Responses of prophenoloxidase system and related defence parameters of <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis> to low salinity

In this study, we investigated the effects of low salinity(26 and 21)on the prophenoloxidase(proPO)system and related defence parameters in the shrimp Litopenaeus vannamei. The results showed that low salinity induced a significant increase of dopamine(DA)concentration in haemolymph at 6h of the experiment; on the other hand, total haemocyte count(THC), differential haemocyte count(DHC)and PO activity decreased over time to the lowest level at 24 h and remained low thereafter. Serine Protease(SP)and Proteinase Inhibitor(PI)activity in the two lower salinity treatments decreased to the lowest level at 12 and 24h, respectively, and both recovered to the control level at 72 h. In contrast, α2-macroglobulin(α2M)activity in the two lower salinity treatments peaked at 24h and then decreased to the control level at 72h. Therefore, it may be concluded that stress-induced DA plays an important temporary role in neurotransmission and causes immune response in L. Vannamei in adapting to salinity changes.     

Effects of hypoxia on dopamine concentration and the immune response of White Shrimp (Litopenaeus vannamei)

Effects of hypoxia on the dopamine concentration and the immune response of White Shrimp Litopenaeus vannamei were studied. The results showed that hypoxia had significant effects on the concentration of dopamine (DA) in the haemolymph, haemocyte count, phenoloxidase activity, phagocytic activity of haemocytes and bacteriolytic and antibacterial activity in the haemolymph (P<0.05). The concentration of the dopamine in haemolymph reached its maximum in the 3.0 and 1.5 mg L⁻¹ DO groups at 12 h and 6 h, and then returned to normal after 24 h and 12 h, respectively. All immune parameters decreased with the reduction of dissolved oxygen. Total haemocyte count (THC), the hyaline cells and semi-granular cells in the 3.0 mg L⁻¹ DO group became stable after 12 h, while granular cells did so after 24 h. The THC and different haemocyte count (DHC) in the 1.5 mg L⁻¹ DO group became stable after 24 h. Phenoloxidase activity and bacteriolytic activity in the 3.0 and 1.5 mg L⁻¹ DO groups reached their stable levels after 24 h and 12 h respectively, while phagocytic activity and antibacterial activity became stable after 24 and 12, and 36 and 24 h, respectively. It was also indicated that the changes of dopamine concentrations in haemolymph, haemocyte count and phenoloxidase activity were obviously related to the exposure time under hypoxic conditions.     

Effect of dopamine injection on the hemocyte count and prophenoloxidase system of the white shrimp Litopenaeus vannamei

Effects of dopamine injection on the hemocyte count, phenoloxidase activity, serine proteinase activity, proteinase in-hibitor activity and α2-macroglobulin-like activity in L. vannamei were studied. Results showed that dopamine injection resulted in a significant effect on the parameters measured (P < 0.05), while no significant difference was observed in the control group (0.85% NaCl). In the experimental groups,the hemocyte count reached the minimum in 3 h;granular and semi-granular cells became stable after 12 h and hyaline cells and the total hemocyte count became stable after 18 h. Phenoloxidase activity reached the minimum in 6 h, and then became stable after 9 h. Serine protease activity and proteinase inhibitor activity reached the minimum in 3 h, and α2-macro-globulin-like activity reached the maximum in 3 h,and all the three parameters became stable after 12 h. The results suggest that the activating mechanisms of the proPO system triggered by dopamine are different from those triggered by invasive agents or sponta-neously activated under a normal physical condition.     

