真江蓠(Gracilaria verrucosa)对网箱养殖海区的生态修复及生态养殖匹配模式

2006年8～9月,在浙江象山港花鲈(Lateolabrax japonicus)养殖网箱中吊养真江蓠(Gracilaria verrucosa)对网箱养殖造成的水体富营养化进行生态修复研究.通过45d内的平面监测、定点跟踪监测和断面监测,结果表明:该网箱养殖区水体呈严重富营养化状态,营养状态指数(E)为32.00,其营养盐分布由高浓度的中心区向周围150m非养殖水域扩散;真江蓠对养殖区的富营养化海水具有较好的修复效果:江蓠生态修复区及其相邻网箱中水体PO4-P、NO2-N、NH4-N和NO3-N含量显著低于非修复区(P＜0.01),修复区海水PO4-P、NO2-N、NH4-N和NO3-N浓度比非修复区分别降低22%～58%、24%～48%、22%～61%和24%～47%.养殖真江蓠45d后,修复区水体DO浓度和透明度显著高于非修复区(P＜0.05),DO平均提高28%,透明度平均提高30%;而修复区水体Chl-a浓度显著低于非修复区(P＜0.05),平均降低49%.通过建立基于N平衡的鱼藻生态养殖模式,每收获1kg花鲈至少需要匹配江蓠4.7 kg wet wt才可实现对鱼类排放N的完全吸收.因此网箱内栽培江蓠的混合生态养殖模式,可平衡因经济动物养殖所带来的额外营养负荷,有利于实现动物养殖环境的自我修复.     

Polymorphic microsatellite markers in the Zhikong scallop Chlamys farreri

The colony hybridization method was presented for isolating microsatellite markers in Zhikong scallop, Chlamys farreri. We designed primers to amplify 26 unique microsatellites. Fourteen primer pairs amplified clearly and were polymorphic. Based on characterization with 60 unrelated individuals, the number of alleles ranges from two to 13, and the expected heterozygosity ranges from 0.22 to 0.91. These markers have the potential for genetic studies in population structure and intraspecific variation.     

The role of Gracilaria lemaneiformis in eliminating the dissolved inorganic carbon released from calcification and respiration process of Chlamys farreri

Respiration and calcification rate were estimated to quantify the effect of Zhikong scallop Chlamys farreri on marine CO₂ system in Sanggou Bay, China. The C. farreri population in Sanggou Bay sequestered 78.06?±?5.76 g C m^?2 y^?1 for shell formation, while the CO₂ fluxes due to calcification and respiration were 53.95?±?3.98 and 71.69?±?6.51 g C m^?2 y^?1, respectively. In order to eliminate the additional CO₂ released from calcification and respiration process of C. farreri, Gracilaria lemaneiformis were introduced into the integrated system and its role was validated by in situ mesocosm methods. Eight mesocosms (1,000 L) were deployed over 42-h period and consisted of four treatments: seaweed-only, scallop-only (SP), seaweed integrated with scallop (SS), and control (C). The aqueous CO₂ concentration and partial pressure of CO₂ in SP treatments were significantly higher than the other three treatments (p?<?0.01), while there were no difference between SS treatments and C treatments (p?>?0.05). Furthermore, compared with the SP treatments, the presence of the G. lemaneiformis can keep the seawater pH stable. These findings suggest that seaweed and shellfish integrated aquaculture practice cannot only reduce dissolved inorganic carbon but also can alleviate ocean acidification.     

栉孔扇贝血液细胞的免疫功能

利用光镜、扫描电镜和透射电镜技术对栉孔扇贝(Chlamysferreri)血细胞与细胞免疫功能相关的几个因素进行了初步研究。对血细胞的数量和不同功能细胞的比例研究结果表明,健康血淋巴中血细胞的平均密度为3.03±0.11×107cell/ml,其中颗粒细胞占42.6%,透明细胞占57.4%;病贝血淋巴中血细胞的平均密度为2.78±0.34×107cell/ml,其中颗粒细胞占40.2%,透明细胞占59.8%。扫描电镜观察表明,血细胞的表面结构主要有表面光滑型,表面松果型和表面褶皱阿米巴型3类。透射电镜观察表明,颗粒细胞吞噬外源性颗粒(Ⅰ型颗粒)通过溶酶体(Ⅱ型颗粒)进行降解。并观察到同心片层结构出现在吞噬泡的降解过程中。利用APIZYM试剂盒对栉孔扇贝血细胞及血清中的19种酶进行检测,结果在血清中检测到了13种酶,在血细胞中检测到10种酶,健康血淋巴中酶的含量高于病贝。对血细胞吞噬活性的研究结果表明,血细胞对大肠杆菌和对类立克次体(RLO)的吞噬率分别为25.4%和21.7%。颗粒细胞的吞噬活性(30%-40%)远远大于透明细胞(4.8%-14%)。环境胁迫对血细胞吞噬活性的影响的研究结果表明,病原菌感染和温度、盐度等环境胁迫因素对血细胞的吞噬活性均有不同程度的影响,其中高温因素影响较大,但未发现贝龄有显著影响     

