Nitrogen Budget in Recirculating Aquaculture and Water Exchange Systems for Culturing <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

In order to investigate the culture characteristics of two indoor intensive Litopenaeus vannamei farming modes, recirculating aquaculture system (RAS) and water exchange system (WES), this study was carried out to analyze the water quality and nitrogen budget including various forms of nitrogen, microorganism and chlorophyll-a. Nitrogen budget was calculated based on feed input, shrimp harvest, water quality and renewal rate, and collection of bottom mud. Input nitrogen retained in shrimp was 23.58% and 19.10% respectively for WES and RAS, and most of nitrogen waste retained in water and bottom mud. In addition, most of nitrogen in the water of WES was TAN (21.32%) and nitrite (15.30%), while in RAS was nitrate (25.97%), which means that more than 76% of ammonia and nitrite were removed. The effect of microalgae in RAS and WES was negligible. However, bacteria played a great role in the culture system considering the highest cultivable cultivable bacterial populations in RAS and WES were 1.03×10¹⁰ cfu mL^?1 and 2.92×10⁹ cfu mL^?1, respectively. Meanwhile the proportion of bacteria in nitrogen budget was 29.61% and 24.61% in RAS and WES, respectively. RAS and WES could realize shrimp high stocking culture with water consuming rate of 1.25 m³ per kg shrimp and 3.89 m³ per kg shrimp, and power consuming rates of 3.60 kw h per kg shrimp and 2.51 kw h per kg shrimp, respectively. This study revealed the aquatic environment and nitrogen budget of intensive shrimp farming in detail, which provided the scientific basis for improving the industrial shrimp farming.     

The effect of three culture methods on intensive culture system of pacific white shrimp (Litopenaeus vannamei)

Different culture methods may affect the intensive culture system of Pacific white shrimp(Litopenaeus vannamei) regarding water quality and growth and economic performance.This study evaluated the potential effects of three culture methods through cultivation of juvenile shrimps under consistent tank management conditions for 84 d.The three methods involved shrimp cultivation in different tanks,i.e.,outdoor tanks with cement bottom(mode-C),greenhouse tanks with cement bottom(mode-G) and outdoor tanks with mud-substrate(mode-M).Results showed that water temperature was significantly higher in mode-G than that in mode-C(P < 0.05).In contrast to the other two treatments,mode-M had stable pH after 50 d cultivation of shrimps.In the mid-late period,the average concentrations of TAN,NO2-N,DIP and COD were significantly lower in mode-M and mode-G compared with those in mode-C(P < 0.05).Despite lack of differences in the final shrimp weight among different treatments(P > 0.05),mode-M had significantly higher shrimp yield,survival rate and feed conversion rate(P < 0.05) than other modes.There were significant differences in revenue and net return among different treatments(P < 0.05).These demonstrated that the treatments of mode-G and mode-M were conductive to the intensive culture system of L.vannamei.     

A comparative study of intensive Litopenaeus vannamei culture on four bottom substrates without water change

The effect of four bottom substrates, oyster shell powder (OP), sugarcane bagasse (SB), a mixture of OP and SB (OS) and fresh soil (FS), on the water quality and bacterial and zooplankton density of intensive shrimp (Litopenaeus vannamei) culture tanks without water change and the growth performance of cultured shrimp were compared in this study. At the end of a 110 days culturing trial, the total ammonium-N (TAN) of the water on SB and the nitrite nitrogen (NO₂-N) on OS was significantly lower than that on the other substrates (P<0.05), which coincided with the high density of ammonium- and nitrite-oxidizing bacteria in the water on SB and OS, respectively. The concentration of chlorophyll a (Chl a) increased slowly on OP, SB and OS but remained low on FS. The density of total bacteria on OP, SB and OS was one order of magnitude higher than that on FS, and the density of zooplankton on SB and OS was significantly higher than that on FS or OP (P<0.05). The improved water quality and increased density of bacteria and zooplankton on SB and OS may have had a synergistic effect on shrimp culture, improving its growth performance (high survival rate and yield and low feed conversion rate). SB and OS were more effective for improving the growth performance of intensively cultured L. vannamei without water change than OP and FS. To our knowledge, this study presents the first evidence regarding the effect of different bottom substrates on intensive shrimp culture.     

