Identification of the Major Allergen in Greasy-Back Shrimp （Metapenaeus ensis） by MALDI-TOF-MS

In order to better understand shrimp allergen,some basic characters of the major allergen of greasy-back shrimp (Metapenaeus ensis)were investigated.The major allergen was extracted and separated,and its peptide mass fingerprint(PMF) was analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).After screening in the NCBI database with Mascot searching engine,the results indicated that the major allergen of greasy-back shirmp was muscle tropomyosin.Database matching search showed that the top protein matched,i.e.the tropomyosin from giant tiger prawn(Penaeus monodon),had a Mowse value of 268.In addition,there were 27 queries matched with the allergen in greasy-back shirmp with an amino acid sequence coverage value of 65%.The matching scores and the sequence coverage values were also high with tropomyosins of other invertebrates,including Tyrophagus putrescentiae and Lepisma saccharina.These results indicated that the allergen of Metapenaeus ensis had high homology with other crustacean allergens,and provided molecular explanations for the high cross-reactivity of the major allergens between crustaceans and some other invertebrates.     

The effect of simulated gastrointestinal digestion on shrimp <Emphasis Type="Italic">Penaeus vannamei</Emphasis> allergenicity

Pen a 1 is a major shrimp allergen that induces an allergy. The aim of this study is to assess the effect of simulated gastrointestinal fluids on the allergenicity of Pen a 1. Purified Pen a 1 from shrimp (Penaeus vannamei) was subjected to digestion in SGF (simulated gastric fluid) and SIF (simulated intestinal fluid) for a set time. The allergenicity of digestive Pen a 1 was analyzed by immunoblotting and Ci-ELISA, using pool sera from patients with shrimp specific IgE. The results showed that Pen a 1 exhibited a decrease in allergenicity with increasing digestion time in the SGF and SIF. However, Pen a l exhibited strong resistance to digestive fluids, and all yielded fragments (33 kD, 23 kD, and 14 kD) showed allergic activity. Therefore, anti-digestion may be an important factor for Pen a 1 to induce an allergy.     

Comparative study on the protease inhibitors from fish eggs

The protease inhibitor was purified from five different fish eggs. The molecular weights of Pacific herring, chum salmon, pond smelt, glassfish, and Alaska pollock egg protease inhibitors were 120, 89, 84.5, 17, and 16.8kDa, respectively. The specific inhibitory activity of glassfish egg protease inhibitor was the highest followed by those of Pacific herring and Alaska pollock in order. The specific inhibitory activity and purity of glassfish egg protease inhibitor were 19.70 U mg^- 1 protein and 164.70 folds of purification, respectively. Glassfish egg protease inhibitor was reasonably stable at 50 - 65℃ and pH 8, which was more stable at high temperature and pH than protease inhibitors from the other fish species. Glassfish egg protease inhibitor was noncompetitive with inhibitor constant （Ki） of 4.44 nmol L^-1     

Enzymatic hydrolysis of defatted mackerel protein with low bitter taste

Ultrasound-assisted solvent extraction was confirmed as a novel,effective method for separating lipid from mackerel pro-tein,resulting in a degreasing rate (DR) of 95% and a nitrogen recovery (NR) of 88.6%.To obtain protein hydrolysates with high ni-trogen recovery and low bitter taste,enzymatic hydrolysis was performed using eight commercially available proteases.It turned out that the optimum enzyme was the ‘Mixed enzymes for animal proteolysis’.An enzyme dosage of 4%,a temperature of 50℃,and a hydrolysis time of 300 min were found to be the optimum conditions to obtain high NR (84.28%) and degree of hydrolysis (DH,16.18%) by orthogonal experiments.Glutamic acid was the most abundant amino acid of MDP (defatted mackerel protein) and MDPH (defatted mackerel protein hydrolysates).Compared with the FAO/WHO reference protein,the essential amino acid chemical scores (CS) were greater than 1.0 (1.0 1.7) in MDPH,which is reflective of high nutritional value.This,coupled with the light color and slight fishy odor,indicates that MDPH would potentially have a wide range of applications such as nutritional additives,functional ingredients,and so on.     

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.     