Modulation by biogenic amines for the hemocyte count and prophenoloxidase exocytosis via receptors in Litopenaeus vannamei

Hemocyte counts and phenoloxidase(PO)activity were examined after hemolymph being incubated in dopamine(DA),noradrenaline(NE)and serotonin(5-HT).Results showed that all the three biogenic amines(BAs)had a significant impact on total hemocyte count(THC),differential hemocyte count(DHC),and intracelluar and extracelluar phenoloxidase(PO)activity.Among these Bas,DA had the strongest effect on the above parameters,whereas 5-HT had the least effect.Preincubation with D1 receptor antagonist SCH23390,D2 receptor antagonist Sulpiride and 1:1 admixture of the two could significantly inhibit the effect of DA on these parameters.SCH23390 showed a stronger inhibitory effect than Sulpiride,and the admixture exhibited the strongest effect.These results suggested that the change of hemocyte count and activation of prophenoloxidase(proPO)system in Litopenaeusvan-namei hemocyte can be regulated by BAs,and DA modulates the two parameters via its receptors.     

Effects of Hypoxia on Dopamine Concentration and the Immune Response of White Shrimp （Litopenaeus vannamei）

Effects of hypoxia on the dopamine concentration and the immune response of White Shrimp Litopenaeus vannamei were studied. The results showed that hypoxia had significant effects on the concentration of dopamine (DA) in the haemolymph, haemocyte count, phenoloxidase activity, phagocytic activity of haemocytes and bacteriolytic and antibacterial activity in the haemolvmph (P<0.05). The concentration of the dopamine in haemolymph reached its maximum in the 3.0 and 1.5mgL-1 DO groups at 12h and 6h, and then returned to normal after 24h and 12h, respectively. All immune parameters decreased with the reduction of dissolved oxygen. Total haemocyte count (THC), the hyaline cells and semi-granular cells in the 3.0mgL-1 DO group became stable after 12h,while granular cells did so after 24h, The THC and different haemocyte count (DHC) in the 1.5mgL-1 DO group became stable alter 24h. Phenoloxidase activity and bacteriolytic activity in the 3.0 and 1.5mgL-1 DO groups reached their stable levels after 24 h and 12 h respectively, while phagocytic activity and antibacterial activity became stable after 24 and 12, and 36 and 24 It, respec-tively. It was also indicated that the changes of dopamine concentrations in haemolymph, haemocyte count and phenoloxidase activ-ity were obviously related to the exposure time under hypoxic conditions.     

A Comparative study on the nonspecific immunity of juvenile Litopenaeus vannamei ever inhabiting freshwater and seawater

A study on the nonspecific immunity of Litopenaeus vannamei ever inhabiting freshwater and seawater was carried out at different molt stages by comparing their total hemocyte count(THC) and respiratory burst(RB) and activity of phenol oxidase(PO), nitric oxide synthase(NOS) and lysozyme(LY). Two-way ANOVA showed that salinity and molt stage independently affected THC and RB and the activity of PO, NOS and LY of juvenile L. vannamei significantly(P 0.05). The THC and RB and the activity of NOS gradually increased from the post-molt stages(A and B) to the pre-molt stages(D0–D3), which were common in shrimps inhabiting freshwater and seawater. The activity of PO peaked at the inter-molt stage(C), and touched the lowest at the post-molt stage in freshwater and pre-molt stage in seawater. The activity of LY was stable over the molt cycle. The RB and the activity of PO, NOS and LY of juvenile L. vannamei were significantly lower in freshwater than in seawater; whereas THC was significantly higher in freshwater than in seawater(P 0.05). It was concluded that the post-molt stage(especially stage A) was critical to shrimp culture, which should be intensively attended when L. vannamei was cultured in freshwater.     

Isolation and Biochemical Characteristics Analyses of Phenoloxidases (POs) in Three Cultured Mollusk Species

