Seasonal Changes of Reproductive Activity and Biochemical Composition of Pen Shell Atrina pectinata Linnaeus,1767 in Bohai Sea,China

Seasonal variations in the biochemical composition and the reproductive cycle of pen shell Atrina pectinata in Bohai Sea were investigated from May 2013 to April 2014. Histological analysis indicated that the reproductive cycle of A. pectinata can be divided into two phases, inactive stage and gametogenesis, which were equally and significantly influenced by seawater temperature and food availability. Gametogenesis began in late autumn(October), and completed in June and July. Spawning took place in August, coinciding with the highest water temperature and the richest phytoplankton. The significantly high glycogen content in adductor muscles sustained throughout the late active and ripeness stages, but plummeted during spawning and inactive stages. The protein content in female gonads exhibited a synchronous increase along with oocyte diameter and lipid content, suggesting that the female gonads could accumulate protein and lipid for vitelline in A. pectinata. Furthermore, the RNA/DNA ratio was found to be a useful index to indicate the level of gonad maturation in both males and females. The findings of the present study provided a foundation for the fishery resource administration and the aquaculture development of this species.     

Zinc and copper bioaccumulation in fish from Laizhou Bay,the Bohai Sea

Abstract Zinc （Zn） and copper （Cu） concentrations were determined in the tissues （muscle, stomach, liver, gills, skin, and gonads） of five commercial fish species （mullet Liza haematocheilus, flathead Platycephalus indicus, mackerel Scomberomorus niphonius, silver pomfret Pampus argenteus, and sea bass Lateolabrax japonicus） from Laizhou Bay in the Bohai Sea. Metal bioaccumulation was highest in the metabolically active tissues of the gonads and liver. Bioconcentration factors for Zn were higher in all tissues （gonads 44.35, stomach 7.73, gills 7.72, liver 5.61, skin 4.88, and muscle 1.63） than the corresponding values for Cu （gonads 3.50, stomach 3.00, gills 1.60, liver 5.43, skin 1.50, and muscle 0.93）. Mackerel tissues accumulated metal to higher concentrations than did other fish species, but bioaccumulation levels were not significantly correlated with the trophic levels of the fish. Zn and Cu concentrations in the tissues were generally negatively correlated with fish length, except for a few tissues of sea bass. Risk assessment based on national and international permissible limits and provisional tolerances for weekly intake of Zn and Cu revealed that the concentrations of these two metals in muscle were relatively low and would not pose hazards to human health.     

Seasonal changes of reproductive activity and biochemical composition of pen shell <Emphasis Type="Italic">Atrina pectinata</Emphasis> Linnaeus, 1767 in Bohai Sea,China

Seasonal variations in the biochemical composition and the reproductive cycle of pen shell Atrina pectinata in Bohai Sea were investigated from May 2013 to April 2014. Histological analysis indicated that the reproductive cycle of A. pectinata can be divided into two phases, inactive stage and gametogenesis, which were equally and significantly influenced by seawater temperature and food availability. Gametogenesis began in late autumn (October), and completed in June and July. Spawning took place in August, coinciding with the highest water temperature and the richest phytoplankton. The significantly high glycogen content in adductor muscles sustained throughout the late active and ripeness stages, but plummeted during spawning and inactive stages. The protein content in female gonads exhibited a synchronous increase along with oocyte diameter and lipid content, suggesting that the female gonads could accumulate protein and lipid for vitelline in A. pectinata. Furthermore, the RNA/DNA ratio was found to be a useful index to indicate the level of gonad maturation in both males and females. The findings of the present study provided a foundation for the fishery resource administration and the aquaculture development of this species.     

Nutrient effects of broodstocks on the larvae in Patinopecten yessoensis

Patinopecten yessoensis is a commercial valuable species. This study deals with the effect of nutrient effects of the broodstock(mainly ovaries) on the larvae. Concentrations of total carbohydrate, total protein and total lipid in the gonads of P. yessoensis from three Hatcheries(Hatchery 1, Hatchery 2, and Hatchery 3) were determined before and after spawning. The relationship between the nutrient concentration in ovaries before spawning(BC) and that of larvae(LC) was assessed as well as the change in nutrient levels in ovaries after spawning(DC). Results indicate that the BC of total carbohydrate(7.66%) and total lipid(14.48%) in ovaries were significantly higher than in testes(5.20%, 5.20% respectively), whereas the BC of total protein in the ovaries was lower(61.76%) than in the testes(81.67%). The different gonadal composition suggests the different nutrient demands between male and female broodstocks in breeding season. P atinopecten yessoensis gonads contained a higher proportion of lipids, in comparison to other bivalves, which might be a response to the low ambient water temperatures. Further analysis of fatty acids showed that the concentrations of n-3PUFA, EPA and DHA in larvae(LC) were positively correlated with BC and DC, indicating the significant nutrient influence of broodstocks on the larvae. As these fatty acids are important in metabolism, and have been demonstrated to be influential to the viability of the larvae, larval growth and the settlement, spat growth, and juvenile survival in many bivalves, they could possibly be used as indexes to evaluate, and predict condition of broodstocks and larvae.     

