Use of antioxidant enzymes of clam Ruditapes philippinarum as biomarker to polycyclic aromatic hydrocarbon pollution

The typical organic pollutant polycyclic aromatic hydrocarbon(PAH) anthracene was selected as a contaminant to investigate its eff ects on the activities of superoxide dismutase(SOD), catalase(CAT) and glutathione peroxidase(GSH-Px) in the clam Ruditapes philippinarum. The results show that SOD, CAT and GSH-Px had diff erent induction and inhibition reactions to anthracene stress, and that three diff erent organs in R. philippinarum(visceral mass, muscle tissue and mantle) had diff erent sensitivities to anthracene stress. This study suggest that SOD activities of the visceral mass, CAT activitities of the mantle and the visceral mass, and GSH-Px activity of the muscle tissue could be used as sensitive indicators of anthracene stress in R. philippinarum.     

Inbreeding depression on growth and survival of full-sib family of Manila clam (<Emphasis Type="Italic">Ruditapes philippinarum</Emphasis>)

In present study, the inbreeding depression (ID) of growth and survival of Manila clam (Ruditapes philippinarum) was investigated at larval and juvenile stages. Nine inbred families (A ₂, B ₂, C ₂, D ₂, E ₂, F ₂, G ₂, H ₂ and I ₂) were established by mating within nine full-sib families with expected inbreeding coefficient of 0.25. Inbred families showed significant differences in shell length and hatching rate of D-larvae (straight-hinged larvae). The larvae of the nine inbred families grew slower than those of control group (CG), and their ID value ranged from 0.81% ± 6.09% to 16.10% ± 1.49%. The ID value of larval survival rate varied between 27.47% ± 9.36% and 70.50% ± 13.66%. The ID was also detected for juvenile growth in A ₂, B ₂, C ₂, and D ₂, which ranged from 4.60 ± 2.21 to 17.71 ± 7.73. The A ₂ family maintained the highest juvenile survival rate, whereas the other inbred families exhibited ID values varying between 62.79% ± 4.54% and 96.14% ± 0.87%. The linear relationship of estimated ID between growth and survival was negatively correlated (R = ?0.434, P < 0.05). The results of this study suggested that the ID of growth was common at the larval stage but was less prevalent at juvenile stage. In contrast, the ID of survival increased from larval to juvenile stage. A better understanding of the effect of inbreeding may aid to selective breeding of Manila clam.     

Growth performance of larval and juvenile manila clam (<Emphasis Type="Italic">Ruditapes philippinarum</Emphasis>) from divergently selected individuals of a full-sib family

In this study, the method of divergent selection was employed to test the larval and juvenile growth performance within a full-sib family of Manila clam Ruditapes philippinarum. The 10% largest and 10% smallest clam individuals (on the basis of shell length) of a full-sib family were selected as parents for the fast and slow growing lines, respectively. The difference in shell length was significant among the three lines (fast, control, and slow) tested. The sequence of shell length were fast line > control line > slow line. The responses to selection, realized heritability, and genetic gain were 0.06%–0.81%, 0.04%–0.47% and 0.58%–18.89% in the fast direction, respectively; and were 0.14%–1.27%, 0.08%–0.73%, and 0.31%–49.03% in the slow direction, respectively. The results suggested that there was a large portion of additive genetic variance affecting the growth in the full-sib family. Selection in the fast direction within the full-sib family would greatly improve the growth of R. philippinarum.     

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)     

<Emphasis Type="Italic">GST</Emphasis> (<Emphasis Type="Italic">phi</Emphasis>) gene from Macrophyte <Emphasis Type="Italic">Lemna minor</Emphasis> is involved in cadmium exposure responses

Reactive oxygen species (ROS) scavengers, including ascorbate peroxidase, superoxide dismutase, catalase and peroxidase, are the most commonly used biomarkers in assessing an organisms’ response to many biotic and abiotic stresses. In this study, we cloned an 866 bp GST (phi) gene in Lemna minor and investigated its characteristics, expression and enzymatic activities under 75 μmol/L cadmium concentrations in comparison with other ROS scavengers. GST (phi) gene expression patterns were similar to those of other scavengers of ROS. This suggests that GST (phi) might be involved in responding to heavy metal (cadmium) stress and that its expression level could be used as a bio-indicator in monitoring cadmium pollution.     