Vaccination in three different ways against vibriosis of <Emphasis Type="Italic">Seriola dumerili</Emphasis> caused by <Emphasis Type="Italic">Vibrio hollisae</Emphasis>

Bacterin was prepared by formalin-inactivating the virulent strain of Vibrio hollisae isolated from diseased Seriola dumerili （amberjack） suffering from vibriosis. Healthy S. dumeriIi were vaccinated by respective procedures of intramuscular injection, immersion, and orally administration. Results of the three different vaccinations were compared. Blood was drawn from the vaccinated fish every 7 days, and the antibody titers and lysozyme activities of the sera were determined. The antibody titer of injected fish was 1：40 at 7 d, and reached its peak of 1：320 at 28 d, while the fish vaccinated by immersion and orally administration exhibited weak antibody responses, the antibody titres of 〈1：10, 1：20, 1：160 were observed at 7 d, 14 d, 35 d respectively. Compared with the control, the vaccinated fish exhibited significantly higher lysozyme activities （P〈0.05）. Upon challenge with virulent strain, the relative percent survival （RPS） of injected, immersed and oral administrated fish were 75%, 45%, and 40% respectively, and the injected fish showed significantly higher RPS than immersed and oral administrated fish. The results suggested that vaccination of S. dumerili by the injection would be the best strategy to prevent the vibriosis in S. dumerili farm.     

Species composition and diversity of pelagic fishes based on a longline fishery catch in the North Pacific Ocean

Evaluation of spatial-temporal variability of species composition and diversity in oceanic ecosystems is not easy because it is usually difficult to obtain sufficient data quantifying such variability.In this study,we examined pelagic species diversity indicators,species richness,Shannon-Wiener index of diversity and Hurlbert’s species evenness,for fish assemblages from two areas(north and south) in the North Pacific Ocean(2°±12°N,178°E±165°W) during May±July 2008.The assemblages were based on data collected by an onboard scientific observer during a commercial longline fishing trip.The species richness and Shannon-Wiener diversity index of fish assemblages in the northern area were slightly higher than those in the southern area,although these differences were not significant(t test,P.0.05).Non-parametric multidimensional scaling and analysis of similarities indicated that there were significant differences in fish assemblages between the two areas(P,0.01).     

An extracellular oligopeptide permease may be a potential virulence factor of Vibrio harveyi

An oligopeptide permease A(OppA)was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58 kDa on SDS-PAGE.The purified protein showed phospholipase C activity at the optimal values of temperature 50℃ and pH 8.0.The enzymatic activity decreased when the temperature increased to 40℃.The N-terminal sequence of the purified protein was determined as ADVPAGTKLA,which is similar to that of OppA.The OppA pre-cursor gene was cloned from th...     

Age and growth of chub mackerel (Xcomber japonicus) in the East China and Yellow Seas using sectioned otolith samples

Although chub mackerel (Scomber japonicus) is a primary pelagic fish species, we have only limited knowledge on its key life history processes. The present work studied the age and growth of chub mackerel in the East China and Yellow Seas. Age was determined by interpreting and counting growth rings on the sagitta otoliths of 252 adult fish caught by the Chinese commercial purse seine fleet during the period from November 2006 to January 2007 and 150 juveniles from bottom trawl surveys on the spawning ground in May 2006. The difference between the assumed birth date of 1st April and date of capture was used to adjust the age determined from counting the number of complete translucent rings. The parameters of three commonly used growth models, the von Bertalanffy, Logistic and Gompertz models, were estimated using the maximum likelihood method. Based on the Akaike Information Criterion (AIC), the von Bertalanffy growth model was found to be the most appropriate model. The size-at-age and size-at-maturity values were also found to decrease greatly compared with the results achieved in the 1950s, which was caused by heavy exploitation over the last few decades.     

Structure analysis of the complete mitochondrial genome in cultivation variety ��Rongfu��

In this report, complete mitochondrial genome sequences of Laminaria cultivation variety ‘Rongfu’ were obtained. The results showed the length of circular molecule of mtDNA was 37 638 bp (64.7% A+T), encoding three rRNAs (23S, 16S and 5S), 25 tRNAs, 35 known mitochondrial proteins and 3 ORFs. Sequence alignment indicated its mtDNA genome was very similar to that of Laminaria japonica. Phylogenetic trees inferred from concatenated 30 mitochondrial genes showed that ‘Rongfu’, Laminaria japonica, Laminaria longipedalis, Laminaria diabolica, Laminaria religiosa and Laminaria ochotensis clustered together. In addition, compared with mitochondrial genome of L. japonica, ‘Rongfu’ mtDNA lacked a non-coding region of 19 nucleotides, which was located between rRNA small subunit gene 3 (rps3) and rRNA small subunit gene 9 (rps9). Seven cultivation varieties of China were divided into two groups based on this non-coding region which was absent in ‘Rongfu’, ‘Fujian’ and ‘Sanhai’ while present in ‘Ailunwan’, ‘Dongfang No.2’, ‘Dongfang No.3’ and ‘Zaohoucheng’. So this variation can be used in germplasm identification of cultivation variety.     

