Genotoxic potential of copper oxide nanoparticles in the bivalve mollusk <Emphasis Type="Italic">Mytilus trossulus</Emphasis>

Copper oxide nanoparticles (CuO-NPs) are among the most widely used metal oxide nanoparticles, which increases the chance of their being released into the marine environment. As the applications of these particles have increased in recent years, their potential impact on the health of marine biota has also increased. However, the toxicological effects of these NPs in the marine environment are poorly known. In the present study, the DNA damaging potential of CuO-NPs in the marine eastern mussel Mytilus trossulus was evaluated and compared to that of dissolved copper exposures. Genotoxicity was assessed by the single cell gel electrophoresis (comet) assay in mussel gill and digestive gland cells. The results showed that copper in both forms (CuO-NPs and dissolved copper) was accumulated to different extents in mussel tissues. The mussel exposed to the dissolved copper attained higher concentrations of copper in the gills than in the digestive gland. In contrast to these results, it was found that CuO-NPs could induce much higher copper accumulation in the digestive gland than in the gills. A clear and statistically significant increase in DNA damage was found in both tissues of the Cu-exposed group compared to the control mussels. Our results indicated that the CuO-NP exposure produced remarkable effects and increased DNA damage significantly in mussel gill cells only. It should be noted that the digestive gland cells were prone to accumulation following CuO-NPs when compared to the gill cells, while the gill cells were more sensitive to the genotoxic effects of CuO-NPs. These results also suggested the need for a complete risk assessment of engineered particles before its arrival in the consumer market.     

Accumulation and depuration of pectenotoxins in brown crab Cancer pagurus

Pectenotoxins (PTXs) are a group of marine algal toxins. In this study, the accumulation and depuration of pectenotoxins in brown crab Cancer pagurus were investigated. Crabs were fed with toxic blue mussels Mytilus edulis for 21 days and then depurated for 42 days. Toxins were extracted with methanol from the digestive glands of contaminated crabs, uncontaminated crabs (control group) and from blue mussels for comparison. Extracts were analyzed by liquid chromatograph coupled with tandem mass spectrometry (LC-MS-MS). The concentrations of PTX-2, PTX-2 SA, 7-epi-PTX-2 SA, and PTX-12 were analyzed in two batches of toxic blue mussels and the crabs. A one-compartment model was applied to describe the depuration of PTXs. The half-life of PTXs was estimated to be 6-7.5 days. After depuration for 42 days, the amount of PTXs measured in the crab digestive glands was less than 1 μg/kg.     

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.     

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)     

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.     

A comparative study of genetic variation between Chinese mitten crab <Emphasis Type="Italic">ericheir sinensis</Emphasis> and Hepu mitten crab <Emphasis Type="Italic">E. hepuensis</Emphasis>

Horizonal starch gel electrophoresis was used to investigate the biochemical genetic structure of Chinese mitten crab Eriocheir sinensis and Hepu mitten crab E. hepuensis. Sixteen putative enzyme-coding loci were examined for forty-nine chinese mitten crabs and thirty-eight Hepu mitten crabs. Nine loci, AAT-1 ^*, AAT-2 ^*, G3PDH ^*, GPI ^*, IDHP-1 ^*, IDHP-2 ^*, MDH-1 ^*, MDH-2 ^* and PGM ^*, are polymorphic in Chinese mitten crab, and seven, AAT-1 ^*, AAT-2 ^*, GPI ^*, IDHP-1 ^*, MDH-1 ^*, MDH-2 ^* and PGM ^*, are polymorphic in Hepu mitten crab. The proportion of polymorphic loci and the expected heterozygosity are 0.562 5 and 0.080 3 for Chinese mitten crab, and 0.437 5 and 0.075 4 for Hepu mitten crab. The Nei’s genetic distance between two species is 0.002 4.     

Occurrence and diversity of <Emphasis Type="Italic">Pichia</Emphasis> spp. in marine environments

A total of 328 yeast strains from seawater, sediments, mud of salterns, the guts of marine fish and marine algae were obtained. The results of routine identification and molecular methods show that five yeast strains obtained in this study belonged to Pichia spp., including Pichia guilliermondii 1uv-small, Pichia ohmeri YF04d, Pichia fermentans YF12b, Pichia burtonii YF11A and Pichia anomala YF07b. Further studies revealed that Pichia anomala YF07b could produce killer toxin against pathogenic yeasts in crabs while Pichia guilliermondii 1uv-small could produce high activity of extracellular inulinase. It is advisable to test if Pichia ohmeri YF04d obtained in this study is related to central-venous-catheter-associated infection.     