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)     

Synedra ulna var. repanda,a new variety of Synedra （Bacillariophyta） from Xinjiang,China

Synedra ulna var, repanda Q. X. Wang ＆ Q. M. You, a new variety of Synedra （Bacillariophyta） from Xinjiang, China, is described and illustrated, and the characteristic of the variety： includes undulate-linear valves and straight pseudoraphe, differs from other species of Synedra.     

Fatty acid composition of <Emphasis Type="Italic">Euphausia superba</Emphasis>, <Emphasis Type="Italic">Thysanoessa macrura</Emphasis> and <Emphasis Type="Italic">Euphausia crystallorophias</Emphasis> collected from Prydz Bay,Antarctica

The information of trophic relationship is important for studying the Southern Ocean ecosystems. In this study, three dominant krill species, Euphausia superba, Thysanoessa macrura and Euphausia crystallorophias, were collected from Prydz Bay, Antarctica, during austral summer of 2009/2010. The composition of fatty acids in these species was studied. E. superba and T. macrura showed a similar fatty acid composition which was dominated by C14:0, C16:0, EPA (eicosapentenoic acid) and DHA (decosahexenoic acid) while E. crystallorophias showed higher contents of C18:1(n-9), C18:1(n-7), DHA and EPA than the former two. Higher fatty acid ratios of C18:1(n-9)/18:1(n-7), PUFA (polyunsaturated fatty acid)/SFA (saturated fatty acid), and 18PUFA/16PUFA indicated that E. crystallorophias should be classified as a typical omnivore with a higher trophic position compared with E. superba and T. macrura.     

Biodegradation of <Emphasis Type="Italic">Enteromorpha</Emphasis> polysaccharides by intestinal micro-community from <Emphasis Type="Italic">Siganus oramin</Emphasis>

Micro-communities are supposed to have more potential functions of biodegradation of polysaccharides than single strain; however, the intestinal micro-communities involved in the biodegradation of Enteromorpha polysaccharides (EP) were seldom reported. In order to obtain the EP-degrading micro-community, the intestines of Siganus oramin was obtained to isolate the micro-communities, which were enriched by 0.3% of EP as the sole carbon source. A stable micro-community with EP degradative capability was achieved after seven generations of subculture, named H1. Results showed that H1 was able to degrade 75% of EP within 24 hours, and the activity of EP lyases reached 500 U mL^?1 in 32 hours. With denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis, ten bacteria closely related to Marinomonas pontica, Microbacterium sp., Leucobacter chironomi, Cyclobacterium sp., Algoriphagus winogradskyi, Pseudoalteromonas sp. and Vibrio sp. were determined. Furthermore, compared with the DGGE bands sequence and the clone library analysis, the dominant bacteria of the EP-biodegrading micro- community were Pseudoalteromonas sp. and Vibrio sp., with the respective proportion of 38% and 46%, and they should play an important role in EP degradation together with other degrading bacteria in the micro-community H1.     

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.     

Genome size of <Emphasis Type="Italic">Alexandrium catenella</Emphasis> and <Emphasis Type="Italic">Gracilariopsis lemaneiformis</Emphasis> estimated by flow cytometry

Flow cytometry(FCM) technique has been widely applied to estimating the genome size of various higher plants. However, there is few report about its application in algae. In this study, an optimized procedure of FCM was exploited to estimate the genome size of two eukaryotic algae. For analyzing Alexandrium catenella, an important red tide species, the whole cell instead of isolated nucleus was studied, and chicken erythrocytes were used as an internal reference. The genome size of A. catenella was estimated to be 56.48 ± 4.14 Gb(1C), approximately nineteen times larger than that of human genome. For analyzing Gracilariopsis lemaneiformis, an important economical red alga, the purified nucleus was employed, and Arabidopsis thaliana and Chondrus crispus were used as internal references, respectively. The genome size of Gp. lemaneiformis was 97.35 ± 2.58 Mb(1C) and 112.73 ± 14.00 Mb(1C), respectively, depending on the different internal references. The results of this research will promote the related studies on the genomics and evolution of these two species.     

