Intestinal disease of scattered mirror carp Cyprinus carpio caused by Thelohanellus kitauei and notes on the morphology and phylogeny of the myxosporean from Sichuan Province,southwest China

The mass mortality of pond-reared scattered mirror carp, Cyprinus carpio, caused by Thelohanellus kitauei, occurred at fish farms in Sichuan Province, southwest China. Morphological and molecular analyses were supplemented with histological evaluation of infected tissues to better understand the route of infection and the pathological effects of T. kitauei on the fish host. The intestine of the diseased host was full of large cysts of the myxosporean. The cysts range from 2 cm to 3.6 cm in diameter. Histopathology indicated that T. kitaue i first invaded the submucosa of the host intestine and then moved into the mucosa layers with the development of their spores, finally entering into the enteric cavity of the hosts after the disruption of mucosa layers. The pyriform spores of T. kitaue i were surrounded by the transparent spore sheath, measuring 25.98 μm±0.95 μm in body length, 8.72 μm±0.51 μm in body width, and 7.86 μm±0.26 μm in body thickness. The single polar capsule was pyriform, measuring 14.73 μm±0.92 μm in length and 6.82 μm±0.45 μm in width, with eight to 10 turns of filament coils winding inside. Phylogenetic analysis based on the 18 S small-subunit ribosomal DNA sequences indicated that minimal genetic differences were present between T. kitauei samples from South Korea and from China. Close affinity was found between the genus Thelohanellus and Myxobolus. Additionally, two polar capsule nuclei were found at the anterior end of the single polar capsule in spores of T. kitaue i stained with hematoxylin and eosin, which suggested the separation of the genus Thelohanellus from Myxobolus.     

Effects of the Dietary Probiotic Clostridium butyricum on Intestine Digestive and Metabolic Capacities,SCFA Content and Body Composition in Marsupenaeus japonicus

A 56-day feeding trial was performed to evaluate the effects of dietary probiotic Clostridium butyricum(CB) on intestine digestive and metabolic capacities, intestine short-chain fatty acids(SCFA) content and body composition of kuruma shrimp Marsupenaeus japonicus. Shrimp s were randomly allocated into 9 tanks, 30 each, and f ed with diets containing different levels of C. butyricum(1×10~9 cfug~(-1)): 0 mgg~(-1) feed(Control), 100 mgg~(-1) feed(CB-100), 200 mgg~(-1) feed(CB-200), while each level was triplicated. The results indicated that compared with the control group, the intestine pepsin(Pep) activity and 5-hydroxytryptamine(5-HT) concentration of two C. butyricum group s were both increased. Amylase(AM Y) and lipase(LPS) activities wer e only induced in CB-200 group. Alanine aminotransferase(ALT) and aspartate aminotransferase(A ST) activities of two C. butyricum group s showed no significant change. The α-amylase(AM Y) gene expression was induced in CB-200 group, and tryp sin gene expression of two C. butyricum treated group s were both induced. Intestine SCFA content and body composition analysis showed that the contents of propionic acid, butyric acid and the crude protein of two C. butyricum group s were all higher than those of control. These results revealed that C. butyricum can modulate intestine digestive and metabolic capacities, improve intestine SCFA content and body crude protein content in M. japonicus.     

Histopathological Observation of Lymphocystis Disease and Lymphocystis Disease Virus （LCDV） Detection in Cultured Diseased Sebastes schlegeli

Lymphocystis nodules occurring in the cultured sting fish Sebastes schlegeli were observed under light and electron microscope. Lymphocystis disease virus (LCDV) in the tissues of diseased fish was detected with indirect immunofluorescence test (IFAT). Results showed that lymphocystis cells had overly irregular nuclei, basophilic intracytoplasmic inclusion bodies with virions budding from the surface, and hyaline capsules outside the cell membrane. Numerous virus particles about 200 nm in diameter scat- tered in the cytoplasm, electron-dense particles 70-80 nm in diameter filled in perinuclear cisterna, and membrane-enveloped parti- cles with electron-dense core of 70-80 nm appeared around cellular nucleus. IFAT using monoclonal antibody against LCDV from Paralichthys olivaceus revealed that specific green fluorescence was present in the cytoplasm of lymphocystis cells, epithelium of stomach, gill lamellae, and muscular fibers under epidermis of S. schlegeli, just as that in the cytoplasm of lymphocystis cells of P. olivaceus, suggesting the presence of LCDV in these tissues.     