Phenoloxidases(POs)are a group of copper proteins including tyrosinase,catecholase and laccase,which play crucial roles in the innate immune response of mollusks.In this research,POs were studied in cultured mollusk species,including scallop Chla-mys farreri,abalone Haliotis discus hannai and clam Scapharca subcrenata.The POs were isolated from hemocytes using linear-gradient native-PAGE combined with catechol staining.The PO activities and their characters were investigated.The molecular mass of PO in C.farreri was 576 kDa,and it was 228 kDa in H.discus hannai.In S.subcrenata,four POs were detected and their mole-cular masses were 391 kDa,206 kDa,174 kDa and<67 kDa,which were named as 391-PO,206-PO,174-PO and s-PO,respectively.Ki-netic analyses indicated that all of the POs,except for 391-PO had higher affinity to L-DOPA and catechol than to hydroquinone and dopamine.However,all of the POs failed to oxidize tyrosine.The effects of divalent metal ions on POs’activities were assayed,in-cluding Fe²⁺,Mg²⁺,Zn²⁺,Mn²⁺,Cu²⁺and Ca²⁺from FeCl₂,MgSO₄,ZnSO₄,MnCl₂,CuSO₄and CaCl₂.The POs were inhibited by Fe²⁺at all determined concentrations.Additionally,the inhibition assay showed that all of the POs were inhibited by cysteine,ascorbic acid,sodium sulfite,citric acid,ethylenediaminetetraacetic acid disodium(EDTA)and sodium diethyldithiocarbamate(DETC).The inhibition effects of critric acid and EDTA are dose-dependent.H.discus hannai PO and 391-PO were slightly inhibited by sodium azide,and H.discus hannai PO,391-PO and 174-PO were slightly inhibited by thiourea.In conclusion,the POs in the three cultured mollusks are copper-containing laccase-type phenoloxidases with similar biochemical characteristics even though their molecular masses are different.     

Effects of several immunostimulants on phenoloxidase and hemocytes of the crab Charybdis japonica

To investigate the stimulating effects of immunostimulants on the autogenous immunocompetence of crabs and the possible mechanisms involved, the immunostimulating effects of β-1,3-glucan, lipopolysaccharide (LPS), inactivated Vibrio harveyi and Vibrio anguillarum on phenoloxidase (PO) and hemocytes of Charybdis japonica were investigated in this study. It was found that the yields and the enzymatic activities of purified PO in C. japonica increased significantly after the crabs were treated with immunostimulants, while the unit enzymatic activities remained almost the same. After treatment with β-1,3-glucan and LPS, the amount of rough endoplasmic reticulum (RER) and the number of mitochondria in both semigranular cells and granular cells increased greatly, and the number of cytoplasmic granules decreased but with enlarged volume. However, the corresponding characteristics of hyaline cells remained almost the same. On the other hand, the number of granules in semigranular cells decreased greatly, and the number of mitochondria of hyaline cells increased greatly, after treatment with inactivated vibrios. It may be concluded that the effect of polysaccharide immunostimulants on the innate immune system of C. japonica is different from that of inactivated vibrio immunostimulants. The immunity-enhancing mechanism of polysaccharides in crab autogenous immunocompetence is probably accomplished by the increased yields of PO and total PO activities, while that of inactivated vibrios is probably accomplished by the partially increased yields of PO and total PO activities as well as the significantly improved phagocytotic abilities of semigranular cells and hyaline cells.     

Structure and Classification of Haemocytes in the Bivalve Mollusc Meretrix meretrix

Light and electron microscopic studies were carried out in order to characterize haemocytes in the bivalve mollusc Meretrix meretrix. According to nucleus and cytoplasm characters, four types of haemocytes were recognized： agranular haemocytes, lymphoid haemocyte, large granular and small granular haemocytes. Agranular hamocyte is the main cell type, accounting for 75%. It is agranular with rich organelles in cytoplasm, including mitochondria, golgi body and endoplasmic reticulum. Glycogen deposits were usually found in this cell type. The number of lymphoid haemocyte accounts for 1% - 2%. This cell type is agranular and shows a high ratio of nucleus to cytoplasm. A few organelles were found. High electrondense granules with diameters of 0.2 - 0.5μm and rich organelles were found in small granular haemocyte. The proportion of this cell type is about 15%. Rich granules of high electron-dense with diameters of 0.8- 2.4μm were found in large granular haemocyte. The proportion of this cell type is about 10%, and the quantity of organelles is the least.     