Non-specific immune response of bullfrog <Emphasis Type="Italic">Rana catesbeiana</Emphasis> to intraperitoneal injection of bacterium <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

Non-specific immune response of bullfrog Rana catesbeiana to pathogenic Aeromonas hydrophila was studied to 60 individuals in two groups. Each bullfrog in bacterium-injected group was injected intraperitoneally （i.p.） with 0.2 ml bacterial suspension at a density of 5.2 × 10^6 CFU/ml, while each one in control group injected i.p. with 0.2 ml sterile saline solution （0.85%, w/v）. Three bullfrogs in both groups were sampled at 0, 1, 3, 7, 11, 15 and 20 days post-injection （dpi） for the evaluation of non-specific immune parameters. It was observed that intraperitoneal injection of A. hydrophila significantly increased the number of leucocytes and that of NBT-positive cells in peripheral blood. Significant increases in serum bactericidal activity and serum acid phosphatase activity were also observed in the bacterium-injected frogs when compared with those in the control group. However, a significant reduction was detected in vitro in phagocytosis activity of peripheral blood phagocytes. No significant difference in changes in the number of peripheral erythrocytes, serum superoxide dismutase （SOD） activity, and lysozyme activity was detected between the two groups. It is suggested that bullfrogs may produce a series of non-specific immune reactions in response to the A. hydrophila infection.     

<Emphasis Type="Italic">Skeletonema</Emphasis> cf. <Emphasis Type="Italic">costatum</Emphasis> biogenic silica production rate determinated by PDMPO method

Diatoms are the only ecological phytoplankton that require silicic acid for growth. They are also the dominant contributor of ocean’s total primary productivity. Generation and circulation with silica walls, which the siliceous organisms form, is an important component of the marine biological pump. It is crucial to the study of the operational mechanisms of biological pump with different sea areas. Moreover, it is the key link to the study of global silicon cycle. This paper introduces the basic mechanism of the formation of diatom silica walls and a new way of researching silicic acid metabolism, namely the 2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)- methoxy)phenyl)oxazole (PDMPO) dyeing method. Under a fluorescence microscope after excitation with bright green fluorescence, it can combine with silicic acid to form a complex into the Si deposition within diatom cells. The advantage of this method is that it can monitor the metabolism of silicate after adding PDMPO. For experimentation and sample collection in each of the specified time points, samples were determinated through the unutilized silicic acid, silica dissoluble intracellular and Si deposition within diatom cells, not only using hot alkaline digestions method but also PDMPO dyeing method. Results showed a good linear relationship between PDMPO fluorescent value and biogenic silica concentration. It was also indicated that PDMPO had no deleterious impact on Skeletonema cf. costatum growth for 34 h and was useful for tracking newly-deposited biogenic silica in diatoms’ frustules.     

Lipid and fatty acid compositions of cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>), haddock (<Emphasis Type="Italic">Melanogrammus aeglefinus</Emphasis>) and halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

This study was conducted to compare lipid and fatty acid composition of cod, haddock and halibut. Three groups of cod (276 g ± 61 g), haddock (538 g ± 83 g) and halibut (3704 g ± 221 g) were maintained with commercial feeds mainly based on fish meal and marine fish oil for 12 weeks prior to sampling. The fatty acid compositions of muscle and liver were determined by GC/FID after derivatization of extracted lipids into fatty acid methyl esters (FAME). Lipids were also fractionated into neutral and polar lipids using Waters silica Sep-Pak?. The phospholipid fraction was further separated by high-performance thin-layer chromatography (HPTLC) and the FAME profile was obtained. Results of the present study showed that cod and haddock were lean fish and their total muscle lipid contents were 0.8% and 0.7%, respectively, with phospholipid constituting 83.6% and 87.5% of the total muscle lipid, respectively. Halibut was a medium-fat fish and its muscle lipid content was 8%, with 84% of the total muscle lipid being neutral lipid. Total liver lipid contents of cod, haddock and halibut were 36.9%, 67.2% and 30.7%, respectively, of which the neutral lipids accounted for the major fraction (88.1%–97.1%). Polyunsaturated fatty acids were the most abundant in cod and haddock muscle neutral lipid. Monounsaturated fatty acid level was the highest in halibut muscle neutral lipid. Fatty acid compositions of phospholipid were relatively constant. In summary, the liver of cod and haddock as lean fish was the main lipid reserve organ, and structural phospholipid is the major lipid form in flesh. However, as a medium-fat fish, halibut stored lipid in both their liver and muscle.     