Cytochemical characterization of yolk granule acid phosphatase during early development of the oyster Crassostrea gigas (Thunberg)

In this study,a cytochemical method and transmission electron microscopy was used to examine acid phosphatase activities of yolk granules throughout the early developmental stages of the Pacific oyster Crassostrea gigas. This study aimed to investigate the dynamic change of yolk granule acid phosphatase,and the mechanisms underlying its involvement in yolk degradation during the early developmental stages of molluscs. Three types of yolk granules(YGI,YGII,and YGIII) that differed in electron density and acid phosphatase reaction were identified in early cleavage,morula,blastula,gastrula,trochophore,and veliger stages. The morphological heterogeneities of the yolk granules were related to acid phosphatase activity and degrees of yolk degradation,indicating the association of acid phosphatase with yolk degradation in embryos and larvae of molluscs. Fusion of yolk granules was observed during embryogenesis and larval development of C. gigas. The fusion of YGI(free of acid phosphatase reaction) with YGII(rich in acid phosphatase reaction) could be the way by which yolk degradation is triggered.     

Change of digestive physiology in sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> (Selenka) induced by corn kernels meal and soybean meal in diets

The present study was conducted to determine the change of digestive physiology in sea cucumber Apostichopus japonicus(Selenka) induced by corn kernels meal and soybean meal in diets. Four experimental diets were tested, in which Sargassum thunbergii was proportionally replaced by the mixture of corn kernels meal and soybean meal. The growth performance, body composition and intestinal digestive enzyme activities in A. japonicus fed these 4 diets were examined. Results showed that the sea cucumber exhibited the maximum growth rate when 20% of S. thunbergii in the diet was replaced by corn kernels meal and soybean meal, while 40% of S. thunbergii in the diet can be replaced by the mixture of corn kernels meal and soybean meal without adversely affecting growth performance of A. japonicus. The activities of intestinal trypsin and amylase in A. japonicus can be significantly altered by corn kernels meal and soybean meal in diets. Trypsin activity in the intestine of A. japonicus significantly increased in the treatment groups compared to the control, suggesting that the supplement of corn kernels meal and soybean meal in the diets might increase the intestinal trypsin activity of A. japonicus. However, amylase activity in the intestine of A. japonicus remarkably decreased with the increasing replacement level of S. thunbergii by the mixture of corn kernels meal and soybean meal, suggesting that supplement of corn kernels meal and soybean meal in the diets might decrease the intestinal amylase activity of A. japonicus.     

Identification of two Skeletonema costatum-like diatoms by fluorescence in situ hybridization

A harmful algae bloom (HAB) is a dense aggregation of algae in a marine or aquatic environment that can result in significant environmental problems. To forecast the occurrence of HAB, development of a rapid and precise detection method is urgently required. In this study, two Skeletonema costatum-like diatoms (SK-1 and SK-2), were identified morphologically under a light microscope, and detected using fluorescent in situ hybridization (FISH). Strain SK-1 was isolated from a frequently HAB affected area of the East China Sea, and strain SK-2 from an aquatic farm in Qingdao, China. Fluorescent DNA probes were designed that were complementary to the ITS sequence (including 5.8S rDNA) of strain SK-1. After hybridization, strong green fluorescence was observed in cells of strain SK-1 under an epifluorescence microscope; however, no such fluorescence was observed with strain SK-2, which indicates that probes hybridized only the DNA of the target strain, SK-1, in species-specific manner, and that the two strains do not belong to a same species. This finding was confirmed by ITS sequence analysis. The FISH technique used in this study was sensitive, simple, and rapid, and is a promising tool for detecting target HAB species in natural environments.     

