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.     

Molecular weight controllable degradation of <Emphasis Type="Italic">Laminaria japonica</Emphasis> polysaccharides and its antioxidant properties

In this study, molecular weight controllable degradation of algal Laminaria japonica polysaccharides（LPS） was investigated by ultrasound combined with hydrogen peroxide. Three main factors, i.e., ultrasonic power（A）, ultrasonic time（B）, and H₂O₂ concentration（C） were chosen for optimizing parameters by employing three-factors, three-levels BBD. The influence of degradation on structure change and antioxidant activities was also investigated. A second-order polynomial equation including molecular weight（Y） of Laminaria japonica polysaccharides and each variable parameter, i.e., ultrasonic power（A）, ultrasonic time（B）, and H₂O₂ concentration（C）, was established: Y=20718.67-4273.13A-4000.38B-1438.75C+2333.25AB+1511.00AC+873.00BC+2838.29A² + 2490.79B²+873.04C². The equation regression coefficient value（R² = 0.969） indicated that this equation was valid. The value of the adjusted determination coefficient（adjusted R² = 0.914） also confirmed that the model was highly significant. The results of selected experimental degradation conditions matched with the predicted value. FT-IR spectra revealed that the structures of LPS before and after degradation were not significantly changed. Antioxidant activities of LPS revealed that low Mws possessed stronger inhibitory than the original polysaccharides. The scavenging effects on superoxide radicals was the highest when IC50 of crude LPS was 4.92 mg mL^-1 and IC50 of Mw 18.576 KDa was 1.02 mg mL^-1, which was fourfold higher than initial polysaccharide.     

Identification and bioinformatics analysis of microRNAs from the sporophyte and gametophyte of <Emphasis Type="Italic">Pyropia haitanensis</Emphasis>

Pyropia haitanensis (T. J. Chang et B. F. Zheng) N. Kikuchi et M. Miyata (Porphyra haitanensis) is an economically important genus that is cultured widely in China. P. haitanensis is cultured on a larger scale than Pyropia yezoensis, making up an important part of the total production of cultivated Pyropia in China. However, the majority of molecular mechanisms underlying the physiological processes of P. haitanensis remain unknown. P. haitanensis could utilize inorganic carbon and the sporophytes of P. haitanensis might possess a PCK-type C₄-like carbon-fixation pathway. To identify microRNAs and their probable roles in sporophyte and gametophyte development, we constructed and sequenced small RNA libraries from sporophytes and gametophytes of P. haitanensis. Five microRNAs were identified that shared no sequence homology with known microRNAs. Our results indicated that P. haitanensis might posses a complex sRNA processing system in which the novel microRNAs act as important regulators of the development of different generations of P. haitanensis.     

The characteristics of <Emphasis Type="Italic">vasa</Emphasis> gene from Japanese sea bass (<Emphasis Type="Italic">Lateolabrax japoni</Emphasis>cas) and its response to the external hormones

The RNA helicase Vasa is an important regulator of primordial germ cell development. Its function in mature fish, especially the hormone-related differences in maturing male fish has seldom been documented. In this study, a full length cDNA sequence of the vasa gene was cloned from Japanese sea bass, Lateolabrax japonicas, and it was named jsb-vasa. Homology analysis showed that jsb-vasa was closely related to its teleost homologs. The spatial distribution of jsb-vasa indicated that it was only highly expressed in testis, showing its germ cell-specific expression pattern. During the testicular development cycle, jsb-vasa was highly expressed during early period of spermatogenesis, and reduced when spermatogenesis advanced. In addition, the jsb-vasa gene expression was significantly inhibited at 6 h, 12 h and 24 h after injecting hCG (human chorionic gonadotropin) and GnRHa (Gonadotropin-releasing hormone analogue), indicating that jsb-vasa gene may play an important role in spermatogenesis of Japanese sea bass, and be under the regulation of external sex hormones.     

Immunogenicity and protective role of antigenic regions from five outer membrane proteins of <Emphasis Type="Italic">Flavobacterium columnare</Emphasis> in grass carp <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>

Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes (IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon (IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.     

Trophic Interaction in a <Emphasis Type="Italic">Portunus rituberculatus</Emphasis> Polyculture Ecosystem Based on Carbon and Nitrogen Stable Isotope Analysis

Trophic interaction among various biomass groups in a swimming crab Portunus trituberculatus polyculture pond was investigated using carbon and nitrogen stable isotope analysis. The polycultured animal species also included white shrimp Litopenaeus vannamei, short-necked clam Ruditapes philippinarum, and redlip mullet Liza haematochila. The mean δ¹³C value for all the biomass groups in polyculture ecosystem ranged from ?25.61‰ to ?16.60‰, and the mean δ¹⁵N value ranged from 6.80‰ to 13.09‰. Significant difference in the δ¹³C value was found between particulate organic matter (POM) and sediment organic matter (SOM) (P < 0.05), indicating that these two organic matter pools have different material sources. Assuming that a ¹³C-enrichment factor of 1.00‰ and a ¹⁵N-enrichment factor of 2.70‰ existed between consumer and prey, diets of the four cultured animals were estimated using a stable isotope mixing model. The estimated model results indicated that P. trituberculatus mainly feed on Aloidis laevis; L. vannamei mainly feed on shrimp feed; while A. laevis, R. philippinarum and L. haematochelia mainly feed on POM. Shrimp feed was also an important food source of R. philippinarum and L. haematochelia. The diets of P. trituberculatus, L. vannamei, R. philippinarum, and L. haematochila showed complementary effects in this polyculture ecosystem. Our finding indicated that the polyculture of these four organisms with suitable farming density could make an effective use of most of the food sources, which can make a highly efficient polyculture ecosystem.     