文蛤Meretrix meretrix血细胞形态与分类

光镜观察湛江南三地区文蛤血细胞的形态与结构特征,并进行分类。按细胞大小、形态和结构特征可将文蛤血细胞分为4类:透明细胞、小颗粒细胞、大颗粒细胞和淋巴细胞。细胞直径有统计学上显著性差异(P<0.05),分别为10.11±0.44μm、10.99±0.32μm、9.63±0.41μm和9.22±0.25μm。透明细胞是血细胞中的主要成分,细胞质中无颗粒;小颗粒细胞的细胞质中存在大量细小颗粒;大颗粒细胞细胞质中的颗粒直径有1.0～2.5μm;淋巴细胞是血细胞中含量最少的,有很高的核质比,大约为0.5。     

Modulation by Biogenic Amines for the Hemocyte Count and Prophenoloxidase Exocytosis via Receptors in Litopenaeus vannamei

Hemocyte counts and phenoloxidase(PO)activity were examined after hemolymph being incubated in dopamine(DA),noradrenaline(NE)and serotonin(5-HT).Results showed that all the three biogenic amines(BAs)had a significant impact on total hemocyte count(THC),differential hemocyte count(DHC),and intracelluar and extracelluar phenoloxidase(PO)activity.Among these Bas,DA had the strongest effect on the above parameters,whereas 5-HT had the least effect.Preincubation with D1 receptor antagonist SCH23390,D2 receptor a...     

Comparison of the respiratory metabolism of juvenile Litopenaeus vannamei cultured in seawater and freshwater

Litopenaeus vannamei, a euryhaline species, can be cultured at a wide range of salinities. The emergence of freshwater pond-culture of L. vannamei is an important prelude to the continued development of shrimp culture in China. In this study, we compared the respiratory metabolism of juvenile L. vannamei cultured in freshwater and saltwater by measuring their oxygen consumption rate (OCR), ammonium-type nitrogen excretion rate (AER) and pyruvate kinase (PK) and lactate dehydrogenase (LDH) activities at different molting stages in order to physiecologically characterize juvenile L. vannamei under freshwater conditions. The results showed that OCR was significantly higher in saltwater than in freshwater at all stages of molting cycle. However, variation of OCR among molting stages in saltwater was similar with that in freshwater, and the highest OCR was observed at post-molting stage. At all stages of molting cycle, AER was significantly higher in freshwater than in saltwater, and the highest was observed at post-molting stage. The activity of PK was significantly higher in saltwater than in freshwater. Conversely, the activity of LDH was higher in freshwater than in saltwater in general. Significant variation of PK and LDH activities in molting cycle was observed in saltwater and freshwater. The results indicated that aerobic metabolism of juvenile L. vannamei was more active in saltwater than in freshwater; while its protein metabolism was more active in freshwater than in saltwater.     

Effect of bioactive peptides (BPs) on the development of Pacific white shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis> Boone, 1931)

The present study was conducted to evaluate the feasibility of replacing fish meal (FM) with bioactive peptides (BPs) in diet of white shrimp (Litopenaeus vannamei). The changes in growth performance, body composition, non-specific immunity, and water quality were examined after the shrimp were fed four diets, in which 0% (control), 33.3%, 66.7% and 100% of FM was replaced by BPs, respectively. The groups were designated as Con, 1/3BPs, 2/3BPs, and 3/3BPs. A total of 720 shrimp with an initial body weight of 1.46 ± 0.78 g were fed the experimental diets for 56 days. The results revealed that: 1) the weight gain rate (WGR) in 1/3BPs, 2/3BPs, and 3/3BPs was significantly higher than that in Con (P < 0.05), while no significant difference was found on survival rate and feed conversion ratio (FCR); 2) the whole-body crude protein (CP) and crude lipids (CL) were significantly different among groups, while there was no significant difference between crude ash and phosphorus contents; 3) the levels of acid phosphatase (ACP), lysozyme (LZM), superoxide dismutase (SOD), phenol oxidase (PO) and bactericidal activity increased significantly with the inclusion of BPs; 4) in terms of water quality, no significant difference was found in pH and dissolved oxygen among diets during the whole experimental period. Moreover, even though nitrite and ammonium levels tended to increase with time, there was no significant difference among groups. The results indicated that BPs is an applicable alternative of protein source, which can substitute FM in the diets of L. vannamei; it is able to effectively promote growth performance and improve immunity. Moreover, BPs in the diets had no negative impact on water quality.     