Seasonal changes in phytoplankton biomass and dominant species in the Changjiang River Estuary and adjacent seas: General trends based on field survey data 1959–2009

The characteristics of seasonal variation in phytoplankton biomass and dominant species in the Changjiang River Estuary and adjacent seas were discussed based on field investigation data from 1959 to 2009. The field data from 1981 to 2004 showed that the Chlorophyll-a concentration in surface seawater was between 0.4 and 8.5 μg dm-3. The seasonal changes generally presented a bimodal trend, with the biomass peaks occurring in May and August, and Chlorophyll-a concentration was the lowest in winter. Seasonal biomass changes were mainly controlled by temperature and nutrient levels. From the end of autumn to the next early spring, phytoplankton biomass was mainly influenced by temperature, and in other seasons, nutrient level(including the nutrient supply from the terrestrial runoffs) was the major influence factor. Field investigation data from 1959 to 2009 demonstrated that diatoms were the main phytoplankton in this area, and Skeletonema costatum, Pseudo-nitzschia pungens, Coscinodiscus oculus-iridis, Thalassinoema nitzschioides, Paralia sulcata, Chaetoceros lorenzianus, Chaetoceros curvisetus, and Prorocentrum donghaiense Lu were common dominant species. The seasonal variations in major dominant phytoplankton species presented the following trends: 1) Skeletonema(mainly S. costatum) was dominant throughout the year; and 2) seasonal succession trends were Coscinodiscus(spring) →Chaetoceros(summer and autumn) → Coscinodiscus(winter). The annual dominance of S. costatum was attributed to its environmental eurytopicity and long standing time in surface waters. The seasonal succession of Coscinodiscus and Chaetoceros was associated with the seasonal variation in water stability and nutrient level in this area. On the other hand, long-term field data also indicated obvious interannual variation of phytoplankton biomass and community structure in the Changjiang River Estuary and adjacent seas: average annual phytoplankton biomass and dinoflagellate proportion both presented increased trends during the 1950 s-2000 s.     

The Combined Effect of Plastic Particles Size and Concentration on Rotifers’ (Brachionus plicatilis) Performance

The presence of microplastics in aquatic ecosystems is of increasing global concern.Nano-sized plastics,in particular,can penetrate the cell membrane and cause biological death.Our study evaluated the combined impacts of several polystyrene mi-crospheres’sizes and nominal concentrations on the overall performance changes of Brachionus plicatilis.Experimental animals were exposed to three microplastic sizes(0.08,0.5 and 6μm)and five nominal concentrations(0,0.5,2,8,32μg mL?1)for 20 d.Our results showed that the toxicological effect of particle size on rotifers did not significantly depend on the nominal concentration.The interaction between the nominal concentration and size occurred only for body length and lorica width.Specifically,high nominal concentrations of microplastics that were close to nanometer size significantly impaired the overall vitality of rotifers,embodied in shortage of body type,delay in the arrival of maturity,reduction in the cumulative number of neonates,and the advance of the death process.In comparison,fair-sized size(0.5 and 6μm)displayed non-significant damage except for individual groups.Most notably,the net reproductive yield was only a third of what it was in the original environment,implying that there was not much fertility left.Besides,with the development of rotifers,the adverse effects of polystyrene microsphere drive had become more and more serious.     

Effect of dietary genistein on growth performance,digestive enzyme activity,and body composition of Nile tilapia Oreochromis niloticus

An 8-week feeding experiment was performed to evaluate the effect of dietary genistein on growth performance,body composition,and digestive enzymes activity of juvenile Nile tilapia(Oreochromis niloticus).Four isonitrogenous and isoenergetic diets were formulated containing four graded supplements of genistein:0,30,300,and 3 000 μg/g.Each diet was randomly assigned in triplicate to tanks stocked with 15 juvenile tilapia(10.47±1.24 g).The results show that 30 and 300 μg/gdietary genistein had no significant effect on growth performance of Nile tilapia,but the higher level of genistein(3 000 μg/g)significantly depressed the final body weight and specific growth rate.There was no significant difference in survival rate,feed intake,feed efficiency ratio or whole body composition among all dietary treatments.An assay of digestive enzymes showed that the diet containing 3 000 μg/ggenistein decreased stomach and hepatopancreas protease activity,and amylase activity in the liver and intestine,while a dietary level of300 μg/g genistein depressed stomach protease and intestine amylase activities.However,no significant difference in stomach amylase activity was found among dietary treatments.Overall,the results of the present study indicate that a high level of dietary genistein(3 000 μg/g,or above) would significantly reduce the growth of Nile tilapia,partly because of its inhibitory effect on the activity of major digestive enzymes.Accordingly,the detrimental effects of genistein,as found in soybean products,should not be ignored when applied as an alternative ingredient source in aquaculture.     

