THE INDUCTION AND EXTRACTION OF METALLOTHIONEINS IN ARTEMIA

A small amount of heavy metal binding protein,identified by BioRad Protein Assay,has been iso-lated from the adult brine shrimp,Artemia franciscanus.This protein has an apparent molecular weight be-tween 6000 to 9000 dalton.a UV absorption peak at 260 instead of 280 nm like most proteins;andhas high affinity towards binding with radioactive labeled ~(109)Cd.These characteristics are similarto that of metallothioneins reported for many vertebrate and invertebrate,marine and terrestrialanimals.After the brine shrimp is exposed to a small amount of Cd~(2+)for 24 h,a large amount ofmetallothionein can be isolated,showing the inducibility of this detoxifying protein in the adult Artemiain a short period of time.     

The brine shrimp (Artemia parthenogenetica) as encapsulation organism for prophylactic chemotherapy of fish and prawn

Brine shrimp (Artemia parthenogenetica) which had ingested three water-insoluble antibacterial drugs i.e. sulfadiazine(SD), oxytetracycline (OTC) and erythromycin estolate (ERY-Es) were fed toTilapia and Mysis IIIof Penaeus orientalis K. The drug contents in the predators were then determined. After administration of drugs toTilapia and Mysis III, through the bio-encapsulation of the brine shrimp, efficacious therapeutical concentration of OTC and ERY-Es (but not SD) in the predators could be reached and maintained for more than 8 hours.     

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.     

Dietary manganese requirement ofP. Vannamei

Graded levels of manganese were supplemented to a semi-purified diet containing 45% crude protein, to provide six levels of manganese (i. e. containing 5, 25, 50, 70, 140 and 210×10⁻⁶, respectively) for two experiments with these experimental diets. The initial weight of shrimp used in the 35–day experiment I was 0.30±0.04 g, and that in the 70–day Experiment II was more than one gram. The results showed that optimum content in the semi-purified diet for the more than 1 gram shrimp ranged from 70 ×10⁻⁶, to 140×10⁻⁶, but supplementation of Mn was not necessary for the small shrimp. Contribution No. 2673 from the Institute of Oceanology, Chinese Academy of Sciences.     

A Study on the contribution of different food sources to shrimp growth in an intensive <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> pond

Stable isotope methods can be used to determine the food sources and prey items of aquatic organisms accurately and reliably. This study examined the relative contribution of artificial foods (the formulated feed and Artemia) and natural foods to shrimp growth in an intensive Fenneropenaeus chinensis pond by using carbon and nitrogen stable isotopes. The results showed that the nutrition utilization efficiency of the harvested shrimp was low, only 33.18% of feed nitrogen and 21.73% of feed carbon being converted to shrimp flesh. Our stable isotope results showed that the shrimp obtained nutrition for maximum growth from artificial foods, whose contribution was 93.5%, with the remaining attributed to the natural foods. However, there was 0.94 t harvested shrimp derived from natural foods (the rest of 13.56 t harvested shrimp derived from artificial foods) in 1ha intensive pond with a shrimp production of 14.50 t ha⁻¹. Therefore, unit area shrimp production can be increased by increasing the contribution proportion of natural foods in intensive shrimp farming.     

Hematological changes in white spot syndrome virus-infected shrimp, Fenneropenaeus chinensis (Osbeck)

The pathological changes of hemocytes in the haemolymph and hepatopancreas were examined in experimentally and naturally WSSV (white spot syndrome virus) infected Fenneropenaeus chinensis. The results showed that the pathological manifestations of hemocytes were similar among moribund shrimps infected via injection, feeding and by nature. Firstly, the total hemocyte counts (THCs) in WSSV-infected shrimp were significantly lower than those in healthy shrimp. Secondly, necrotic, broken and disintegrated cells were often observed, and a typical hematolysis was present in the haemolymph smear of WSSV-infected shrimp. Thirdly, necrosis and typical apoptosis of hemocytes were detected with TEM in the peripheral haemolymph of WSSV-infected shrimp. Hyalinocytes and semi-granulocytes with masses of WSSVs in their nuclei often appeared, whereas no granular hemocytes with WSSV were found in the hepatopancreas of moribund infected shrimps. All our results supported that hemocytes were the main target cells of WSSV, and hyalinocytes and semigranular hemocytes seemed to be more favorable for WSSV infection in F. chinensis.     

Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp (Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK’s (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.     

Amylase production by the marine yeast Aureobasidium pullulans N13d

The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pacific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identification methods. The optimal sea water medium for amylase production by this yeast strain was 1.0% peptone and 1.0% soluble starch with pH 4.0. The optimal conditions for amylase production by this yeast strain were with temperature 28 °C, aeration rate 6 Lmin⁻¹ and agitation speed 250 rmin⁻¹. Under these conditions, 58.5 units of amylase activity per mg protein were produced within 56 h of fermentation.     