Species- and tissue-specific mercury bioaccumulation in five fish species from Laizhou Bay in the Bohai Sea of China

Mercury (Hg) concentrations in the tissues (muscle, stomach, liver, gills, skin, and gonads) of five fish species (mullet Liza ha em atocheil us, flathead fish Platycephalus indicus, sea bass Lateolabrax japonic u s, mackerel Scomberomorus niphonius and silver pomfret Pampus argenteus) collected from Laizhou Bay in the Bohai Sea of China were investigated. The results indicate that Hg bioaccumulation in the five fish was tissue-specific, with the highest levels in the muscle and liver, followed by the stomach and gonads. The lowest levels were found in the gills and skin. Fish at higher trophic levels (flathead fish and sea bass) exhibited higher Hg concentrations than consumers at lower trophic levels. Mercury bioaccumulation tended to be positively correlated with fish length in mullet, silver pomfret, mackerel, and flathead fish, but was negatively correlated with fish length in sea bass. The Hg concentrations in the muscles of all fish species in Laizhou Bay were within the permissible limits of food safety set by national and international criteria. However, the suggesting maximum consumption of sea bass is 263 g per week for human health.     

Age and growth characteristics of crimson sea bream <Emphasis Type="Italic">Paragyrops edita</Emphasis> Tanaka in Beibu Gulf

Abstact  Age and growth characteristics of crimson sea bream Paragyrops edita Tanaka in Beibu Gulf were studied through bottom trawling and gillnet fleets fishing from July 2006 to December 2007. A total number of 1155 individuals, ranging from 49 to 249 mm in standard length was examined. The age of the fish was determined from sagittal otoliths. One year growth was made up of one translucent and one opaque zone. A maximum likelihood estimation procedure was used to fit the Von Bertalanffy, Logistic and Gompertz growth functions to the length-at-age data. ARSS indicated that there were no significant differences in growth between sexes in the three growth models (P>0.05), and the Von Bertalanffy growth function L _t =292.8{1−exp [−0.167(t+1.116)]} was selected as the most appropriate growth model according to Akaike’s information criterion (AIC).     

Amino acid composition and functional properties of giant red sea cucumber (Parastichopus californicus) collagen hydrolysates

Giant red sea cucumber (Parastichopus californicus) is an under-utilized species due to its high tendency to autolysis.The aim of this study was to evaluate the functional properties of collagen hydrolysates from this species.The degree of hydrolysis (DH),amino acid composition,SDS-PAGE,emulsion activity index (EAI),emulsion stability index (ESI),foam expansion (FE),and foam stability (FS) of hydrolysates were investigated.The effects of pH on the EAI,ESI FE and FS of hydrolysates were also inves-tigated.The results indicated that the β and α1 chains of the collagen were effectively hydrolyzed by trypsin at 50℃ with an En-zyme/Substrate (E/S) ration of 1:20 (w:w).The DH of collagen was up to 17.3% after 3 h hydrolysis with trypsin.The hydrolysates had a molecular weight distribution of 1.1 17 kDa,and were abundant in glycine (Gly),proline (Pro),glutamic acid (Glu),alanine (Ala) and hydroxyproline (Hyp) residues.The hydrolysates were fractionated into three fractions (< 3 kDa,3 10 kDa,and > 10 kDa),and the fraction of 3 10 kDa exhibited a higher EAI value than the fraction of > 10 kDa (P<0.05).The fraction of > 10 kDa had higher FE and FS values than other fractions (P<0.05).The pH had an important effect on the EAI,ESI,FE and FS.All the fractions showed undesirable emulsion and forming properties at pH 4.0.Under pH 7.0 and pH 10.0,the 3 10 kDa fraction showed higher EAI value and the fraction of > 10 kDa showed higher FE value,respectively.They are hoped to be utilized as functional ingredients in food and nutraceutical industries.     

A study on the meat and bone meal and poultry by-product meal as protein substitutes of fish meal in practical diets for Litopenaeus vannamei juveniles

A study was conducted to evaluate the effects of meat and bone meal (MBM) and poultry by-product meal (PBM) as the replacement of fish meal in the diets on the growth performance, survival and apparent digestibility coefficients (ADC) of Litopenaeus vannamei. The basal diets were formulated with 22% fish meal and other ingredients which provided about 40% protein and 9% lipid in the diet. The experimental diets included MBM or PBM to replace 0, 20%, 40%, 60% and 80% of total fish meal respectively. All diets were iso-nitrogenous and isocaloric in gross terms. The results showed that there were no significant differences (Pτ;0.05) in growth performance and ADC among the treatments fed with the diets in which 0–60% fish meal had been replaced with MBM, while the percent weight gain (WG, %), body length gain (BLG, %) and ADC significantly decreased when the MBM was up to 80% of the fish meal. There were no significant differences (Pτ;0.05) in growth performance and ADC among all the treatments fed with the diets in which 0–80% fish meal had been replaced with PBM.     