Development of fatty acid biomarkers for the identification of wild and aquacultured sea cucumber (Apostichopus japonicus)

In this study,the fatty acids(FAs) of the organs and tissues of sea cucumber(Apostichopus japonicus) were profiled in order to compare the FA composition of sea cucumber collected from natural habitat(wild) and cages(cultured).The differences in FA contents in dermomuscular tube,peripharyngeal annulus,gonad and intestine(with or without content) between the wild and the cultured were determined.The main fatty acids in all organs and tissues were 20:5n-3,16:1n-7,20:4n-6,22:6n-3,18:0,and 18:1n-7.The basically different FAs of body wall and digestive tube were 16:1n-7,18:1n-9 and 20:1n-11.The ratio of saturated to mono-and polyunsaturated FAs in digestive tube was independent on inside content while there was a redistribution of the total amount of n-3 and n-6 fatty acids.The comparison of FA composition of the wild and the cultured sea cucumber showed that 20:5n-3,16:1n-7 and 18:1n-7 predominated the wild while 20:4n-6 predominated the cultured.The content of branched-chain fatty acids in the wild was 3%–4% and about 9% in the cultured.The possible FAs for identifying the wild and the cultured sea cucumbers were selected.It was suggested that the indexes such as the ratio of either(n-3:n-6) to(n-7:n-6) or(n-3) +(n-7) to(n-6) may serve as the biomarkers distinguishing the wild and the cultured sea cucumber.     

Studies on the Tolerating Mechanism for Sulfide in Urechis unicinctus (Echiura: Urechidae) ──Cytological Observation on Urechis unicinctus in Different Hydrogen Sulfide Environment

ＩＮＴＲＯＤＵＣＴＩＯＮＵｒｅｃｈｉｓａｎｉｍａｌｓａｒｅｏｆｔｅｎｐｒｅｓｅｎｔｉｎＨ2 Ｓｒｉｃｈｓｅａａｒｅａｗｈｅｒｅｍｏｓｔａｎｉｍａｌｓｃａｎｎｏｔｌｉｖｅ .ＩｔｃａｎｔｏｌｅｒａｔｅｔｈｅｓｔｒｏｎｇｔｏｘｉｃｉｔｙｏｆＨ2 Ｓ .ＭａｎｙｓｃｈｏｌａｒｓｗｅｒｅｉｎｔｅｒｅｓｔｅｄｉｎｔｈｅｔｏｌｅｒａｔｉｎｇｍｅｃｈａｎｉｓｍｏｆＨ2 ＳｉｎｔｈｅＵｒｅｃｈｉｓａｎｉｍａｌ (ＰｏｗｅｌｌａｎｄＡｒｐ ,1 989;Ａｒｐｅｔａｌ.,1 992 ) .Ａｒｐｅｔａｌ.(1 992 )ｉｎｄｉｃａｔｅｄｔｈａｔｔｈｅ…     

The antitumor effect of bromophenol derivatives <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">Leathesia nana</Emphasis> extract <Emphasis Type="Italic">in vivo</Emphasis>