An integrative study of larval organogenesis of American shad <Emphasis Type="Italic">Alosa sapidissima</Emphasis> in histological aspects

We describe organogenesis at a histological level in American shad (Alosa sapidissima) larvae from 0 until 45 days after hatching (DAH). Larval development was divided into four stages based on the feeding mode, external morphological features, and structural changes in the organs: stage 1 (0–2 DAH), stage 2 (3–5 DAH), stage 3 (6–26 DAH) and stage 4 (27–45 DAH). At early stage 2 (3 DAH), American shad larvae developed the initial digestive and absorptive tissues, including the mouth and anal opening, buccopharyngeal cavity, oesophagus, incipient stomach, anterior and posterior intestine, differentiated hepatocytes, and exocrine pancreas. The digestive and absorptive capacity developed further in stages 2 to 3, at which time the pharyngeal teeth, taste buds, gut mucosa folds, differentiated stomach, and gastric glands could be observed. Four defined compartments were discernible in the heart at 4 DAH. From 3 to 13 DAH, the excretory systems started to develop, accompanied by urinary bladder opening, the appearance and development of primordial pronephros, and the proliferation and convolution of renal tubules. Primordial gills were detected at 2 DAH, the pseudobranch was visible at 6 DAH, and the filaments and lamellae proliferated rapidly during stage 3. The primordial swim bladder was first observed at 2 DAH and started to inflate at 9 DAH; from then on, it expanded constantly. The spleen was first observed at 8 DAH and the thymus was evident at 12 DAH. From stage 4 onwards, most organs essentially manifested an increase in size, number, and complexity of tissue structure.     

Accumulation and depuration of pectenotoxins in brown crab Cancer pagurus

Pectenotoxins (PTXs) are a group of marine algal toxins. In this study, the accumulation and depuration of pectenotoxins in brown crab Cancer pagurus were investigated. Crabs were fed with toxic blue mussels Mytilus edulis for 21 days and then depurated for 42 days. Toxins were extracted with methanol from the digestive glands of contaminated crabs, uncontaminated crabs (control group) and from blue mussels for comparison. Extracts were analyzed by liquid chromatograph coupled with tandem mass spectrometry (LC-MS-MS). The concentrations of PTX-2, PTX-2 SA, 7-epi-PTX-2 SA, and PTX-12 were analyzed in two batches of toxic blue mussels and the crabs. A one-compartment model was applied to describe the depuration of PTXs. The half-life of PTXs was estimated to be 6–7.5 days. After depuration for 42 days, the amount of PTXs measured in the crab digestive glands was less than 1 μg/kg. Supported by Norwegian Education Funding “Quata”(2005)     

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.     

<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.     

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.     

The effect of simulated gastrointestinal digestion on shrimp <Emphasis Type="Italic">Penaeus vannamei</Emphasis> allergenicity

Pen a 1 is a major shrimp allergen that induces an allergy. The aim of this study is to assess the effect of simulated gastrointestinal fluids on the allergenicity of Pen a 1. Purified Pen a 1 from shrimp (Penaeus vannamei) was subjected to digestion in SGF (simulated gastric fluid) and SIF (simulated intestinal fluid) for a set time. The allergenicity of digestive Pen a 1 was analyzed by immunoblotting and Ci-ELISA, using pool sera from patients with shrimp specific IgE. The results showed that Pen a 1 exhibited a decrease in allergenicity with increasing digestion time in the SGF and SIF. However, Pen a l exhibited strong resistance to digestive fluids, and all yielded fragments (33 kD, 23 kD, and 14 kD) showed allergic activity. Therefore, anti-digestion may be an important factor for Pen a 1 to induce an allergy.     

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.     

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.     

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)     

Influence of Probiotics on the Growth and Digestive Enzyme Activity of White Pacific Shrimp （Litopenaeus vannamel）

The influence of Bacillus probiotics on the digestive enzyme activity and the growth of Litopenaeus vannamei were de-termined in this study. The shrimp was treated with five percentages (1.5, 3.0, 4.5, 6.0 and 7.5) of probiotics (Bacillus spp.) supple-mented to the feed and cultured for 45d. The growth measured as the weight gain at the end of culturing was significantly (P<0.05) higher in probiotic-treated shrimps than that of the control (without receiving probiotics). Activities of protease and amylase, two digestive enzymes of the midgut gland and the intestine were significantly (P<0.05) higher in probiotic-treated shrimp than in the control.     

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.     

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.