Lipid class and fatty acid composition of phospholipids from the gonads of skipjack tuna

ABSTRACT:   Lipid class and fatty acid composition of phospholipids from the gonads of skipjack tuna were examined to evaluate effective utilization of the processing of by-products. The predominant phospholipids in the ovaries were phosphatidylcholine (PC; 47.9%), phosphatidylethanolamine (PE; 19.3%) and lyso-phosphatidylcholine (LPC; 19.1%). In contrast, those in the testes were PC (40.1%), PE (29.3%) and phosphatidylserine (PS; 9.6%). The percentage of docosahexaenoic acid (DHA) was markedly high at more than 50% in LPC of the ovaries, and PE and PS of the testes were also high. The percentages of DHA at sn-position 2 of the predominant phospholipids, except for PC in the testes, were more than 60%, in particular PE in the testes was remarkably high at 81.9%. After storage for 2 days at 5°C, the LPC content in the ovaries increased twofold and the DHA level of LPC was the same as before, though the contents of other phospholipids decreased.     

Purification and characterization of phospholipase A2 isoforms from the hepatopancreas of red sea bream, Pagrus major1

Two phospholipase A₂ (PLA₂) isoforms, tentatively denoted as DE-1 and DE-2 PLA₂s, were purified from the hepatopancreas of red sea bream (Pagrus major) to near homogeneity by sequential column chromatography on S-Sepharose fast flow, DEAE-Sepharose fast flow and butyl-Cellulofine, and by ion-exchange, gel-filtration and reversed-phase HPLC. The purified DE-1 and DE-2 PLA₂s both showed a single band with the apparent molecular mass of approx. 13.5 kDa by SDS-polyacrylamide gel electrophoresis, and were found to be both related to group I PLA₂ based on the N-terminal amino acid sequences. DE-1 PLA₂ had a pH optimum in the alkaline region at around pH 10 and required approximately 10 mM of Ca²⁺ and 4-10 mM of sodium deoxycholate for its maximal activity, using 2 mM of phosphatidylcholine and phosphatidylethanolamine as substrates. DE-2 PLA₂ also had a pH optimum in the alkaline region at around pH 8-9 and required >10 mM of Ca²⁺ and approximately 6 mM of sodium deoxycholate for its maximum activity with 2 mM of phosphatidylcholine as a substrate; its enzymatic activity towards phosphatidylethanolamine was greatly inhibited by the addition of sodium deoxycholate. The results demonstrate that red sea bream hepatopancreas contains two enzymatically distinct group I PLA₂ isoforms.     

Purification and characterization of phospholipase A2 from the pyloric caeca of red sea bream, Pagrus major

A phospholipase A₂ was purified 55,000-fold in a yield of 10% from the lipid-free extract of powder of the pyloric caeca of red sea bream to near homogeneity by sequential column chromatography on S-sepharose fast flow, butyl-cellulofine, Asahipak ES-502C cation-exchange HPLC, TSK gel G3000SW gel-filtration HPLC, and Asahipak ODP-50 reversed-phase HPLC. The final preparation showed a single band with the apparent molecular mass of 14 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and an estimated specific activity was 717 µmol min^-1 mg^-1 protein. The purified enzyme had a pH optimum in the range of pH 8.0–9.0 and required the presence of both 8 mM of Ca²⁺ and from 2 to 10 mM of sodium deoxycholate for its maximal activity, using 2 mM of phosphatidylcholine as a substrate. The purified enzyme preferentially hydrolyzed the 2-acyl ester bonds of both phosphatidylglycerol and phosphatidylcholine in the presence of sodium deoxycholate, followed in order by phosphatidylethanolamine and phosphatidyl-serine. In contrast to porcine pancreatic PLA₂, pyloric caeca PLA₂ hydrolyzed mixed-micellar phosphatidylcholine substrate effectively, regardless of the kinds of bile salts used. These results indicate that Ca²⁺-dependent low molecular mass PLA₂, so called secretory PLA₂, occurs in the pyloric caeca of red sea beam.     