Effect of copper on the growth of shrimps Litopenaeus vannamei: water parameters and copper budget in a recirculating system

Shrimps( Litopenaeus vannamei) were intensively cultured in a recirculating aquaculture system for 98 days to investigate effects of 0.3 mg/L Cu on its performance, Cu budget, and Cu distribution. Shrimps in Cu-treated systems had greater mean final weight(11.10 vs 10.50 g), body length(107.23 vs 106.42 mm), survival rate(67.80% vs 66.40%), and yield(6.42 vs 5.99 kg/m 3), and lower feed conversion ratio(1.20 vs 1.29) than those in control systems but the differences were not significant. Vibrio numbers remained stable(10 4 –10 6 colony forming units/mL) in the rearing tanks of both control and treated systems. Total ammonium-N, nitrite-N, nitrate-N, pH, chemical oxygen demand, 5-day carbonaceous biochemical oxygen demand, and total suspended solids were similar in controls and treatments. Dissolved Cu concentration in the treated systems decreased from 0.284 to 0.089 mg/L while in the control systems it increased from 0.006 2 to 0.018 mg/L. The main sources of Cu in the treated systems were the artificially added component(75.7% of total input), shrimp feed(21.0%), water(2.06%), and shrimp biomass(1.22%). The major outputs of Cu occurred via the mechanical filter(41.7%), water renewal(15.6%), and draining of the sediment trap(15.1%). The foam fractionator removed only 0.69% of total Cu input. Harvested shrimp biomass accounted for 11.68% of Cu input. The Cu concentration of shrimps in the Cu-treated systems(30.70 mg/kg wet weight) was significantly higher than that in control systems(22.02 mg/kg). Both were below the maximum permissible concentration(50 mg/kg) for Cu in seafood for human consumption in China. Therefore, recirculating systems can be used for commercial on-growing of Litopenaeus vannamei without loss of shrimp quality, even in water polluted by 0.30 mg/L Cu. The mechanical filter is the main route for Cu removal.     

Effects of Several Immunostimulants on Phenoloxidase and Hemocytes of the Crab Charybdis japonica

To investigate the stimulating effects of immunostimulants on the autogenous immunocompetence of crabs and the possible mechanisms involved, the immunostimulating effects of β-1,3-glucan, lipopolysaccharide (LPS), inactivated Vibrio harveyi and Vibrio anguillarum on phenoloxidase (PO) and hemocytes of Charybdis japonica were investigated in this study. It was found that the yields and the enzymatic activities of purified PO in C. japonica increased significantly after the crabs were treated with immunostimulants, while the unit enzymatic activities remained almost the same. After treatment with β-1,3-glucan and LPS, the amount of rough endoplasmic reticulum (RER) and the number of mitochondria in both semigranular cells and granular cells increased greatly, and the number of cytoplasmic granules decreased but with enlarged volume. However, the corresponding characteristics of hyaline cells remained almost the same. On the other hand, the number of granules in semigranular cells decreased greatly, and the number of mitochondria of hyaline cells increased greatly, after treatment with inactivated vibrios. It may be concluded that the effect of polysaccharide immunostimulants on the innate immune system of C. japonica is different from that of inactivated vibrio immunostimulants. The immunity-enhancing mechanism of polysaccharides in crab autogenous immunocompetence is probably accomplished by the increased yields of PO and total PO activities, while that of inactivated vibrios is probably accomplished by the partially increased yields of PO and total PO activities as well as the significantly improved phagocytotic abilities of semigranular cells and hyaline cells.     