To investigate the antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo, six bromophenol derivatives 6-(2,3-dibromo-4,5-dihydroxybenzyl)-2,3-dibromo-4,5-dihydroxy benzyl methyl ether (1), (+)-3-(2,3-dibromo-4,5-dihydroxyphenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroisobenzofuran (2), 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)-5-methoxymethyl-pyrocatechol (3), 2,2′,3,3′-tetrabromo-4,4′,5,5′-tetrahydroxy-diphenylmethane (4), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (5), 2,2′,3-tribromo-3′,4,4′,5-tetrahydroxy-6′-ethyloxymethyldiphenylmethane (6) were isolated from brown alga Leathesia nana, and their cytotoxicity were tested by MTT assays in human cancer cell lines A549, BGC-823, MCF-7, B16-BL6, HT-1080, A2780, Bel7402 and HCT-8. Their inhibitory activity against protein tyrosine kinase (PTK) with over-expression of c-kit was analyzed also by ELISA. The antitumor activity of ethanolic extraction of Leathesia nana (EELN) was evaluated on S₁₈₀-bearing mice. All compounds showed very potent cytotoxicity against all of the eight cancer cell lines with IC₅₀ below 10 μg/mL. In PTK inhibition study, all bromophenol derivatives showed moderate inhibitory activity and compounds 2, 5 and 6 showed significant bioactivity with the inhibition ratio of 77.5%, 80.1% and 71.4%, respectively. Pharmacological studies reveal that EELN could inhibit the growth of Sarcoma 180 tumor and increase the indices of thymus and spleen to improve the immune system remarkably in vivo. Results indicated that the bromophenol derivatives and EELN can be used as potent antitumor agents for PTK over-expression of c-kit and considered in a new therapeutic strategy for treatment of cancer. Supported by the National High Technology Research and Development Program of China (863 Program, No. 2007AA09Z410) and Knowledge Innovation Program of Chinese Academy of Sciences (No. KZCX2-YW-209)     

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 <Emphasis Type="Italic">Urechis unicinctus</Emphasis> juveniles on chemical characteristics of organically contaminated coastal sediment

Biological activities of marine benthos such as burrowing and feeding may change sediment characteristics. We conducted three experiments to examine the potential of using juveniles of a spoon worm Urechis unicinctus to improve the quality of organically contaminated coastal sediment. Sediment samples were collected from a site that was heavily contaminated with organic matter (Seonso) and two sites that were clean (Myo-do, Dolsan-do). Urechis juveniles, obtained by artificial fertilization and cultured in the laboratory, were introduced to the sediment (weight 3 kg, depth 10 cm) at a density of 500 individuals per aquarium (length 50 cm, width 35 cm, height 30 cm) (Experiment 1), or at densities ranging from 100 to 900 individuals per beaker (Experiment 2). To examine how sediment contamination can be modified by the effects of Urechis, 500 individuals (per aquarium) were exposed to the Seonso contaminated sediment that had been mixed with 0–100% clean sand (Experiment 3). Each experiment lasted two months and sediment samples were collected every 15 d to determine the several indexes of sediment quality, which included acid volatile sulfide (AVS), chemical oxygen demand (COD) and total ignition loss (TIL). In Experiment 1, the existence of Urechis did not result in significant changes in quality indexes in the sediments collected from Myo-do, Dolsan-do. However, AVS, COD and TIL of the Seonso sediment all decreased significantly after co-incubation with Urechis juveniles for 30 to 45 d. Experiment 2 showed that a density of at least 300 juveniles per beaker was necessary to significantly reduce all three quality indexes, and the magnitude of reduction was positively correlated with juvenile density. Experiment 3 revealed that Urechis juveniles were effective in reducing the AVS, COD and TIL of the Seonso sediment that had been mixed with 60%, 80%, and 80% of clean sand, respectively. The results of the present study therefore indicated that juveniles of this spoon worm have the potential to be used to improve the quality of organically contaminated sediment in coastal waters.     

Structure and function of the digestive system of <Emphasis Type="Italic">solen grandis</Emphasis> dunker

Structure and function of the digestive system of a bivalve mollusc, Solen grandis, were studied using light microscopy and histochemical methods. The wall of digestive tube consists of four layers: the mucosal epithelium, connective tissue, muscular and fibrosa or serosa (only in the portion of rectum) from the inner to the outer. The ciliated columnar epithelial cells, dispersed by cup-shaped mucous cells, rest on a thin base membrane. There are abundant blood spaces in connective tissue layer. The digestive diverticula are composed of multi-branched duct and digestive tubules. The digestive tubules are lined with digestive and basophilic secretory cells, and surrounded by a layer of smooth muscle fibers and connective tissues. Activities of acid and alkaline phosphatases, esterase and lipase are detected in the digestive cells, and the epithelia of stomach and intestine, suggesting that these cells are capable of intracellular digesting of food materials and absorbing. Besides, acid phosphatase and esterase activities are present in the posterior portion of esophagus. Phagocytes are abundant in blood spaces and the lumens of stomach and intestine, containing brown granules derived from the engulfed food materials. The present work indicates that phagocytes play important roles in ingestion and digestion of food materials, which is supported as well by the activities of acid phosphatase, esterase and lipase detected in blood spaces.     