Impact of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and white spot syndrome virus (WSSV) co-infection on survival of penaeid shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

White spot syndrome virus (WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect Litopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus (WSSV+Vp), or we first infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection (Vp+WSSV). The eff ect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of Vibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction (PCR) method. LvMyD88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the diff erent mortality rates. Our results show that (1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection; (2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed significant increases in the numbers of Vibrio (P<0.05); (3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10⁵ to 10⁷ /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.     

Distribution and elimination of Norfloxacin in Fenneropenaeus chinensis larvae

This study examined the distribution and elimination of Norfloxacin(NFLX) in Fenneropenaeus chinensis ovary and egg and newly hatched larvae.Mature parental shrimp were exposed to 4 or 10 mg L 1NFLX for 2 or 5 d.Ovary and eggs of the shrimp were sampled after spawning in order to detect NFLX residue using high-performance liquid chromatography(HPLC).Results showed that NFLX residue accumulated in F.chinensis eggs after the parental exposure,with the highest residue detected in ovary.To examine the fate of NFLX residue in larvae,we further determined the concentration of NFLX residue in F.chinensis eggs and larvae at 4 different developmental stages after 24-h exposure.From the newly metamorphosed larvae(0 h post-metamorphosis,h.p.m),samples were taken at different time intervals to 72 h.p.m.HPLC assay showed that the concentrations of NFLX residue in zoea exposed to 4 and 10 mg L 1NFLX were the highest at 1.5 h,i.e.,0.332 and 0.454 μg g 1,respectively.At the two NFLX exposure levels,the elimination time of half NFLX(half life) in nauplius was 45.36 and 49.85 h,respectively,followed by that in zoea(31.68 and 33.13 h),mysis larvae(42.24 and 47.28 h) and postlarvae(24.48 and 30.96 h).Both NFLX exposure levels had a germicidal effect.The distribution and elimination of NFLX residue in F.chinensis tissue,eggs and larvae correlated well with the drug exposure level.The disappearance of NFLX residue coincided with the larval growth,and the half-life of NFLX decreased with the larval development.     

Effect of dissolved oxygen on swimming ability and physiological response to swimming fatigue of whiteleg shrimp (Litopenaeus vannamei)

The swimming endurance of whiteleg shrimp(Litopenaeus vannamei, 87.66 mm ± 0.25 mm, 7.73 g ± 0.06 g) was examined at various concentrations of dissolved oxygen(DO, 1.9, 3.8, 6.8 and 13.6 mg L-1) in a swimming channel against one of the five flow velocities(v1, v2, v3, v4 and v5). Metabolite contents in the plasma, hepatopancreas and pleopods muscle of the shrimp were quantified before and after swimming fatigue. The results revealed that the swimming speed and DO concentration were significant factors that affected the swimming endurance of L. vannamei. The relationship between swimming endurance and swimming speed at various DO concentrations can be described by the power model(ν·tb = a). The relationship between DO concentration(mg L-1) and the swimming ability index(SA∫ 9000I), defined as SAI =vdt( cm), can be described as SAI = 27.947 DO0.137(R2 = 0.9312). The 0level of DO concentration directly affected the physiology of shrimp, and exposure to low concentrations of DO led to the increases in lactate and energetic substrate content in the shrimp. In responding to the low DO concentration at 1.9 mg L-1 and the swimming stress, L. vannamei exhibited a mix of aerobic and anaerobic metabolism to satisfy the energetic demand, mainly characterized by the utilization of total protein and glycogen and the production of lactate and glucose. Fatigue from swimming led to severe loss of plasma triglyceride at v1, v2, and v3 with 1.9 mg L-1 DO, and at v-11 with 3.8, 6.8 and 13.6 mg L DO, whereas the plasma glucose content increased significantly at v3, v4 and v5 with 3.8 and 6.8 mg L-1 DO, and at v5 with 13.6 mg L-1 DO. The plasma total protein and hepatopancreas glycogen were highly depleted in shrimp by swimming fatigue at various DO concentrations, whereas the plasma lactate accumulated at high levels after swimming fatigue at different velocities. These results were of particular value to understanding the locomotory ability of whiteleg shrimp and its physiological changes, further contributing to the improvement of capture and rearing technique.     

