV) can cause the most serious viral disease of shrimp and has a wide host range among crustaceans. Although researches show a lot about its genome and structure, information concerning the mechanism of how WSSV infects' cells is lacking. In this study, some experiments were applied to confirm the biological meaning of the protein–protein interaction between WSSV envelope protein, VP53A, and Penaeus monodon chitin-binding protein (PmCBP). Immunofluorescent study indicated that PmCBP is located on the cell surface of host cells. PmCBP amounts of about 34 kDa can be detected in both P. monodon and Litopenaeus vannamei tissues by Western blotting. In the in vivo neutralization experiment, both rVP53A and rPmCBP that were produced by Esherichia coli can promote resp. a 40% and 20% survival rate of the shrimp which were challenged by WSSV. Furthermore, a yeast-two-hybrid result revealed that PmCBP could interact with at least 11 WSSV envelope proteins. Those findings suggest that PmCBP may be involved in WSSV infection.     

Whiteleg shrimp (Litopenaeus vannamei, Boone, 1931) isotrypsins: Their genotype and modulation

Based on information about isotrypsins found by substrate-SDS-PAGE in the whiteleg shrimp Litopenaeus vannamei and their segregation, an explanation for the presence of three-trypsin phenotype was sought. Isotrypsins A, B, and C depend on two loci: locus β, which is homozygous, yielding isoenzyme C, and locus α, which is heterozygous, yielding isoenzymes A and B. Segregation in locus α in offsprings from 20 families was analyzed. Results demonstrated that isotrypsins were segregated according to Mendelian rules, and hence, external stimuli did not affect the phenotype. To corroborate this, the phenotype was evaluated in relation to changes in trypsin activity during a digestion process. Trypsin phenotypes found in shrimp feces were also analyzed. Results showed that phenotypes were conserved by individuals. Therefore, changes in trypsin activity found in previous investigations must be related to changes in the concentration of the established isotrypsins.     

Cleavage and gastrulation in the shrimp Penaeus (Litopenaeus) vannamei (Malacostraca, Decapoda, Dendrobranchiata)

While most malacostracan crustaceans develop through superficial cleavage, in the Amphipoda, Euphausiacea, and Dendrobranchiata (Decapoda) cleavage is complete. Euphausiaceans and dendrobranchiate shrimp share a similar early cleavage pattern, early cleavage arrest and ingression of mesendoderm progenitor cells, a ring of crown cells (prospective naupliar mesoderm) around the blastopore, and hatching as a nauplius larva. Yet recent phylogenies do not support a close relationship between Euphausiacea and Decapoda. In addition, some variation is reported in the timing of mesendoderm cell arrest and number of crown cells for a number of dendrobranchiates. To determine the representative pattern of development in the Dendrobranchiata, embryos of the Pacific white shrimp Penaeus (Litopenaeus) vannamei were stained with Sytox Green to label chromosomes and nuclei and examined with confocal microscopy. The early cleavage pattern, mesendoblast arrest and subsequent ingression at the 32-cell stage, presence of 8 initial crown cells, and fates of the mesendoblasts are the same for P. vannamei (family Peneaeidae) and Sicyonia ingentis (family Sicyoniidae). The lineage of the primordial endoderm cells differs from that reported for P. kerathurus. These characters were discussed in the context of the evolution of development in the Dendrobranchiata and in comparison to the Euphausiacea.     