Endoenzymes associated with haemocyte types in the scallop (Chlamys farreri)

Haemocyte types of the scallop (Chlamys farreri) were identified by Giemsa stain and flow cytometry (FCM). Additionally, the activities of peroxidase (POD), phenoloxidase (PO) and alkaline phosphatase (ALP) in haemocytes were analysed by immunocytochemical and biochemical methods. The results indicate that there were two types of haemocytes in the scallop, hyalinocytes and granulocytes, and that POD, PO and ALP were more abundant and more active in granulocytes than in hyalinocytes.     

Essential role of seaweed cultivation in integrated multi-trophic aquaculture farms for global expansion of mariculture: an analysis

Ecologically friendly aquaculture crops, such as seaweeds, herbivores, omnivores, and detritivores can be cultured using relatively less of our limited natural resources and produce relatively less pollution. They also top FAO’s estimates of aquaculture crops for the 21st century. These crops already comprise nearly 90% of global aquaculture tonnage, >90% of all aquaculture production in China and >60% of production even in North America. Consumers prefer them, most likely due to their low prices. Production costs of organisms low on the food chain are low due to the ability of these organisms to efficiently utilize low-cost, mostly plant-based diets and to recycle their own waste. Thus, ecologically friendly aquaculture is not a dream but a dominant global reality. The less ecologically-friendly aquaculture of salmon, sea bream, fed shrimp, among others, has attracted public opposition to aquaculture, but these crops totaled approximately only 10% of global production in 2004. The profitability of industrialized monocultures of these crops is threatened further by rising costs of energy and feed, environmental regulation compliance, disease, and public opposition. Current monoculture practices and perceptions intrinsic to the aquaculture industry can be turned around into a vision of sustained profitable expansion of carnivores production with trophically lower organisms in ecologically-balanced aquaculture farms. This category of aquaculture, which is the modern intensive form of polyculture practiced in Asia, feeds the waste of carnivore culture to lower trophic level organisms, primarily algae and mollusks. Species are selected based on their ecological functions in addition to their economic potential. Ecologically-balanced farms turn the costly treatment of carnivore waste outside the farm to a revenue-generating process of biofiltration, conversion, and resource recovery into plant and mollusk crops inside the farm. In doing so, they solve several of the major problems faced by modern aquaculture. The aquaculture industry can protect its own interests – and reap major benefits – by understanding the importance of ecological balance, the potential of seaweeds as components in feeds, and the importance of the culture and R&D of low trophic level organisms. The industry should also accept the relevance of environmental, social, and image aspects of aquaculture to its success. Governments have the tools to reward multi-trophic farms with seaweeds by means of tax credits and nutrient credits and to penalize unbalanced monoculture approaches by means of ‘polluter pays’ fines, thereby providing the multi-trophic farms with a significant economic advantage. Such measures have been discussed, but their implementation has been slow.     