Plasma biochemical responses of the omnivorous crucian carp (<Emphasis Type="Italic">Carassius auratus</Emphasis>) to crude cyanobacterial extracts

Healthy crucian carp (Carassius auratus) were treated by intraperitoneal (i.p.) injection of crude cyanobacterial extracts at two doses, 50 and 200 μg MC-LR equiv kg⁻¹ BW. High mortality (100%) was observed within 60 h post injection in the high-dose group. In the treated fish, activities of four plasma enzymes, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), all showed substantial increases, with both dose and time-dependent effects. These increases of enzyme activity indicate severe impairment occurred in the liver of crucian carp over time. Plasma concentrations of energy-related biomolecules including glucose (GLU), cholesterol (CHO), triglyceride (TG), and total protein (TP) showed marked changes in the high-dose group, possibly a nutritional imbalance correlated with the liver injury caused by intraperitoneal exposure to crude cyanobacterial extracts.     

长江口雌性成体纹缟虾虎鱼不同组织的脂肪酸组成分析

实验测定和比较了性腺发育至Ⅴ期的雌性成体纹缟虾虎鱼的肌肉、肝脏和卵巢组织中水分、总脂含量及其脂肪酸组成。结果表明: (1) 肌肉、肝脏和卵巢中的水分含量依次降低, 且差异显著(P<0.05)。肌肉、肝脏和卵巢中的总脂含量分别为1.32%±0.08%、7.75%±0.38%和26.73%±0.59%。3种组织间的总脂含量具有显著性差异(P<0.05), 其中卵巢中总脂含量最高, 其次为肝脏中的含量, 肌肉中含量最低。(2) 肌肉中饱和脂肪酸(SFA)总量明显高于肝脏和卵巢, 3种组织中SFA含量呈显著性差异(P<0.05); 肝脏中单不饱和脂肪酸(MUFA)C 16:1含量最高,肌肉、肝脏和卵巢中的含量分别为11.90%±0.71%、15.28%±2.42%和9.49%±0.37%, 3种组织间含量呈显著性差异(P<0.05); 多不饱和脂肪酸(PUFA)、w3PUFA和w6PUFA在卵巢中的含量显著高于肌肉和肝脏中含量 (P<0.05), 而肌肉和肝脏间的差异不显著 (P>0.05)。肌肉、肝脏和卵巢中二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)的总量依次升高, 在3种组织间的含量分别为30.17%±1.02%、30.53%±0.50%和34.33%±0.71%, 卵巢中的含量显著高于肌肉和肝脏中的含量(P<0.05), 而后两者间的差异不显著 (P>0.05)。3种组织中DHA/EPA的比值分别为1.50、1.75和1.59, w3PUFA/∑w6PUFA的比值分别为6.44、8.05和7.00。DHA/EPA和w3PUFA/∑w6PUFA均在肝脏中最高, 其次为卵巢, 肌肉中最低。     

Development of digestive enzyme activity in larvae of spotted sand bass <Emphasis Type="Italic">Paralabrax maculatofasciatus</Emphasis>. 1. Biochemical analysis

Spotted sand bass Paralabrax maculatofasciatus is a potential aquaculture species in Northwest Mexico. In the last few years it has been possible to close its life cycle and to develop larviculture technology at on pilot scale using live food, however survival values are low (11%) and improvements in growth and survival requires the study of the morpho-physiological development during the initial ontogeny. In this research digestive activity of several enzymes were evaluated in larvae, from hatching to 30 days after hatching (dah), and in live prey (rotifers and Artemia), by use of biochemical and electrophoretic techniques. This paper, is the first of two parts, and covers only the biochemical analysis. All digestive enzyme activities were detected from mouth opening; however the, maximum activities varied among different digestive enzymes. For alkaline protease and trypsin the maximum activities were detected from 12 to 18 dah. Acid protease activity was observed from day 12 onwards. The other digestive enzymes appear between days 4 and 18 after hatching, with marked fluctuations. These activities indicate the beginning of the juvenile stage and the maturation of the digestive system, in agreement with changes that occur during morpho-physiological development and food changes from rotifers to Artemia. All enzymatic activities were detected in rotifers and Artemia, and their contribution to enhancement the digestion capacity of the larvae appears to be low, but cannot be minimised. We concluded that the enzymatic equipment of P. maculatofasciatus larvae is similar to that of other marine fish species, that it becomes complete between days 12 and 18 after hatching, and that it is totally efficient up to 25 dah.     