EFFECTS OF LOW MOLECULAR WEIGHT CHITOSAN (LMC-1) ON SHRIMP PRESERVATION

This study on the effects of low molecular weight chitosan (LMC- 1) and shrimp preserving agentssuch as phytic acid (PA), sodium bisulfite (SB), and crustacean preservative (CP) on the preservation ofshrimp (Trachypenaeus curvirostris) and the bacteriostasis of LMC-1 showed that: (1) Different LMC-1conontration has different bacteriostasis on E. coIi, B. subtilis and S. aureou: (2) LMC-1 and CP arebetter than PA and SB for preserving the freshness of shrimp stored at 4℃.     

Influence of white spot syndrome virus infection on hepatopancreas gene expression of ‘Huanghai No. 2’ shrimp (<Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis>)

To elucidate the molecular response of shrimp hepatopancreas to white spot syndrome virus (WSSV) infection, microarray was applied to investigate the differentially expressed genes in the hepatopancreas of ‘Huanghai No. 2’ (Fenneropenaeus chinensis). A total of 59137 unigenes were designed onto a custom-made 60K Agilent chip. After infection, the gene expression profiles in the hepatopancreas of the shrimp with a lower viral load at early (48–96 h), peak (168–192 h) and late (264–288 h) infection phases were analyzed. Of 18704 differentially expressed genes, 6412 were annotated. In total, 5453 differentially expressed genes (1916 annotated) expressed at all three phases, and most of the annotated were either up- or down-regulated continuously. These genes function diversely in, for example, immune response, cytoskeletal system, signal transduction, stress resistance, protein synthesis and processing, metabolism among others. Some of the immune-related genes, including antilipopolysaccharide factor, Kazal-type proteinase inhibitor, C-type lectin and serine protease encoding genes, were up-regulated after WSSV infection. These genes have been reported to be involved in the anti-WSSV responses. The expression of genes related to the cytoskeletal system, including β-actin and myosin but without tubulin genes, were down-regulated after WSSV infection. Astakine was found for the first time in the WSSV-infected F. chinensis. To further confirm the expression of differentially expressed genes, quantitative real-time PCR was performed to test the expression of eight randomly selected genes and verified the reliability and accuracy of the microarray expression analysis. The data will provide valuable information to understanding the immune mechanism of shrimp’s response to WSSV.     

Water wave solutions obtained by variational method

Abstract Variational problem for irrotational, incompressible inviscid fluid in finite water depth is considered. Based on the variational principle, a special solution of the problem is presented under the assumption that the dispersion /u and the nonlinearity ?satisfied e = O(fj2) as the Lagrange function is expanded up to O(//). It is shown that the elevation of the free surface should be expanded to // order to ensure the Lagrange function is in fj* order. Comparison the nonlinear free surface profiles obtained from the solution with the corresponding ones obtained from linear solutions showed that the wave crest of the nonlinear wave is steepened but the trough is flattened compared to the linear wave as expected.     

Purification and identification of a clotting protein from the hemolymph of Chinese shrimp (Fenneropenaeus chinensis)

The clotting protein (CP) plays important and diverse roles in crustaceans, such as coagulation and lipid transportation. A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis (named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography. Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca²⁺, suggesting that the clotting reaction is catalyzed by a Ca²⁺-dependent transglutaminase in shrimp hemocytes. The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE. CP exists as disulfide-linked homodimers and oligomers. The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon, Farfantepenaeus paulensis and Litopenaeus vannamei; and similar to that of other decapods. The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.     

Effects of dietary β-glucan and glycyrrhizin on non-specific immunity and disease resistance of the sea cucumber (<Emphasis Type="Italic">Apostichopus japonicus</Emphasis> Selenka) challenged with <Emphasis Type="Italic">Vibrio splendidus</Emphasis>