The immunomodulation of inducible nitric oxide in scallop Chlamys farreri

Nitric oxide (NO) is an important signalling molecule which plays an indispensable role in immunity of all vertebrates and invertebrates. In the present study, the immunomodulation of inducible NO in scallop Chlamys farreri was examined by monitoring the alterations of haemocyte behaviours and related immune molecules in response to the stimulations of LPS and/or with S-Methylisothiourea Sulphate (SMT), an inhibitor of inducible NO synthase (NOS). The total activity of NOS and NO concentration in the haemolymph of scallop C. farreri increased significantly at 3, 6 and 12 h after LPS stimulation respectively, whereas their increases were fully repressed when scallops were treated in the collaborating of LPS and SMT. Meanwhile, some cellular and humoral immune parameters were determined after the stimulation of LPS and SMT to investigate the role of inducible NO in innate immunity of scallop. After LPS stimulation, the highest levels of haemocytes apoptosis and phagocytosis were observed at 24 h (38.5 ± 2.5%, P < 0.01) and 12 h (38.6 ± 0.2%, P < 0.01), respectively, and the reactive oxygen species (ROS) level (5.88 ± 0.90%, P < 0.01) of haemocytes and anti-bacterial activity of haemolymph (10.0 ± 2.2%, P < 0.01) all elevated dramatically at 12 h. Although the activity of lysozyme and phenoloxidase (PO) in haemolymph both declined at 48 h (93.0 ± 6.3 U mgprot^?1, 0.40 ± 0.06 U mgprot^?1, P < 0.01), superoxide dismutase (SOD) activity and GSH concentration both increased to the highest level at 24 h post treatment (99.2 ± 8.1 U mgprot^?1, 93.0 ± 6.3 nmol mgprot^?1, P < 0.01). After the collaborating treatment of LPS and SMT, the apoptosis index increased much higher from 48 h, while the increase of haemocytes phagocytosis, ROS level and haemolymph anti-bacteria activities were suppressed completely at 12 h. The declines of lysozyme and PO activity in haemolymph were reversed at 48 h, and the rise of SOD activity and GSH concentration started earlier from 3 h. These results indicated clearly that NO could participate in the scallop immunity and play a crucial role in the modulation of immune response including haemocytes apoptosis and phagocytosis, anti-bacterial activity and redox homeostasis in the haemolymph of scallop.     

Catecholaminergic responses to environmental stress in the hemolymph of Zhikong scallop Chlamys farreri

Catecholamines have long been known to play essential roles in several physiological processes in invertebrates. In this study, enzyme immunoassays were used to examine the levels of catecholamines (including adrenaline (AD), noradrenaline (NA) and dopamine (DA)) in the hemolymph of the scallop, Chlamys farreri, in responses to selected environmental stress. These stressors include high temperature (acute transfer from 17 to 28 degrees C), low salinity (acute transfer from salinity 31 to 20 psu) and air exposure, which are commonly encountered in aquaculture practices or in their natural habitat. The results showed that both AD and NA concentrations increased significantly in response to all the stressors. The levels of these catecholamines peaked 72 hr after acute temperature or salinity stresses, and then decreased gradually. For scallops subjected to air exposure, the duration of AD concentration elevation depended on the exposure temperature with a higher air temperature resulting in a shorter time to return to control level. In contrast, the NA responses were long lasting and no obvious decreases were observed after reaching the peak value. Interestingly, the DA concentration showed an inverse trend in response to stresses compared with AD or NA. These results suggested that the catecholaminergic responses in bivalve hemolymph will provide useful references in predicting the success of aquaculture practices and monitoring the environmental stresses.     

Development of an eco-friendly agar extraction technique from the red seaweed Gracilaria lemaneiformis

The red seaweed, Gracilaria lemaneiformis growing as an aquaculture bioremediator along the coasts of Liaodong Peninsula, China, was investigated for the agar production. An eco-friendly method called agar photobleaching extraction process was developed for the benefit of workers' health and safety of the environment. The native agar (NA), alkali-modified agar (AA), chemical-bleached agar (CA) and photobleached agar (PA), which were extracted using different processes, were evaluated for their physical and chemical properties. The PA showed most desirable performances in terms of gel strength, gelling temperature, sulfate content and 3,6-anhydro-l-galactose content. Among the different processed agars, PA gel strength was 1913 g/cm2, the highest among the different processed agars, which increased 8.6% on the basis of the AA. Further we applied this new technique to extract agars from Gracilaria asiatica, and similar results were obtained with that of G. lemaneiformis. This indicates that the agar photobleaching extraction process is a feasible method for Gracilaria species and has a potential application. During the whole agar photobleaching extraction process the pigment content of G. lemaneiformis declined gradually and the TOC concentration in photobleaching solution increased along with the increase in the irradiation time. The mechanism of agar photobleaching could be elucidated by the photolysis theory.     