Detection of hybrids between masu salmon <Emphasis Type="Italic">Oncorhynchus masou masou</Emphasis> and amago salmon <Emphasis Type="Italic">O. m. ishikawae</Emphasis> occurred in the Jinzu River using a random amplified polymorphic DNA technique

Identification of masu salmon Oncorhynchus masou masou and amago salmon O. m. ishikawae was accomplished using a random amplified polymorphic DNA (RAPD) technique. Of 80 primers screened, three primers amplified subspecies-specific fragments (OPA11-1095 ^*, OPB5-1618^* and OPD5-2038 ^*). Based on fragment patterns, 33 hybrids were detected among 150 upstream-migrating individuals in the Jinzu River, central Japan, a long-time habitat of masu salmon, to which amago salmon were recently introduced. All of the individuals examined in the 2000–2002 cohorts were identified as F ₁ and F _x as well as pure masu and amago forms. These results indicated continuing hybridization, the genetic pollution of the native masu salmon population by amago salmon possibly being a serious problem in the Jinzu River. Both standard length and body weight in F ₁ hybrids tended to be less than in pure masu salmon. However, F _x hybrids showed similar body sizes to masu salmon.     

Partial characterization of the digestive enzymes of Pacific bluefin tuna <Emphasis Type="Italic">Thunnus orientalis</Emphasis> under culture conditions

The digestive enzyme activities of Pacific bluefin tuna Thunnus orientalis were evaluated for specific activity and characterized for pH and temperature optima in crude extracts of stomach, caecal mass, and proximal, middle and distal intestine. A higher level of alkaline proteolytic activity was detected in the caecal mass than in the proximal intestine. Total alkaline proteases, trypsin, chymotrypsin and leucine aminopeptidase (LAP) were tested. The temperature and pH analyses showed that proteolytic activity as well as lipase were maximal in the alkaline range, with a maximum at pH 9.0 and at temperatures between 35 and 60°C, except for the pepsin, which showed maximum activity at the same temperatures but in the acid range (pH 3.0). The α-amylase activity showed a broader range in activity, both for pH and temperature, with higher activity over the alkaline pH values and higher temperature. The lipase activity seems to be nondependent on bile salts under our assay conditions, resulting in a significant activity reduction in the presence of bile salts. This knowledge will allow the development of a gastrointestinal model (everted intestine) where food or feed will be hydrolysed with the fish’s own enzymes, a project that is being undertaken in our laboratory as a contribution to the development of novel diets for tuna fish.     

Effects of dietary thiamin on the physiological status of the grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

This study was conducted to evaluate the effects of dietary thiamin on the physiological status of the juvenile grouper, Epinephelus coioides. Graded levels of thiamin (0.08, 0.50, 2.12, 3.15, 4.63, 12.37 mg thiamin kg⁻¹ diet) were fed to grouper juveniles (mean weight: 16.97 ± 0.14 g) for 10 weeks. Although fish fed the thiamin-deficient (TD) diet showed no obvious symptoms of thiamin deficiency or increased mortality, those fed the lowest doses of thiamin (0.08 and 0.50 mg thiamin kg⁻¹ diet) had significantly decreased transketolase activity in the liver. In addition, the level of liver thiobarbituric acid reactive substances in fish fed the TD diet was 33–67% higher than that in fish with the thiamin-supplemented diet. There were no significant differences in superoxide dismutase activity between the different groups of fish.     