Sea cucumbers, Apostichopus japonicus Selenka, were fed diets containing non-immunostimulant (basal diet), 0.2% β-glucan and 0.02% glycyrrhizin in a recirculatory water system for 45 days, and subsequently challenged with Vibrio splendidus by injection at 1.0×10⁸ cfu / sea cucumber for 15 days. Phagocytic capacity (PC), intracellular superoxide anion production (ISAP), lysozyme (LSZ) activity and superoxide dismutase (SOD) activity in the coelomic fluid were analyzed on the 0^th, 5^th, 10^th and 15^th days after injection. Results showed that after the 45-day feeding period, PC, ISAP, LSZ activity and SOD activity in sea cucumbers fed with dietary β-glucan or glycyrrhizin were significantly higher than in those fed with the basal diet. On the 5th day after infection, all the immune parameters examined in the sea cucumbers injected with V. splendidus decreased in value significantly. On the 15^th day, PC, ISAP and LSZ activity returned to levels similar to those on the 0^th day. For the sea cucumbers injected with saline, there were no significant differences in all the immune parameters examined and in the cumulative morbidity during the 15-day challenging trial. After injecting with V. splendidus, the cumulative morbidity of sea cucumbers fed with the basal diet was significantly higher than those fed with dietary β-glucan or glycyrrhizin when challenged with V. splendidus challenged sea cucumber fed with the basal diet was significantly higher than those fed with dietary β-glucan or glycyrrhizin. There was no significant difference in cumulative morbidity between the dietary β-glucan and glycyrrhizin treatments over time.     

Glutamate dehydrogenase and Na+-K+ ATPase expression and growth response of Litopenaeus vannamei to different salinities and dietary protein levels

Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture.The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp(L.vannamei) were investigated.This involved an examination of growth performance,glutamate dehydrogenase(GDH) and Na+-K+ ATPase mRNA expression,,and GDH activity in muscles and gills.Three experimental diets were formulated,containing 25%,40%,and 50% dietary protein,and fed to the shrimp at a salinity of 25.After 20 days,no significant difference was observed in weight gain,though GDH and Na+-K+ ATPase gene expression and GDH activity increased with higher dietary protein levels.Subsequently,shrimp fed diets with 25% and 50% dietary protein were transferred into tanks with salinities of 38 and 5,respectively,and sampled at weeks 1 and 2.Shrimp fed with 40% protein at 25 in salinity(optimal conditions) were used as a control.Regardless of the salinities,shrimp fed with 50% dietary protein had significantly higher growth performance than other diets;no significant differences were found in comparison with the control.Shrimp fed with 25% dietary protein and maintained at salinities of 38 and 5 had significantly lower weight gain values after 2 weeks.Ambient salinity change also stimulated the hepatosomatic index,which increased in the first week and then recovered to a relatively normal level,as in the control,after 2 weeks.These findings indicate that in white shrimp,the specific protein nutrient and energy demands related to ambient salinity change are associated with protein metabolism.Increased dietary protein level could improve the osmoregulation capacity of L.vannamei with more energy resources allocated to GDH activity and expression.     

Trophic Interaction in a <Emphasis Type="Italic">Portunus rituberculatus</Emphasis> Polyculture Ecosystem Based on Carbon and Nitrogen Stable Isotope Analysis

Trophic interaction among various biomass groups in a swimming crab Portunus trituberculatus polyculture pond was investigated using carbon and nitrogen stable isotope analysis. The polycultured animal species also included white shrimp Litopenaeus vannamei, short-necked clam Ruditapes philippinarum, and redlip mullet Liza haematochila. The mean δ¹³C value for all the biomass groups in polyculture ecosystem ranged from ?25.61‰ to ?16.60‰, and the mean δ¹⁵N value ranged from 6.80‰ to 13.09‰. Significant difference in the δ¹³C value was found between particulate organic matter (POM) and sediment organic matter (SOM) (P < 0.05), indicating that these two organic matter pools have different material sources. Assuming that a ¹³C-enrichment factor of 1.00‰ and a ¹⁵N-enrichment factor of 2.70‰ existed between consumer and prey, diets of the four cultured animals were estimated using a stable isotope mixing model. The estimated model results indicated that P. trituberculatus mainly feed on Aloidis laevis; L. vannamei mainly feed on shrimp feed; while A. laevis, R. philippinarum and L. haematochelia mainly feed on POM. Shrimp feed was also an important food source of R. philippinarum and L. haematochelia. The diets of P. trituberculatus, L. vannamei, R. philippinarum, and L. haematochila showed complementary effects in this polyculture ecosystem. Our finding indicated that the polyculture of these four organisms with suitable farming density could make an effective use of most of the food sources, which can make a highly efficient polyculture ecosystem.