The enhanced immune protection of Zhikong scallop Chlamys farreri on the secondary encounter with Listonella anguillarum

It is well known that invertebrates are devoid of adaptive immune components and rely primarily on innate immunity to defend against pathogens, but recent studies have demonstrated the existence of enhanced secondary immune protection in some invertebrates. In the present study, the cumulative mortality of scallops received two successive Listonella anguillarum stimulations was recorded, and variations of immune parameters including phagocytosis (phagocytic rate and phagocytic index), phenoloxidase-like enzyme, acid phosphatase and superoxide dismutase activities were also examined. The scallops received a previous short-term L. anguillarum stimulation were protected against a long-term stimulation of L. anguillarum. Significantly higher level of phagocytic activities and acid phosphatase activity were observed in the scallops received twice stimulations compared with those only received the secondary stimulation. These results indicated that a short-term immersion with L. anguillarum modulated the scallops' immune system and endowed the scallops with enhanced resistance to the secondary bacterial stimulation; phagocytosis and acid phosphatase were suspected to be involved in the protection.     

A preliminary study of the bioremediation potential of Codium fragile applied to seaweed integrated multi-trophic aquaculture (IMTA) during the summer

In integrated multi-trophic aquaculture (IMTA), seaweeds have the capacity to reduce the environmental impact of nitrogen-rich effluents in coastal ecosystems. To establish such bioremediation systems, selection of suitable seaweed species is important. The distribution and productivity of seaweeds vary seasonally based on water temperature and photoperiod. In Korea, candidate genera such as Pophyra, Laminaria, and Undaria grow from autumn to spring. In contrast, Codium grows well at relatively high water temperatures in summer. Thus, aquaculture systems potentially could capitalize on Codium’s capacity for rapid growth in the warm temperatures of late summer and early fall. In this study, we investigated ammonium uptake and removal efficiency by Codium fragile. In laboratory experiments, we grew C. fragile under various water temperatures (10, 15, 20, and 25°C), irradiances (dark, 10, and 100 μmol photons m⁻² s⁻¹), and initial ammonium concentrations (150 and 300 μM); in all cases, C. fragile exhausted the ammonium supply for 6 h. At 150 μM of , ammonium removal efficiency was greatest (99.5 ± 2.6%) when C. fragile was incubated at 20°C under 100 μmol photons m⁻² s⁻¹. At 300 μM of , removal efficiency was greatest (86.3 ± 2.1%) at 25°C under 100 μmol photons m⁻² s⁻¹. Ammonium removal efficiency was significantly greater at 20 and 25°C under irradiance of 100 μmol photons m⁻² s⁻¹ than under other conditions tested.     

Isolation and characterization of 150 novel microsatellite markers for Zhikong scallop (Chlamys farreri)

We isolated and characterized 150 novel microsatellite markers of Zhikong scallop (Chlamys farreri) from three simple sequence repeat‐enriched libraries constructed with (GA)₁₅ and (CA)₁₅. The polymorphism was assessed with 48 individuals, and the result showed the number of allele ranged from two to 30, with an average of 8.4 alleles/locus. The values of observed and expected heterozygosities ranged from 0.0791 to 0.9878 and from 0 to 1.0000, respectively. Sixty‐five loci showed significant departure from Hardy–Weinberg equilibrium, and 14 locus pairs displayed linkage disequilibrium. These markers are therefore potentially useful for conservation studies, population structure assessment, ecological analyses and linkage map construction.     

Monoclonal antibodies developed for detection of an epizootic virus associated with mass mortalities of cultured scallop Chlamys farreri

Recently, an enveloped, spherical RNA virus was identified as the causative agent of mass mortalities among adult scallop Chlamys farreri, which is cultured on the northern coast of China. Hybridomas were prepared from mice immunized with highly purified virions. Four stable hybridomas secreting monoclonal antibodies (MAbs) of IgG isotype were obtained after screening by means of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). The specificity of the MAbs to this virus was confirmed by immunogold electron microscopy (IEM). All the selected MAbs recognized epitopes on the envelope spikes of the virions. Subsequently, the MAbs were used for in situ immunofluorescent detection of the virus in Davidson's fixed tissue sections. The results showed that the fluorescent cells were mostly observed in epithelia of different organs, but not in the epithelium of the digestive diverticulae. Cytopathological changes and focal lesions corresponding to virus-positive cells were clearly recognized in the affected epithelia, revealing a potential role of this virus in pathogenesis.     