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.     

马氏珠母贝热休克蛋白HSP60基因的克隆与表达分析

运用cDNA末端快速扩增（RACE）技术,克隆马氏珠母贝（Pinctadamαrtensii）热休克蛋白HSP60基因 cDNA全序列。序列全长2495坤,开放阅读框（ORF） 1734坤,编码577个氨基酸,预测的分子量约为61.79阳, 理论等电点为5.4905＇非翻译区（ 5＇UTR）长150坤, 3＇非翻译区（3＇UTR）长611 bp。同源性比对分析结果,马 氏珠母贝HSP60基因与与太平洋长牡妨（Crωsos仰a gIgω）和光滑双蹄螺（Biomphalaria glabrata）的同源性较 高,达到75%。氨基酸序列分析显示,马氏珠母贝HSP60氨基酸序列具有典型的rnt-HSP60特征序列、C-末端典型的GGM重复基序和一个ATP结合结构域。荧光定量数据分析发现,该基因在闭壳肌、外套腹、血淋巴、肝膜腺、性腺、躁、等6个组织中具有表达,在肝膜腺中表达量最高,腮中次之,而在血液和闭壳肌中仅有少量表达;在脂多糖剌激后,该基因表达水平上调,12 h后达到最大值,之后又逐渐下调,均高于对照组,差异具统计学意 义（P〈0.05）。     

Higher Dietary Arachidonic Acid Levels Improved the Growth Performance,Gonad Development,Nutritional Value,and Antioxidant Enzyme Activities of Adult Sea Urchin(Strongylocentrotus intermedius)

The gonads of sea urchins(Strongylocentrotus intermedius) are characterized by high levels of arachidonic acid(ARA, 20:4 n-6) and eicosapentaenoic acid(EPA, 20:5 n-3). However, to our knowledge, little information is available regarding the physiological response of adult sea urchins to dietary ARA. In the present study, four dietary feeds were formulated with graded ARA(0, 0.5%, 1%, and 2% dry diet). Each diet was randomly allocated to three cages during a 56-day feeding experiment. The results showed that the sea urchin weight gain rate(WGR) and the gonadosomatic index(GI) significantly increased as ARA was equal to or above 1.0% of dry diet(P 0.05). The activities of superoxide dismutase(SOD), catalase(CAT), and total anti-oxidative capacity(T-AOC) were the highest in the coelomic fluid of sea urchins that were fed diets with 1% ARA. The total essential amino acid(TEAA) and its ratio to total non-essential amino acid(TNEAA) showed a similar tendency to WGR and GI as dietary ARA increased, and the highest TEAA and TEAA/TNEAA were observed in the gonads of sea urchins that were fed diets with 1% ARA. Levels of ARA and ARA/EPA of the gonads increased while n-3/n-6 polyunsaturated fatty acid(PUFA) decreased with the increase of dietary ARA(P 0.05). EPA in the gonads of experimental animals fed with formulated feeds showed no significant differences(P 0.05), but was significantly lower than those fed with kelp(P 0.05). These results suggested that relatively higher levels of ARA(1% dry diet) significantly promoted growth, gonad development, activities of antioxidant enzymes, as well as nutritional values(TEAA, TEAA/TNEAA, and PUFA) of adult S. intermedius.     

Nutrient compositions of cultured Skeletonema costatum,Chaetoceros curvisetus,and Thalassiosira nordenskildii

We cultured different-sized fractions of dominant phytoplankton species,Skeletonema costatum,Chaetoceros curvisetus,and Thalassiosira nordenski ldii,collected in different sea areas in various seasons,and measured and compared their C,N,P,Si contents.The N content of these species is similar,while the C,P,and Si contents of S.costatum from eutrophic Changjiang (Yangtze River) estuary are higher than those from Jiaozhou Bay (JZB),particularly the content of Si.The C,N,P,and Si contents of cultured phytoplank...     