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.     

Interspecific competition and allelopathic interaction between <Emphasis Type="Italic">Karenia mikimotoi</Emphasis> and <Emphasis Type="Italic">Dunaliella salina</Emphasis> in laboratory culture

Algal allelopathy is a manifold ecological/physiological phenomenon that is focused on chemical interactions and autotoxicity. We investigated the allelopathic interactions between Karenia mikimotoi and Dunaliella salina in laboratory cultures based on diff erent temperature (15°C, 20°C, and 25°C) and lighting (40, 80, and 160 μmol/(m²·s)) conditions. The growth of D. salina in bi-algae culture (1:1 size/density) was significantly restrained. The results of cell-free filtrate culture indicate that direct cell-tocell contact was not necessary in interspecific competition. Further experimental results demonstrated that allelochemicals released from K. mikimotoi were markedly influenced by both temperature (P =0.013) and irradiance (P =0.003), resulting in diff erent growth characteristics of D. salina in filtrate mediums. Compared with the plateau period, K. mikimotoi exudates in the exponential phase had a stronger short-term inhibition effect on D. salina in normal conditions. A clear concentration-dependent relationship was observed in the effect of allelochemicals released from K. mikimotoi with low-promoting and high-repressing effects on D. Salina in a short time-scale. In addition, allelopathic substances remain stable and effective under high temperature and pressure stress. Many flocculent sediments adhering with D. salina cells were observed in all filtrate mediums, while the quantity and color depended on the original culture conditions.     

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.     

Photosynthetic response of <Emphasis Type="Italic">Scirpus validus</Emphasis> and <Emphasis Type="Italic">Typha orientalis</Emphasis> to elevated temperatures in Dianchi Lake,Southwestern China

Understanding the effects of simulated warming on photosynthetic performance of aquatic plants may provide strong supports for predicting future dynamics of wetland ecosystems in the context of climate change. The plateau wetlands located in Yunnan province are highly sensitive to climate warming due to their high altitude and cold temperature. Here, we conducted a temperaturecontrolled experiment using two temperature manipulations (ambient temperature as the control and 2°C higher than ambient temperature as the warmed treatment) to determine the photosynthetic characteristics of two lakeside dominant species (Scirpus validus Vahl and Typha orientalis C. Presl.) in Dianchi Lake. Net photosynthetic rate (P_n), stomatal conductance (G_s), intercellular CO₂ concentration (C_i), and transpiration rate of S. validus that grew under warmed treatment were all significantly higher than those under the control. G_s and C_i of T. orientalis showed similar patterns as S. validus did. For the response curves of P_n to photosynthetic active radiation (Pn-PAR) and intercellular CO₂ concentration (P_n-C_i), S. validus had higher P_n values under elevated temperatures than the control, while P_n-PAR and P_n-C_i curves of T. orientalis did not separate clearly under two temperature scenarios. Both S. validus and T. orientalis had higher maximum net photosynthetic rate, light saturation point, dark respiration rate, the maximum rate of RuBP carboxylation (V_cmax), maximum electron transport rate driving RuBP regeneration (J_max), the ratio of V_cmax to J_max, triosephosphate utilization, and 1, 5-bishosphate carboxylase ribulose content under warmed treatment than those under the control. This study provides a preliminary step for predicting the future primary production and vegetation dynamics of plateau wetlands in Yunnan province.