海带养殖在桑沟湾多营养层次综合养殖系统中的生态功能

采用现场和实验生态学方法研究了大型经济海藻——海带(Saccharina japonica)的生长、光合作用和氮营养盐的吸收特性。实验结果表明:在1个生长周期内(约200d),海带的湿重与养殖天数呈明显的幂函数(W=1.3886 t~(1.362),R~2=0.9611),海带湿重是长度的幂函数(W=0.0071 L~(2.0882),R~2=0.9392);海带的光合作用放氧速率(O_2mg/h)与湿重(g)具有明显的线性相关(R~2范围为0.950—0.981),直线斜率(反应单位时间单位重量光合作用放氧速率)的变化范围为0.096—0.195(平均0.191),养殖初期单位鲜重的光合放氧能力较弱,后期趋于稳定;不同部位海带藻片对TIN的吸收速率不同,中带部上部(60—110cm)和基部(20—50cm)的吸收速率大于中带部下部(150—200cm)和边缘部,氮饥饿后最初0.5—1h对TIN的吸收速率最高(0.6μmol/g WW),培养24h可去除介质中TIN(初始浓度24.2μmol/L,密度4g/L)的64.2%—97.1%,10℃条件下藻片对营养盐的吸收率和去除率均大于4℃。海带藻片对NO_3-N的吸收速率大于对NH_4-N的吸收速率,24h后对NO_3-N的收速率趋于稳定。结果显示,海带具有较高的生长速度、光合作用产氧和营养盐吸收能力。海带养殖后期,每天可以增加氧气28.8g/m2(光周期按14h计算),收获时海带的平均碳氮含量分别为33.1%和1.8%,以桑沟湾海带养殖产量8.45万t计算,每年可移除2.8万t碳和1538t氮,海带在多营养层次综合养殖系统中具有较高的生态功能。     

Effects of benzo(k)fluoranthene exposure on the biomarkers of scallop Chlamys farreri

Scallops (Chlamys ferrari) were cultured for 30 days in seawater containing benzo(k)fluoranthene (BkF) at 0.5, 1.0 and 10.0 microg/L. No effects were noted on 7-ethoxyresorufin O-deethylase (EROD) activity in digestive gland at low concentrations (0.5 and 1 microg/L) of BkF, but BkF increased the glutathione-S-transferase (GST) activity. At 10 microg/L BkF increased EROD activity significantly, and depressed GST activity. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in digestive gland increased significantly in 0.5 and 1 microg/L BkF. In 10 microg/L concentrations of BkF, the activity of three antioxidant enzymes increased first and reached a peak after a few days, before tapering off towards the end of the 30 day exposure. In high concentrations of BkF, activity of three antioxidant enzymes in gill showed an early peak (12 h), before dropping off. Lipid peroxidation (LPO) levels increased along with sampling times, and there were time- and concentration-effects between LPO levels and BkF. The responses of the gills and the digestive gland were not always parallel which can be explained by differences in the bioavailability of the toxicant. The performance of each biomarker is assessed in the context of the role and advantages of selecting a battery of biomarkers for detecting contamination problems. The use of C. ferrari as a sentinel species for biomonitoring potential toxic effects in situ is discussed as well as mechanisms of BKF toxicity and alexipharmic strategies of C. ferrari.     

Molecular cloning, characterization and expression of a serine protease with clip-domain homologue from scallop Chlamys farreri

Serine proteases play critical roles in a variety of invertebrate immune defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization. The first mollusk serine protease with clip-domain (designated CFSP1) cDNA was obtained from the scallop Chlamys farreri challenged with Vibrio anguillarum by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the C. farreri serine protease was 1211bp, consisting of a 5'-terminal untranslated region (UTR) of 72bp, a 3'-terminal UTR of 77bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1062bp. The CFSP1 cDNA encoded a polypeptide of 354 amino acids with a putative signal peptide of 19 amino acids and a mature protein of 335 amino acids. The deduced amino acid sequence of CFSP1 contained an amino-terminal clip domain, a low complexity region, and a carboxyl-terminal serine protease domain. CFSP1 mRNA was mainly expressed constitutively in the hemocytes and was up-regulated and increased 2.9- and 1.9-fold at 16h after injury and injection of bacteria.