Detection of lymphocystis disease virus in Japanese flounder Paralichthys olivaceus and other marine teleosts from northern China

We isolated a strain of lymphocystis disease virus (LCDV) from Japanese flounder (Paralichthys olivaceus) cultured in northern China. Based on published sequences of major capsid protein (MCP) gene of LCDV-cn (GenBank: AF126405), we designed two primer sets P1/P2 and P3/P4. We then used one-step or nested PCR and in-situ hybridization (ISH) to detect LCDV and identify the target tissues or cells in infected Japanese flounder. The PCR products were positive in purified viral supernatant, skin nodules, gut, gill, kidney, spleen, stomach, heart, and liver of Japanese flounder. We compared the DNA sequence with 14 MCP nucleotide sequences from GenBank, including Megalocytivirus (OFIV and RSIV), Iridovirus (CzIV and WIV), Ranavirus (TFV and FV3), and Lymphocystivirus (8 LCDV). Based on the alignment, we confirmed the PCR product was from Lymphocystivirus (GenBank accession number DQ279090 (LCDV-HD)). Using ISH, we noted the presence of LCDV in the skin nodules, gut, gill, spleen, stomach, and heart of spontaneously infected Japanese flounders. We successfully amplified LCDV fragments from Schlegel’s black rockfish (Sebastes schlegeli Higendorf), redwing sea robin (Lepidotrigla microptera Günther) and turbot (Scophthalmus maximus) using the one-step and nested PCR, suggesting the target genes can be widely detected in fish using this method.     

Serum immunoglobulin of the mandarin fish, Siniperca chuatsi with development of polyclonal antibody

Serum immunoglobulin from the mandarin fish, or the so-called Chinese perch,Siniperca chuatsi (Basilewsky), was successfully purified using affinity chromatography. Heavy and light chains were detected on electrophoresis gel, with molecular weights being estimated at 72 and 29 kDa, respectively. The tetrameric IgM ofS. chuatsi was calculated to be 808 kDa. The rabbit polyclonal antisera against the purifed immunoglobulin were developed and tested by Western blot analysis. The antisera reacted strongly with the heavy chains ofS. chuatsi immunoglobulin. Humoral immune responses of the mandarin fish can then be examined using the developed polyclonal antibody. Project No. 30025035 supported by NSFC, State Key Laboratory of Freshwater Ecology and Biotechnology (2000FB02), and the Chinese Academy of Sciences (KZ9511-B1-111).     

Effect of lead on cytoskeletal protein stability in crucian carp <Emphasis Type="Italic">Carassius auratus</Emphasis>

Inorganic lead （Pb） is one of the most common environmental pollutants. Much evidence indicates that Pb exposure could directly affect fish growth and development. In this study, we investigated the cytotoxic effects of Pb on cytoskeletal protein stability at both protein and mRNA level in crucian carp Carassius auratus. Pb（NO3）2 treatment in concentration of 100 μmol/L resulted in decreased expression of both α- and β-tubulin but γ-tubulin as assayed with SDS-PAGE, Western Blot, and ELISA. In vivo and in vitro analyses on protein expression of tubulins are consistent. The effect of Pb on mRNA expression varied among different tissues. Our results suggest that cytotoxicity of Pb at protein translation level is stronger than at mRNA expression level.     

Occurrence of two distinct types of tissue inhibitors of metallo-proteinases-2 in Fugu rubripes

In this study, genes of two distinct tissue inhibitors of metalloproteinases-2 (TIMP-2) from Japanese puffer fish Fugu rubripes, Fugu TIMP-2a and TIMP-2b, were cloned. The open reading frames of Fugu TIMP-2a and TIMP-2b cDNAs are composed of 660 and 657 nucleotides and 220 and 219 amino acids, respectively. Both Fugu TIMP-2s contain 12 cysteine residues, which might form six disulfide bonds as in other animals’ TIMP-2s. Reverse-transcribed polymerase chain reaction analysis showed the mRNAs of Fugu TIMP-2a and TIMP-2b to be expressed in some tissues examined with different expression patterns. These findings suggest that the two distinct Fugu TIMP-2s might perform different functions in Fugu tissues.     

Expression and localization of a novel phosducin-like protein from amphioxus <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>

A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri. Supported by the Pilot Projects of Knowledge Innovation Project of Chinese Academy of Sciences (No. KZCX2-YW-211-03 and MGE2008KG06)