Genotype and Phylogenetic Diversity of <Emphasis Type="Italic">Symbiodinium</Emphasis> ITS2 Sequences Within Clade C in Three Typical Coral Species from Luhuitou Fringing Reef of the South China Sea

Dinoflagellates in the genus Symbiodinium, including nine clades (A–I), mainly form mutualistic symbioses with corals. More than 100 Symbiodinium molecular types have been identified by the ITS2-based genotype method within any given clade, and specifically within Symbiodinium clade C. However, the genotype identification method using the ITS2 sequence is likely to lead to high diversity estimates due to the intra-genomic variations in the ITS2 space; thus, further validation is essential for a correct identification. In this study, the molecular diversity of Symbiodinium ITS2 sequences cloned from two stone corals, Acropora sp. SY-01 and Pocillopora sp. SY-05, and one soft coral, Sarcophyton sp. SY-07, living in the northern part of South China Sea (SCS), were analyzed and compared using the ITS2-based genotype identification method, coupled with ITS2-based secondary structural and phylogenetic analyses. As the result, 12 Symbiodinium ITS2 genotypes were identified, while only six and three Symbiodinium ITS2 genotypes were supported by ITS2-based secondary structural and phylogenetic analyses, respectively. In addition, no shared Symbiodinium ITS2 genotypes were observed among the three coral species, suggesting coral species-dependent Symbiodinium genotypes were within clade C. In summary, the present study provides a theoretical basis for validating the molecular diversity of Symbiodinium ITS2 genotypes in corals.     

Effects of a Potential Autochthonous Probiotic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> 2-1 on the Growth and Intestinal Microbiota of Juvenile Sea Cucumber, <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> Selenka

The effects of Bacillus subtilis 2-1 from the intestine of healthy sea cucumber on the growth, digestive enzyme activities and intestinal microbiota of juvenile sea cucumber (Apostichopus japonicus) were determined in the present study. Sea cucumber was fed with Sargassum thunbergii powder supplemented with B. subtilis 2-1 at different concentrations varying among 0 (control), 10⁵, 10⁷, and 10⁹ CFU g^?1 for 8 weeks. Results showed that the growth performance and intestinal amylase and trypsin activities were significantly increased by dietary B. subtilis 2-1 at 10⁹ CFU g^?1 (P < 0.05). However, dietary B. subtilis 2-1 had no significant influence on the lipase activity in sea cucumber (P > 0.05). The polymerase chain reaction denaturing gradient gel electrophoresis and 16S rRNA gene sequencing analysis indicated that dietary B. subtilis 2-1 at 10⁵ and 10⁷ CFU g^?1 inhibited most of the Proteobacteria including those in genus Vibrio. Dietary B. subtilis 2-1 at 10⁹ CFU g^?1 not only decreased the abundance and species of genus Vibrio, but also increased the intensity of genera Psychrobacter and Bacillus. A specific dosage of dietary B. subtilis 2-1 could increase the growth and modulate the intestinal microbiota of sea cucumber; thus it might be a novel probiotic for keeping the health of sea cucumber.     

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.     

Temporal and spatial variations of abundance of phycocyanin- and phycoerythrin-rich <Emphasis Type="Italic">Synechococcus</Emphasis> in Pearl River Estuary and adjacent coastal area

Three surveys were carried out in Pearl River Estuary and adjacent coastal area in May, August, and November, 2013, to investigate the temporal and spatial variations of abundance of phycoerythrin-rich Synechococcus (PE-rich SYN) and phycocyanin-rich Synechococcus (PC-rich SYN). The effects of environmental factors on the alternation of the different Synechococcus groups were also elucidated. PE-rich SYN was detected in three surveys, whereas PC-rich SYN was detected in May and August, but not in November. The highest abundances of PE-rich SYN and PC-rich SYN were recorded in August and May, with mean values of 74.17×10³ and 189.92×10³ cells mL^?1, respectively. From May to November, the relative abundance of PE-rich SYN increased, whereas that of PC-rich SYN declined. PE-rich and PC-rich SYN presented similar horizontal distributions with high abundance in the southern estuary in May, and in the western estuary in August. The abundances of PE-rich and PC-rich SYN were high at 27–32°C and salinity of 10–20. PC-rich SYN was not detected at < 24°C, and PC:PE-rich SYN decreased in abundance with salinity increase. When less than 20 mg L^?1, suspended particulate matter (SPM) was helpful for Synechococcus growth. PE-rich SYN decreased in abundance when the concentration of dissolved inorganic nitrogen increased in May and November, and the concentration of phosphate increased in November. However, PC-rich SYN abundance and nutrients showed no correlation. Principal component analysis and regression analysis indicated that PE-rich SYN significantly correlated with the principal components that were affected by environmental factors.     