Interaction of haematopoietic tissue cultures of the Dublin Bay Prawn, Nephrops norvegicus (L.), with the causal agent of luminous vibriosis Vibrio harveyi

Vibrio harveyi cells (dose—<10³ cells mL⁻¹) and extracellular products (ECP; >25 μmg mL⁻¹ of total protein concentration) destroyed haematopoietic cultures of Nephrops norvegicus within 24 h of exposure. Cytopathic effects (CPE) started after 4h of exposure to the bacterial cells, with some granularity in the cytoplasm, mostly in cells in the outer periphery of the explant growth. At the end of the infection, a considerable number of nuclei remained attached to the substrate, apparently unaffected. Following exposure to ECP, initial deterioration was observed at 2 h with the presence of granularity in the cytoplasm of<20% cells, and few cells displayed small vacuoles around the nuclei. Parallel results were obtained using whole animal experiments, with V. harveyi cells being lethal to nephrops within 24 h.     

Purification and characterization of an alkaline protease from Acetes chinensis

An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca²⁺ could enhance the proteolytic activity of the protease, while Cu²⁺, EDTA and PMSF could inhibit its activity.     

Isolation of protoplasts from <Emphasis Type="Italic">undaria pinnatifida</Emphasis> by alginate lyase digestion

The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28°C for 2h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5g fresh thalline with NaCl 50 and at the shaking speed of 150 r min⁻¹ during digestion. The protoplast yield can reach 2.62±0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca²⁺ and slightly enhanced by Fe²⁺ and Mn²⁺ at concentrations of 0.05, 0.08 and 0.10 mol L⁻¹.     

The Complete Sequence of Mitochondrial COⅡ Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit Ⅱ （COⅡ） gene of Penaeinae shrimp Fenneropenaeus chinensis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus Japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A＋T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COⅠ and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COⅡ gene has a faster evolutionary rate than that of the COⅠ gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

Effects of Dietary Protein Level on Growth and Utilization of Protein and Energy by Juvenile Mangrove Red Snapper （ Lutjanus argentimaculatus ）

A feeding trial was conducted in a recirculating water system to investigate the effects of dietary protein levels on growth, feed utilization, hepatosomatic index and liver lipid deposition of juvenile red snapper, Lutjanus argentimaculatus (average initial wet weight 8.0 ± 0.39 g and total length 3.14 ± 0.3 cm). In the experiment, six fishmeal-based diets were formulated to contain various protein levels (20% to 45% in 5% increments), with dietary energy ranging from 2210.7kJ lOOg to 2250.2kJlOOg dry matter. The protein to energy ratios of diets ranged from 8.58 mg protein kJ⁻¹ to 20.03 mg protein kJ⁻¹. Diets were fed for 90d to triplicate groups of fish stocked in 0.128m³ seawater tanks, 25 individuals each. The daily ration of 2% wet body weight was offered to the fish thrice a day. The fish at the end of the study had more than ten-fold (77.0g) increase in weight compared to the initial (8.0g). Fish fed diets of 40% and 45% protein produced significantly (P<0.05) higher weight gain of 77.2g and 76.5g, and specific growth rate (SGR) of 2.65% and 2.62% than those of 67.0 g and 68.3g, and 2.49% and 2.51% of the other diets. The broken-line regression of SGR against dietary protein level yielded an optimum dietary protein requirement of 42.6% (Y=−1.6295 + 0.1114 X ²,P<0.05). Survival remained 100% among groups. Feed conversion ratio decreased from 0.45 for fish fed 20% dietary protein to 0.35 for fish fed 45% dietary protein. Nitrogen intake increased with an increase in dietary protein, which in turn resulted in an increase in nitrogen gain of fish whole body. Fish fed 40% and 45% protein diets showed higher (P<0.05) nitrogen gain (0.27g and 0.26g) than those (0.23g and 025g) fed all other diets. Gross energy intake (GEI) in fish fed 45% protein was lower (600.67kJ) than that (607.97 kJ) of 40% protein diet, though the differences were not statistically significant (P>0.05); GEI ranging from 677.31 kJ to 663.20 kJ at remaining four diets (20% to 35% protein) did not appear to differ significantly (P>0.05). The highest energy gain of 518.33 kJ was obtained with fish fed 40% protein, resulting in the highest energy retention efficiency of 85.26%. The hepatosomatic index of fish fed diets of 20%, 25%, 30% and 35% protein were significantly (P<0.05) higher (2.09% to 2.57%) than those (1.44% and 1.41%) of fish fed diets containing 40% and 45% protein. Liver lipid contents decreased from 8.72% to 7.0% in fish fed dietary protein of 20% to 45% in 5% increments. Results suggest that the diet containing 40% to 42.6% protein with a P/E ratio of 17.6 mg protein kJ⁻¹ is required for good growth of L. argentimaculatus weighing between 8.0 g and 85.2 g under the culture conditions of the present study.     