Effects of soy sauce <Emphasis Type="Italic">koji</Emphasis> and lactic acid bacteria on the fermentation of fish sauce from freshwater silver carp <Emphasis Type="Italic">Hypophthalmichthys molitrix</Emphasis>

The effects of soy sauce koji and the lactic acid bacterium, Tetragenococcus halophilus, were studied on the fermentation of fish sauce prepared from Chinese silver carp. The fish sauce prepared without koji and the lactic acid bacterium contained low levels of organic materials, total nitrogen, and organic acids. The use of koji was effective in increasing these qualitative parameters and further improved the amino acid score of the fish sauce. Addition of T. halophilus had an effect on lowering the pH value during the initial period of fermentation when the soy sauce koji was also supplemented. In contrast, T. halophilus-like bacteria were found to be predominant for all tanks fermented under the different starting conditions. Although it was not examined whether the T. halophilus-like bacteria observed after fermentation were the same as the starter-bacterium or not, it was suggested that T. halophilus plays an important role in the successful fermentation of silver carp fish sauce. Sensory evaluation conducted with Japanese and Chinese panelists also suggested the superiority of the use of koji for fermentation of silver carp fish sauce.     

Intraspecific diversity of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Harvey) Suringar (Laminariales,Phaeophyta) from Japan,China and Korea,based on the <Emphasis Type="Italic">cox1</Emphasis> gene and ITS2 sequences

As a trial to develop a method of authenticating the place of origin of circulated Undaria pinnatifida products, we investigated their intraspecific genetic diversity using the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) and the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA (rDNA) sequence. Four dried U. pinnatifida products labeled with their origins (one from Japan, one from China and two from Korea), natural plants collected from three locations (two from Japan and one from China), and cultivated plants collected from two locations (one from Japan and one from China) were used in the present study. The amplified fragments of cox1 were 664 bp in length, and the aligned sequences were highly homologous. Among the nine sequences, no insertions or deletions were found and six substitution positions were detected, and they were classified into five haplotypes. In contrast, multiple highly variable regions were found in ITS2, and some of them carried a restriction site for Mbo II. Polymerase chain reaction-restriction fragment length polymorphism analysis showed different restricted profiles among the tested samples. The availability of molecular markers for authenticating food products of U. pinnatifida is discussed.     

The effects of dietary lipid sources and lecithin on the production of giant freshwater prawn <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> larvae in the Mekong Delta region of Vietnam

The giant freshwater prawn, Macrobrachium rosenbergii, is cultured widely in the Mekong Delta region of Vietnam but it is often difficult or expensive for hatchery operators to purchase commercial diets used as a feeding supplement to Artemia nauplii. Therefore, in the present study, the effects of lipid sources and lecithin on the growth and survival rate of M. rosenbergii larvae were examined in order to develop suitable hand-prepared larval diets for seed production of M. rosenbergii in this area. Six egg custard diets consisting of various ratios of lipid (originating from soybean oil and squid oil) and lecithin were used for rearing Macrobrachium rosenbergii larvae. Treatments in which larvae were fed diets containing squid oil exhibited the highest body length and survival rates (7.14–7.43 mm and 51.1–68.1%, respectively), and differed significantly from other treatments (P<0.05). Use of dietary soybean oil yielded the lowest body length and survival rates (6.29–6.75 mm and 22.0–48.7%), respectively). The supplementation of dietary lecithin did not increase final body weight but did improve larval survival rates. The n-3 HUFA content of prawns fed dietary squid oil was higher than those of animals provided with other diets. These results indicated that the most appropriate diet for rearing M. rosenbergii larvae is the diet containing 3% squid oil and 1.5% lecithin.     

Age and growth of the lizardfish <Emphasis Type="Italic">Saurida elongata</Emphasis> from the Tsushima/Korea Strait

We studied the age and growth of the lizardfish Saurida elongata using ground thin sections of otoliths (sagittae) from specimens collected in the Tsushima/Korea Strait between May 1999 and June 2001. A total of 695 individuals with fork lengths (FL) ranging from 189 to 478 mm were examined. The frequency of translucent zone occurrence at the outer margin of the otoliths indicated that translucent zones formed once a year between November and February. Most of the males examined were estimated to be 2–7 years old and the females, 3–9 years old. The maximum estimated age of a male specimen was 10 years and that of a female, 11 years. The estimated von Bertalanffy growth curves were FL _t  = 451{1 − exp[−0.172(t + 2.50)]} and FL _t  = 515{1 − exp[−0.151(t + 2.47)]} for males and females, respectively. At all ages, the FL at a specific age for females was greater than that for males, suggesting that females of this species grow faster than males.     