Isolation of immune-relating 185/333-1 gene from Sea Urchin (Strongylocentrotus intermedius) and Its expression analysis

The 185/333 gene family involved in the immune response of sea urchin.One 185/333 c DNA was isolated from Strongylocentrotus intermedius,and named as Si185/333-1.Its full-length c DNA was 1246 bp in length with a 906 bp open reading frame encoding a protein of 301 aa.The molecular weight of the deduced protein was approximately 33.1 k D with an estimated PI of p H 6.26.Si185/333-1 had high identities(70%–86%) to most of Sp185/333.An extraordinary identity of 92% was found between Si185/333-1 and Sp185/333 C5 alpha(ABR22474).Moderate identities(63%–64%) were displayed between Si185/333-1 and He185/333.Si185/333-1 had similar structure to Sp185/333.A signal-peptide,a gly-rich region and a his-rich region were found in its secondary structure.RGD motif was found in gly-rich region at position 116–118aa.There was no transmembrane region in Si185/333-1.The element pattern of Si185/333-1 is different from any available pattern that identified in Sp185/333.Si185/333-1 clustered together with pattern C Sp185/333 in phylogenetic tree.The Si185/333-1 m RNA could be detected in tissues including peristomial membrane,coelomocytes,muscle of Aristotles lantern,gut and tube feet,with the highest expression level detected in peristomial membrane and a relatively low expression in ovary and testis.The temporal expression of Si185/333-1 in peristomial membrane and coelomocytes were up-regulated after bacterial,β-D-glucan and ds RNA challenges,reaching the maximum at 12 h post-stimulation.The up-regulation was more obvious in coelomocytes,and bacterial challenge triggered the highest response.These results proved that 185/333-1 gene was involved in the immune defense of S.intermedius,while more studies were necessary for its function in S.intermedius immunity.     

An Evaluation on the Ratio of Plant to Animal Protein in the Diet of Juvenile Sea Cucumber(Apostichopus japonicus): Growth,Nutrient Digestibility and Nonspecific Immunity

This study was conducted to evaluate the effects of plant/animal(P/A) protein ratios(viz.1:4, 1:3, 1:2, 1:1,2:1, 3:1, 4:1) on growth performance, body composition, apparent digestibility of diets, and nonspecific immunity of juvenile sea cucumber(Apostichopus japonicus). Sea cucumbers were divided into 21 plastic tanks, and each tank was stocked with 15 individuals(initial weight: about 23.73 g). Each feed was allocated to three replicates of sea cucumbers. The feeding experiment lasted for 50 days. Results indicated that weight gain rate(WGR) and body wall weight(BWW) significantly increased as dietary ratio of P/A increased from 1:4 to 3:1, and then decreased significantly with further increase of this ratio(P 0.05). The body wall coefficient(BWC) showed a similar tendency to WGR and BWW, but no significance was detected among dietary treatments(P 0.05). The apparent digestibility of dry matter, protein and lipid increased with ratio of P/A increasing from 1:4 to 2:1(P 0.05), and then decreased with further increase of this ratio. Correspondingly, activities of trypsin and amylase were significantly increased as P/A increased from 1:4 to 2:1(P 0.05). The activities of SOD and CAT showed a similar trend with WGR, with the highest value observed in the ratio of 1:2 and 1:1, respectively. Results above showed that moderate or relatively higher ratio of P/A protein(1:1-3:1) significantly increased the growth performance, apparent digestibility, and nonspecific immunity of sea cucumber. This will contribute to improving the feed formulation for juvenile cucumbers.     

Silicon limitation on primary production and its destiny in Jiaozhou Bay, China V： Silicon deficit process

Silicon is a necessary nutrient for diatoms, silicon uptake in diatom reproduction decreased seawater silicon content. This paper clarified the characteristics of silicon transferring in the sea, which plays an important role in phytoplankton growth, zooplankton graze and marine ecosystem. Analysis revealed that silicate is supplied by terrestrial sources, through plankton uptake, death, and eventually deposits to the sea bottom, and cannot diffuse upward. This is a general silicon deficit process. Many global marine waters showed the same silicon transfer route: land→silicon biogeochemical process→sea bottom. River flow brings abundant silicate into marine waters, silicate concentration in the waters decreased in the distance away from the river estuaries. In discussion of silicon characteristics and its transfer route, it was considered that the main factor controlling the mechanism of diatom and non-diatom red tides occurrence is silicon, and the changes in silicon source. Human activities, such as sea-route cutting by building embankment and dam, and silicon supplement by the sea, such as sandstorm, rainstorm and storm tide, have largely impaired the earth ecosystem and hugely threatened the human existence. It is suggested in this paper that man should resume the original face of the Si input into the sea to keep natural ecosystem in sustainable pattern.     