EFFECT OF Cu, Cd AND Cu:Cd MIXTURE ON THE BIOCHEMICAL COMPOSITION OF TWO PENAEID SHRIMP POSTLARVAE

IwrRODUcrIONBiochewhcalindiasrnaybeusedtoreboresponsestospedccontandnantSincludingheaVyrnetaIs.ChineseshrimpfisheriesarebasedonpenaeidsofWhichPenaeuspoiciIlatusistheddrinantsthe(Xuetal.,l993)incoastalwatersofFujianProvince.Larvalpenaeids(Andelsonetal.,l976)likeotherplanktoniccrustaonns(Clarkl992),arepartioulariysensitivetoheaVymetalcontandnatedwaterduetothefortnationofmetalbindingproteinsintheirbodies.Cytoplasndcproteins,dondnatedbythestilphydrygrouphave~reportedinvariousdassesofphylog…     

Bioaccumulation of Pb<Superscript>2+</Superscript> and its effects on growth,morphology and pigment contents of <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

A laboratory experiment was conducted to assess the bioaccumulation of Pb²⁺ and its effects on growth, morphology and pigment contents of Spirulina (Arthrospira) platensis. The specimen cultured in Zarrouk liquid medium was treated with various initial metal concentrations (0, 5, 10, 30, 50 and 100 μg mL⁻¹). The growth of S. platensis was adversely affected by Pb²⁺ at high concentrations (30, 50 and 100 μg mL⁻¹). However, at low concentrations (5 μg mL⁻¹), Pb²⁺ could stimulate its growth slightly. The pigment contents (chlorophyll α and β carotene) were decreased in a dose-dependent manner. The highest reductions (67% and 53% respectively in chlorophyll α and β carotene) were observed in 100 μg mL⁻¹ treatment group. The LC₅₀ (96 h) of Pb²⁺ was measured as 75.34 μg mL⁻¹. Apart from a few cases of filament breakages at elevated concentrations (50 and 100 μg mL⁻¹), morphological abnormalities are not specific. Metal bioaccumulation increased with Pb²⁺ concentrations, but decreased with exposure time. The maximum accumulated amount was 188 mg g⁻¹ dry weight. The bioconcentration factor (BCF) reached to a peak at day 2, followed by a gradual reduction for all the exposure concentrations. S. platensis is able to tolerate considerably high Pb²⁺ concentrations. Consequently it can be used as a potential species to remove heavy metal from contaminated waters.     

Effects of salinity fluctuation frequency on the osmolarity, Na+-K+-ATPase activity and HSP70 expression in juvenile chinese shrimp, Fenneropenaeus chinensis

Experiments were conducted to examine the effects of salinity fluctuation frequency on the osmolarity, Na⁺-K⁺-ATPase activity and HSP70 of Chinese shrimp Fenneropenaeus chinensis with initial wet body weight of 1.460 g ± 0.091 g. The salinity in the control group (D0) was 28 throughout the experiment, whereas treatments D2, D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8 d, respectively. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8 d, respectively, decreased abruptly to salinity 24, lasted for another 2 d, and then was raised to its initial value 28. This was a complete salinity fluctuation cycle that afterwards repeated itself. After 32 days, the osmolarity in treatments D2, D4, D6 and D8 was significantly lower than that in treatment D0 (P<0.05). There were significant differences in both muscle and eyestalks HSP70 expression among groups. The HSP70 expressions in muscle and eyestalks in group D4 were 61.4% and 57.0% higher, respectively, than that in the control group D0 (P<0.05). There were, however, no significant differences in gill or hepatopancreas Na⁺-K⁺-ATPase activity between the treatments and the control.     

Requirements of shrimp,Penaeus chinensis O'sbeck for potassium,sodium, magnesium and iodine

Potassium, sodium, magnesium and iodine requirements of shrimp,Penaeus chinensis were studied. Orthogonal design was employed in this experiment. The composition of the basal diet consisted of fish meal, peanut cake, corn meal, soybean cake, wheat bran, vitamin mix and mineral mix, and supplementations of potassium, sodium, magnesium and iodine in the basal diet were made according to the L₉(3⁴) orthogonal table. The results indicated that iodine supplementation improved growth of the shrimp significantly and raised survival very significantly, iodine requirement of the shrimp was 0.003%; sodium requirement was 0.87% or less; and that 1.1–1.3% potassium and 0.18–0.38% magnesium in the diet were proper nutrition supplements for the shrimp. Contribution No. 2342 from the Institute of Oceanology, Chinese Academy of Sciences.     

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.     

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.