Isolation and characterization of fucoidans from five brown algae and evaluation of their antioxidant activity

In this study, we evaluated the chemical property and antioxidant activity of fucoidans isolated from brown algae, Laminaria japonica(LJF), Lessonia nigrescens(LNF), Lessonia trabeculata(LTF), Ascophyllum mackaii(AMF), and Ecklonia maxima(EMF). LJF was less in sulfate content(14.16%) and more in galactose and mannose content(1.08 and 0.68) than the documented early. EMF contained 20%–30% of sulfate and fucose, 0.97 in molar ratio which was lower than that of sulfate to other four fucoidans(1.21–1.41). AMF(162 kDa) and EMF(150 kDa) were the first two largest in molecular weight, which were followed by LJP(126 kDa), LNF(113 kDa) and LTF(105 kDa). The fucoidans isolated these algae showed a wide range of antioxidant activity in vitro. It was found that the reducing power of the isolated fucoidans was positively correlated with their sulfate content and molecular weight. In addition, LNF and LTF at low concentrations exhibited high superoxide and hydroxyl radical scavenging activity. This demonstrated that low molecular weight fucoidans may perform a high antioxidant activity.     

ZResponse to selection, heritability and genetic correlations between body weight and body size in Pacific white shrimp, Litopenaeus vannamei

To quantify the response to selection,heritability and genetic correlations between weight and size of Litopenaeus vannamei,the body weight (BW),total length (TL),body length (BL),first abdominal segment depth (FASD),third abdominal segment depth (TASD),first abdominal segment width (FASW),and partial carapace length (PCL) of 5-month-old parents and of offspring were measured by calculating seven body measurings of offspring produced by a nested mating design.Seventeen half-sib families and 42 full-sib families ofL.vannamei were produced using artificial fertilization from 2-4 dams by each sire,and measured at around five months post-metamorphosis.The results show that heritabilities among various traits were high:0.515士0.030 for body weight and 0.394士0.030 for total length.After one generation of selection,the selection response was 10.70% for offspring growth.In the 5"’ month,the realized heritability for weight was 0.296 for the offspring generation.Genetic correlations between body weight and body size were highly variable.The results indicate that external morphological parameters can be applied during breeder selection for enhancing the growth without sacrificing animals for determining the body size and breed ability;and selective breeding can be improved significantly,simultaneously with increased production.     

Effect of dietary genistein on growth performance,digestive enzyme activity,and body composition of Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

An 8-week feeding experiment was performed to evaluate the effect of dietary genistein on growth performance, body composition, and digestive enzymes activity of juvenile Nile tilapia (Oreochromis niloticus). Four isonitrogenous and isoenergetic diets were formulated containing four graded supplements of genistein: 0, 30, 300, and 3 000 μg/g. Each diet was randomly assigned in triplicate to tanks stocked with 15 juvenile tilapia (10.47±1.24 g). The results show that 30 and 300 μg/g dietary genistein had no significant effect on growth performance of Nile tilapia, but the higher level of genistein (3 000 μg/g) significantly depressed the final body weight and specific growth rate. There was no significant difference in survival rate, feed intake, feed efficiency ratio or whole body composition among all dietary treatments. An assay of digestive enzymes showed that the diet containing 3 000 μg/ggenistein decreased stomach and hepatopancreas protease activity, and amylase activity in the liver and intestine, while a dietary level of 300 μg/g genistein depressed stomach protease and intestine amylase activities. However, no significant difference in stomach amylase activity was found among dietary treatments. Overall, the results of the present study indicate that a high level of dietary genistein (3 000 μg/g, or above) would significantly reduce the growth of Nile tilapia, partly because of its inhibitory effect on the activity of major digestive enzymes. Accordingly, the detrimental effects of genistein, as found in soybean products, should not be ignored when applied as an alternative ingredient source in aquaculture.