Studies on the phosphorus requirement and proper calcium/phosphorus ratio in the diet of the black sea bream (Sparus macrocephalus)

An expriment on the phosphorus requirement and the proper Ca/P ratio in the diet of the black sea bream using the phosphorus gradient method (with casein as basic diet, sodium dihydrogen phosphate as source of phosphorus, and calcium lactate as source of calcium) showed that growth was greatly affected by the diet's phosphorus content and Ca/P ratio. Inadequate phosphorus in the diet resulted in slow growth and poor food conversion ratio (FCR). Analyses of the fish body showed it contained a high level of lipid but a low level of moisture, ash, calcium and phosphorus. The optimal values of phosphorus and Ca/P ratio in the black sea bream diet are 0.68% and 1∶2 respectively. Phosphorus in excess of this optimum value resulted in slow growth or even death. The results of this experiment clearly indicated that phosphorus is the principal mineral additive affecting black sea bream growth. Contribution No. 1972 from the Institute of Oceanology, Academia Sinica.     

Localization and Dynamic Expression of a 27.8kDa Receptor Protein for Lymphocystis Disease Virus Infection in Sea Bass(Lateolabrax japonicus) Tissues

Lymphocystis disease virus(LCDV) infects target cells by attaching to a 27.8 k Da receptor(27.8R) protein in flounder Paralichthys olivaceus, and anti-27.8R monoclonal antibodies(MAbs) have been developed. However, the 27.8R existence in tissues of sea bass(Lateolabrax japonicus) and its role in LCDV infection have remained unclear. In this study, the results of western blotting demonstrated that the same 27.8R was shared by flounder and sea bass. LCDV-free sea bass individuals were intramuscularly injected with LCDV, and viral copies were detected in tissues from 3 h post infection and showed a time-dependent increase during 9 days infection. Distribution and synthesis of 27.8R in sea bass tissues were investigated by using anti-27.8R MAbs as probes. It was found that 27.8R was distributed in all the tested tissues. The levels of 27.8R protein were highest in gill and skin, then a bit lowly in stomach, head kidney and heart, followed by spleen, intestine, blood cells, gonad and liver, and least in kidney and brain in healthy sea bass. Upon LCDV infection, 27.8R synthesis was up-regulated in each tissue, and higher in the tissues with higher LCDV copies. The 27.8R and LCDV were detected in some peripheral blood leukocytes but not in red blood cells. These results suggested that 27.8R was widely distributed in sea bass tissues, and it served as a receptor and correlated with tissue tropism of LCDV infection. Furthermore, leukocytes had the potential of being a LCDV carrier and were responsible for a systemic infection of LCDV in sea bass.     

STUDIES ON THE PHOSPHORUS REQUIREMENT AND PROPER CALCIUM/PHOSPHORUS RATIO IN THE DIET OF THE BLACK SEA BREAM(SPARUS MACROCEPHALUS)

An experiment on the phosphorus requirement and the proper Ca/P ratio in the diet of the black sea bream using the phosphorus gradient method (with casein as basic diet, sodium dihydrogen phosphate as source of phosphorus, and calcium lactate as source of calcium) showed that growth was greatly affected by the diet's phosphorus content and Ca/P ratio. Inadequate phosphorus in the diet resulted in slow growth and poor food conversion ratio (FCR). Analyses of the fish body showed it contained a high level of lipid but a low level of moisture, ash, calcium and phosphorus. The optimal values of phosphorus and Ca/P ratio in the black sea bream diet are 0.68% and 1:2 respectively. Phosphorus in excess of this optimum value resulted in slow growth or even death. The results of this experiment clearly indicated that phosphorus is the principal mineral additive affecting black sea bream growth.     