Mitochondrial DNA sequence variation of Japanese anchovy <Emphasis Type="Italic">Engraulis japonicus</Emphasis> from the Yellow Sea and East China Sea

Sequence variation of a fragment of the mitochondrial cytochrome b (cyt b) and cytochrome c oxidase subunit I (COI) gene was examined using polymerase chain reaction and direct sequencing among three populations of Japanese anchovy Engraulis japonicus from the Yellow Sea and East China Sea. A total of 24 and 47 nucleotide sites were detected variable defining 29 and 32 haplotypes in cyt b and COI data, respectively. All variable sites except one in COI were silent in the two sets of sequences. The Ewens-Watterson test indicated that the observed allelic configurations in both data sets were in full agreement with neutral expectations. The variation level was high, with h=0.957±0.018, π=0.644±0.387 (%) in cyt b data set and h=0.958±0.021, π=0.640±0.386 (%) in COI data set, respectively. However, at the population level, Fst values between pairs of populations were not significantly different from zero (P>0.05) in both data sets. The analysis of haplotype frequency distribution showed no significant differences among populations. Similarly, the analysis of the partitioning of molecular variance indicated that all or almost all of the genetic variation was distributed within populations. Based on the data from this study, the existence of separate genetic stocks in this area were not detected. Mixing of stocks to some extent in migration cycle and dispersal capacity of anchovy’s planktonic larvae could be the reasons for genetic homogeneity in this species in the Yellow Sea and East China Sea.     

High water-temperature tolerance in photosynthetic activity of <Emphasis Type="Italic">Zostera marina</Emphasis> seedlings from Ise Bay,Mie Prefecture,central Japan

Photosynthetic activities of seedlings of Zostera marina were successively measured using a gas volumeter for 6 days at seven light (0–400 μmol photons/m² per s) and 11 water temperature conditions (5–35°C). The seedlings were collected from mature plants (Ise Bay, central Japan), and stored and cultured in incubators accurately controlled at each test temperature. The maximum gross photosynthesis (P _maxg) was recorded at an optimal water temperature of 29°C after 0 days. After 6 days, P _maxg appeared at 25°C and most plants cultured at 29–30°C bleached and withered after the drastic increase of light compensation point (I _c). On the contrary, at 5–28°C, the photosynthetic activities either changed little (5–25°C) or recovered after a temporal reduction (26–28°C); seedlings survived and looked healthy after being cultured for 6 days. The recovery was thought to be an acclimation to tolerate higher water temperature. As a result, the critical upper water temperature for Z. marina seedlings was proposed as 28°C. The temperature was consistent with the previously reported maximum water temperature in habitats around the southern boundary of Z. marina in the northern hemisphere.     

Phylogeography of the Japanese eight-barbel loach <Emphasis Type="Italic">Lefua echigonia</Emphasis> from the Yamagata area of the Tohoku district,Japan

The Japanese eight-barbel loach Lefua echigonia, which is a freshwater fish native to Japan, is distributed from the Tohoku to Kinki districts and is divided into six regional populations according to mtDNA analysis. In this study, we investigated L. echigonia collected from several locations in Yamagata Prefecture and neighboring prefectures using mtDNA control region sequences and confirmed the spatial distribution pattern among the new regional population (Yamagata population). The new population was limited to the Mogami river system in the inland area of Yamagata Prefecture and is distinguished from other regional populations by high sequence divergences.     

Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

The sea cucumber (Stichopus japonicus) is able to undergo autolysis in response to a variety of environmental and mechanical cues. Within the framework of a long-term study of this phenomenon we have purified a protease from the body wall of the sea cucumber by means of ion-exchange chromatography with DE-52 cellulose and gel filtration chromatography with Sephadex G-100. The final enzyme preparation was nearly homogeneous on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 35.5 kDa. The purified enzyme exhibited a maximum activity for the hydrolysis of casein at pH 7.0 and 50°C and a remarkable stability at pH 4.0–7.0 and 40–60°C. Based on the inhibition and activation profiles obtained with numerous specific protease inhibitors and an activator, the protease purified from the body wall of the sea cucumber was defined to be a cysteine-like protease.