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.     

Effects of different phosphorus concentrations and N/P ratios on the growth and photosynthetic characteristics of <Emphasis Type="Italic">Skeletonema costatum</Emphasis> and <Emphasis Type="Italic">Prorocentrum donghaiense</Emphasis>

The effects of different phosphorus (P) concentrations (0.36, 3.6, and 36 μmol/L corresponding to low-, middle-, and high-P concentration groups, respectively) and nitrogen (N)/P ratios on the growth and photosynthetic characteristics of Skeletonema costatum and Prorocentrum donghaiense were studied. For both species, the high-P (HP) concentration group showed the greatest algal density and highest specific growth rate. Changes in the maximum efficiency of photosystem II (F _v /F _m ) were monitored under the various P and N/P conditions. The largest decrease in F _v /F _m was in the low-P (LP) group in S. costatum and in the HP group in P. donghaiense. There were high rapid light curves and photochemical quantum yields (Φ _PSII) for S. costatum in the HP group, while the actual photosynthetic capacity was higher in P. donghaiense than in S. costatum in the MP group. Under eutrophic but relatively P-restricted conditions, P. donghaiense had higher photosynthetic activity and potential, which could cause this dinoflagellate to increasingly dominate the phytoplankton community in these conditions. Under the same P concentration and N/P ratio, P. donghaiense had a larger relative maximum rate of electron transport and higher Φ _{PS II} values than those of S. costatum. These differences between P. donghaiense and S. costatum may explain the interaction and succession patterns of these two species in the Changjiang (Yangtze) River estuary from a photosynthesis perspective.     

Variations in leaf functional traits and physiological characteristics of <Emphasis Type="Italic">Abies georgei</Emphasis> var. <Emphasis Type="Italic">smithii</Emphasis> along the altitude gradient in the Southeastern Tibetan Plateau

Variations in leaf functional traits of Abies georgei var. smithii at 3700, 3900, 4100, 4300, and 4390 m altitude were investigated in 15 typical plots in the Southeastern Tibetan Plateau. In each plot, three seedlings were selected, of which functional leaves in current-year sunny branches were chosen for the measurement of morphological, photosynthetic, and physiological and biochemical characteristics, and their variations were analyzed. Results showed that significant variations existed among the leaf functional traits of A. georgei var. smithii along the altitudinal gradient, as well as their physiological adaption indicators. Leaf area decreased, while the mass per area and thickness of leaf increased at an altitude above 4,100 m. The maxima of pigment, total nitrogen concentration, net photosynthesis rate during light-saturated, and when water use efficiency appeared at 4100 m altitude. In addition, A. georgei var. smithii seedlings regulated the activities of superoxide dismutase and ascorbate peroxidase to resist abiotic stress under 4100 m altitude. Meanwhile, malondialdehyde concentration and the dark respiration rate rapidly increased, which indicates that A. georgei var. smithii seedlings suffered from heavy abiotic stress from 4100 m to 4390 m altitude. Basing on variations in leaf functional traits along the altitude gradient, we inferred that 4100 m altitude was the suitable region for A. georgei var. smithii growth in the Sygera Mountain. Moreover, the harsh environment was the main limiting factor for A. georgei var. smithii population expansion to high altitude.     

<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.     

Effects of dietary genistein on GH/IGF-I axis of Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

There is considerable concern that isoflavones, such as genistein in fish feed composed of soybean protein, aff ects somatic growth in fish. Our previous works demonstrated that 30 and 300 μg/g dietary genistein had no significant eff ect on growth performance in Nile tilapia (Oreochromis niloticus), but the higher level of genistein (3 000 μg/g) significantly depressed growth. This study was conducted to further examine the eff ects of dietary genistein on the endocrine disruption on growth hormone/insulin-like growth factor-I (GH/IGF-I) axis in Nile tilapia (O. niloticus). Juvenile fish were fed by hand twice daily to satiation with one of four isonitrogenous and isoenergetic diets, each containing either 0, 30, 300 or 3 000 μg/g genistein. Following an 8-week feeding period, plasma GH and IGF-I levels were investigated by radioimmunoassay and gene expression levels of gh, ghrelin, gnrhs, ghr, npy, npyrs, pacap, ghrs, igf-I, igf-Ir, and igfbp3 were examined by real-time PCR. The results show that no significant change in plasma GH and IGF-I levels in fish fed with diets containing 30 μg/g and 300 μg/g genistein. mRNA expression of genes along the GH/IGF-I axis remained unaff ected, except for igf-Ir, which was stimulated by the 300 μg/g genistein diet. While in fish fed the 3 000 μg/g genistein diet, the plasma GH and IGF-I levels decreased, and mRNA expression of gh, ghr2, npyr1, igf-I, and igf-Ir were also significantly depressed. In contrast, npy and igfbp3 mRNA expression were enhanced. This study provides convincing evidence for growth impediment by genistein by disturbing the GH/IGF-I axis in Nile tilapia O. niloticus.