Metabolic rates and biochemical compositions of Apostichopus japonicus (Selenka) tissue during periods of inactivity

Estivation, hibernation, and starvation are indispensable inactive states of sea cucumbers Apostichopus japonicus in nature and in culture ponds. Generally, temperature is the principal factor that induces estivation or hibernation in the sea cucumber. The present study provided insight into the physiological adaptations of A. japonicus during the three types of inactivity (hibernation, estivation, and starvation) by measuring the oxygen consumption rates (Vo₂) and biochemical compositions under laboratory conditions of low (3°C), normal (17°C) and high (24°C) temperature. The results show that the characteristics of A. japonicus in dormancy (hibernation and estivation) states were quite different from higher animals, such as fishes, amphibians, reptiles, and mammals, but more closely resembled a semi-dormant state. It was observed that the shift in the A. japonicus physiological state from normal to dormancy was a chronic rather than acute process, indicated by the gradual depression of metabolic rate. While metabolic rates declined 44.9% for the estivation group and 71.7% for the hibernation group, relative to initial rates, during the 36 d culture period, metabolic rates were not maintained at constant levels during these states. The metabolic depression processes for sea cucumbers in hibernation and estivation appeared to be a passive and an active metabolic suppression, respectively. In contrast, the metabolic rates (128.90±11.70 μg/g h) of estivating sea cucumbers were notably higher (107.85±6.31 μg/g h) than in starving sea cucumbers at 17°C, which indicated that the dormancy mechanism here, as a physiological inhibition, was not as efficient as in higher animals. Finally, the principle metabolic substrate or energy source of sea cucumbers in hibernation was lipid, whereas in estivation they mainly consumed protein in the early times and both protein and lipid thereafter.     

Localization and dynamic expression of a 27.8 kDa receptor protein for lymphocystis disease virus infection in sea bass (<Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>) tissues

Lymphocystis disease virus (LCDV) infects target cells by attaching to a 27.8 kDa receptor (27.8R) protein in flounder Paralichthys olivaceus, and anti-27.8R monoclonal antibodies (MAbs) have been developed. However, the 27.8R existence in tissues of sea bass (Lateolabrax japonicus) and its role in LCDV infection have remained unclear. In this study, the results of western blotting demonstrated that the same 27.8R was shared by flounder and sea bass. LCDV-free sea bass individuals were intramuscularly injected with LCDV, and viral copies were detected in tissues from 3 h post infection and showed a time-dependent increase during 9 days infection. Distribution and synthesis of 27.8R in sea bass tissues were investigated by using anti-27.8R MAbs as probes. It was found that 27.8R was distributed in all the tested tissues. The levels of 27.8R protein were highest in gill and skin, then a bit lowly in stomach, head kidney and heart, followed by spleen, intestine, blood cells, gonad and liver, and least in kidney and brain in healthy sea bass. Upon LCDV infection, 27.8R synthesis was up-regulated in each tissue, and higher in the tissues with higher LCDV copies. The 27.8R and LCDV were detected in some peripheral blood leukocytes but not in red blood cells. These results suggested that 27.8R was widely distributed in sea bass tissues, and it served as a receptor and correlated with tissue tropism of LCDV infection. Furthermore, leukocytes had the potential of being a LCDV carrier and were responsible for a systemic infection of LCDV in sea bass.     

末次冰期深切谷的识别及其在生物气勘探中的意义──以钱塘江深切谷为例

本文通过钱塘江深切谷的论述对末次冰期深切谷的恢复主要依据下列证据进行：（1）谷底为侵蚀不整合面，为末次冰期海平面下降，河流侵蚀切割所致；（2）谷内为异常厚的河流沉积物所充填，是冰后期海平面上升时形成，其中河漫滩沉积年代约14000～7500a以前；（3）深切谷之上被海相地层覆盖。超浅层生物气田分布在深切谷内，当今河流及全新世晚期河口湾与末次冰期深切谷既有区别又有继承性，其流域是浅层生物气藏分布的有利地段。     

Quantitative Evaluation of the Larval Density of Sea Potato Acaudina spp. with Mitochondrial Molecular Marker

The sea potato Acaudina spp. is a species of sea cucumber, belonging to the class Holothuroidea under Phylum Echinodermata. It becomes blooming recently in the East Sea of China, causing serious ecological problems. However, until now there are no molecular data for its larval identification and population genetic analysis. In this study, we firstly screened a mtDNA fragment and demonstrated that it was the species-specific molecular marker for the identification of Acaudina spp. We then developed a quantitative polymerase chain reaction(PCR) method to evaluate the larval density of Acaudina spp. based on this molecular probe. Utilizing this method, we examined 116 plankton samples collected in four seasons from 13 stations along the coastal region in Fujian province, China. The results showed that the high larval density was presented at stations 1, 2, and 3, which were near a quay in the coast. The larval density increased from April and reached the highest value in June and July, suggesting temperature might be the main environmental factor on the effects of its population distribution and density. Our work provides an important molecular tool for species identification and risk evaluation of a potentially invasive species.