Metabolic response to feeding in the Chinese striped-necked turtle, Ocadia sinensis

We measured oxygen consumption in juvenile Chinese striped-necked turtles (Ocadia sinensis) after they ingested food, either as a single meal or as double meals, to examine the influence of meal type and feeding frequency on specific dynamic action (SDA). Temporal variation in oxygen consumption after feeding was evident in the ingesting turtles but not in the unfed control turtles. In the single-meal experiment, the peak metabolic rate and the integrated SDA response (the whole energetic cost for the processes of digestion) both did not differ between turtles ingesting mealworms and shrimps when the influence of variation in ingested energy was removed, and the time to reach peak metabolic rate was not affected by meal type and the amount of food ingested. Turtles in the double-meal experiment ingested more energy and hence had a prolonged duration of SDA response than did those in the single-meal experiment, but the integrated SDA response did not differ between both experimental treatments when the influence of variation in ingested energy was removed. Our results show that meal type and feeding frequency have important consequences on the SDA response of juvenile O. sinensis. As the integrated SDA response remained remarkably constant either between turtles ingesting different food or between turtles ingesting the same food but at different frequencies when the influence of variation in ingested energy was removed, we therefore conclude that the energetic cost associated with ingestion is primarily determined by energy content of food ingested in juvenile O. sinensis.     

An energetic and conceptual model of the physiological role of dietary carbohydrates and salinity on Litopenaeus vannamei juveniles

We are reporting results directed to explain the relation between carbohydrates (CHO), protein metabolism, and the energetic balance of Litopenaeus vannamei juveniles. The interaction of dietary CHO and salinity was measured to try to understand the relation between osmotic control and metabolism, both from a biochemical and energetic point of view. Two experiments were done. In the first experiment, shrimp were fed with 0%, 5%, 33%, and 61% CHO and maintained at 15‰ and 40‰ salinity. Glucose, lactate protein, hemocyanin, ammonia concentration, and osmotic pressure were measured in blood. Digestive gland glycogen (DGG) was measured also. In the second experiment, shrimp were fed with 0% and 38% dietary CHO and maintained at 15‰ and 40‰ salinity. From that shrimp, absorbed energy (Abs) was calculated as: Abs=respiration (R)+ammonia excretion (U) and production (P); assimilated energy (As) was calculated as the product of R×P. Osmotic pressure, hemocyanin, protein, lactate, and blood ammonia increased with the reduction in dietary CHO. In contrast, an increase in blood glucose was observed with an increase in dietary CHO. Digestive gland glycogen (DGG) increased following a saturation curve with a DGG maximum at 33% dietary CHO. Blood metabolites of fasting and feeding shrimp showed the same behavior. Energy balance results showed that shrimp maintained in low salinity and fed without CHO waste more energy in U production than for shrimp maintained in high salinity and fed with high CHO levels. Notwithstanding, the production efficiency was higher in shrimp fed without CHO than that observed in shrimp fed with high CHO independent of salinity. A scheme trying to integrate the relation between CHO and protein metabolism and the way in which both are modulated by salinity is presented. From published and present results, there are two factors that apparently control the use of high dietary CHO levels; α-amylase enzyme-dietary CHO level capacity and glycogen saturation in DG. Production of glucose is limited in shrimp because of saturation of α-amylase when shrimp are fed with diets above 33% CHO. This is the first control point of starch metabolism. The digestive gland is saturated with glycogen in shrimp fed with dietary CHO levels >33%. This is apparently the second control point of CHO metabolism that limits growth rate in such conditions. The high metabolic cost related to high CHO diets could explain why shrimp are well adapted to use protein as a source of energy.     

Metabolic depression during aestivation in Cyclorana alboguttata

The green striped burrowing frog, Cyclorana alboguttata, spends, on average, nine to ten months of every year in aestivation. Recently, C. alboguttata has been the focus of much investigation regarding the physiological processes involved in aestivation, yet our understanding of this frog's capacity to metabolically depress remains limited. This study aimed to extend our current knowledge of metabolic depression during aestivation in C. alboguttata. C. alboguttata reduced whole animal metabolism by 82% within 5 weeks of aestivation. The effects of aestivation on mass specific in vitro tissue metabolic rate (VO₂) varied among individual organs, with muscle and liver slices showing significant reductions in metabolism; kidney VO₂ was elevated and there was no change in the VO₂ of small intestine tissue slices. Organ size was also affected by aestivation, with significant reductions in the mass of all tissues, except the gastrocnemius. These reductions in organ size, combined with changes in mass specific VO₂ of tissue slices, resulted in further energy savings to aestivating animals. This study shows that C. alboguttata is capable of selectively down- or up-regulating individual tissues, using both changes in metabolic rate and morphology. This strategy allows maximal energy savings during aestivation without compromising organ functionality and survival at arousal.     