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.     

Immune response of <Emphasis Type="Italic">Pseudosciaena crocea</Emphasis> to the injection of <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

For the investigation of anti-infection immune response of Pseudosciaena crocea, 160 healthy fish samples were categorized into infected and control groups. Each individual fish in the infected group was injected intraperitoneally (i.p.) with 0.2 ml bacterial suspension of Vibrio alginolyticus in density of 2×10⁷ CFU/ml, while each individual in the control group was injected i.p. with 0.2 ml sterile saline solution (0.85%). It was observed that the artificial injection of V. alginolyticus significantly increased the number of erythrocytes, leucocytes, lymphocytes in peripheral blood as well as peripheral serum antibacterial activity and antibody titer of large yellow croaker, and significantly reduced the number of peripheral blood granulocytes as compared with those in the control group. No significant difference in acid phosphytase and superoxide dismutase activity of serum was detected between the two groups. It is suggested that non-specific immune factors including leucocytes and anti-bacteria substance in peripheral blood played important role at the initial stage of infection, and specific immune factors such as antibody then played important role in response to anti-infection at the latter stage. Supported by the National High Technology Research and Development Program of China (863 program, No. 2007AA09Z115) and Technology Program of Xiamen (No. 3502Z73019)     

Variations of ion-transport enzyme activities during early development of the shrimps <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> and <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

The ion-transport enzyme activities were studied in the nauplii 1–2, zoea 1–3, mysis 1–3, and postlarva 1–7 of the shrimps Marsupenaeus japonicus and Fenneropenaeus chinensis. The results showed that total ATPase, Na⁺-K⁺-ATPase, and V-ATPase (V-type H⁺-ATPase) activities increased during the early development for both the species, from zero in nauplii to stable levels after zoea stage. The enzyme activities of the latter species were significantly higher than those of the former species after zoea stage (F>F _0.05). The contributions of Na⁺-K⁺-ATPase, V-ATPase and HCO₃ ⁻-ATPase to the total ATPase activity of the two species varied in different developmental stages and accounted for 40%–70%, 22%–46% and 2%–13% in M. japonicus from zoea to postlarva stage, whilst the shares of them were 42%–69%, 28%–44% and 2.5%–22%, respectively in F.chinensis. These findings suggest the possible culture of the two species with varying water quality, especially the salinity and pH.     

Genetic diversity analysis with ISSR PCR on green algae <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis>

In the present study, genetic polymorphism and diversity in unicellular clones of Chlorella vulgaris Beijerinck and Chlorella pyrenoidosa Chick were studied with Inter Simple Sequence Repeats PCR （ISSR PCR）. Samples including four clones of C. vulgaris and three clones of C. pyrenoidosa were purified by single-clone-choice method. For four C. vulgaris unicellular clones, the total number of the bands scored for 18 primers was 298; and the number of the polymorphic bands was 118, of which 39.6% were polymorphic. The size of PCR products ranged from 200 to 2 500 bp. The total number of bands scored for 18 primers, the number of polymorphic bands and the percentage of three C. pyrenoidosa unicellular clones was 194.83 and 30.8%, respectively. POPGENE analysis show that the average Nei genetic diversity （h^＊） and Shannon index of diversity （I^＊） in the four C. vulgaris unicellular clones was 0.2181 and 0.3208, respectively, which is slightly higher than those of the three C. pyrenoidosa unicellular clones （0.190 3 and 0.274 8）, which agreed with the percentage of polymorphic bands in the mixed samples of the two species. The results suggest that ISSR is a useful method to Chlorella for intra-species genetic analysis.