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.     

High Frequency and Large Number of Polymorphic Microsatellites in Cultured Shrimp, <Emphasis Type="Italic">Penaeus (Litopenaeus) vannamei</Emphasis> [Crustacea:Decapoda]

A total of 1479 recombinant clones were obtained from a Sau3A-digested genomic library of Penaeus (Litopenaeus) vannamei and used for probe hybridization. Of the 251 clones that tested positive to one or more of the probes and were sequenced, 173 (69%) contained 573 simple sequence repeats, or microsatellites, with 3 or more repeats. The frequency of microsatellites with 3, 5, and 10 or more repeats was 1 in 0.94 kb, 1 in 2.78 kb, and 1 in 5.94 kb, respectively. To increase the number of polymorphic markers for mapping, 136 primer sets that flanked microsatellites containing single or multiple motifs with 3 or more repeats were designed and tested. Of the 136 primers, 93 (68.0%) were polymorphic in cultured shrimp, with polymorphism information content (PIC) values ranging from 0.195 to 0.873, and observed heterozygosities ranging from 10% to 100%. These markers are being used along with other markers to construct a linkage map for P. vannamei.     

Influence of highly unsaturated fatty acids on the responses of white shrimp (Litopenaeus vannamei) postlarvae to low salinity

Salinity stress tests are commonly applied in shrimp hatcheries to estimate the quality of postlarvae (PL) to be used during growout. Higher larval survival during culture and to a salinity stress test in both fish and crustaceans have been reported when specimens were offered a diet containing high levels of highly unsaturated fatty acids (HUFA). However, it is not clear if increased survival is a result of better overall physiological condition resulting from the diet or a specific effect of HUFA on osmoregulatory mechanisms. This study analyzed if HUFA-rich diets could modify the fatty acid composition of membranes in gills, and if this change in composition could affect the activity of the Na⁺/K⁺ ATPase pump and carbonic anhydrase in relation to changes in salinity. One-day-old postlarvae (PL1) pooled from different spawns were fed for 20 days with Artemia sp. nauplii enriched with three levels of HUFA: low, medium and high. At PL20, survivals during culture and to salinity stress test (tap water for 30 min) were evaluated. Also at this stage, Na⁺/K⁺-ATPase and carbonic anhydrase activity, morphometric variables, and fatty acid composition in the hepatopancreas and gills were measured after they were submitted to a salinity challenge in dilute seawater (10 ppt) for 3 h. No significant differences were observed in survival rates during culture, but survival to a salinity stress test was higher and gill area was larger in PL20 fed the Artemia sp. nauplii enriched with medium HUFA levels, probably as a result of an increased 22:6n-3 content and higher 22:6n-3/20:5n-3 ratio in this diet and in the tissues of the organisms fed this diet. Na⁺/K⁺-ATPase specific activity was significantly higher in posterior gills, while the specific activity of the carbonic anhydrase was higher in anterior gills. Enzymatic activities increased significantly in PL20 submitted to a salinity challenge, and HUFA levels in the diet affected both. The proportion of fatty acids in hepatopancreas and gills were significantly affected not only by diet, but also by exposure to dilute media. This effect is discussed in relation to an increase in gill surface and changes in fatty acid composition in the phospholipids present in gill membranes, which can modify the permeability and the activity of the Na⁺/K⁺-ATPase pump. The beneficial effect of HUFA supplementation in the diet on survival to salinity stress test is partially related to modification of fatty acid composition of gills and to a larger gill area, which in turn enhances osmoregulatory mechanisms, namely Na⁺/K⁺-ATPase and carbonic anhydrase activities.