Unusual hydrocarbon waxes detected in <Emphasis Type="Italic">Salix oritrepha</Emphasis> leaf from Nianbaoyeze Mountains,eastern Qinghai-Tibetan Plateau

Plant biomarkers, such as hydrocarbon waxes, are frequently found in various sediments and could be adopted as paleovegetation and paleoclimate indicators. Nevertheless, scarce researches have focused on leaf waxes in higher plants of alpine region. Herein, hydrocarbon leaf wax components of Salix oritrepha, which flourish in Nianbaoyeze Mountains in eastern Tibetan Plateau were fully discussed. The n-alkane distribution in leaves ranges from n-C₂₁ to n-C₂₉ with maxima at n-C₂₅, which were entirely different with Salix taxa displayed in previous surveys in non-alpine regions. The unusual even carbon nalkenes from n-C_22:1 to n-C_30:1, which were thought to appear only in aquatic organisms, were firstly reported in an alpine plant. Additionally, iso-(2-methyl) alkanes, ranging from i-C₂₃ to i-C₂₉ with maxima at i-C₂₅, which have been commonly reported in microorganisms, were also identified in an alpine plant for the first time. Unusual hydrocarbon distribution detected in Salix oritrepha leaf from Nianbaoyeze Mountains is most likely due to the extreme environment in such alpine region.     

Temporal variation in biodeposit organic content and sinking velocity in long-line shellfish culture

We measured the organic content and sinking velocities of biodeposits from two scallop species (Chlamys farreri, Patinopecten yessoensis) and abalone (Haliotis discus hannai) that were cultured on suspended long-lines. Measurements were conducted every two months from April 2010 to February 2011. The shellfish were divided into three size groups (small, middle, and big sizes). At each sample point, we assessed biodeposit organic content, average sinking velocity, the frequency distribution of sinking velocities, and the correlation between organic content and sinking velocity. The organic content of biodeposits varied significantly among months (P<0.05) and the pattern of change varied among species. Sinking velocities varied significantly, ranging from <0.5 cm/s to >1.9 cm/s. The sinking velocities of biodeposits from C. farreri and P. yessoensis were 0.5–1.5 cm/s and from H. discus hannai were <0.7 cm/s. The organic content was significantly negatively correlated to the sinking velocity of biodeposits in C. farreri (P<0.001) and P. yessoensis (P<0.05).     

Effects of Anthropogenic Disturbance on Sediment Organic Carbon Mineralization Under Different Water Conditions in Coastal Wetland of a Subtropical Estuary

The changes in soil organic carbon(C) mineralization as affected by anthropogenic disturbance directly determine the role of soils as C source or sink in the global C budget. The objectives of this study were to investigate the effects of anthropogenic disturbance(aquaculture pond, pollutant discharge and agricultural activity) on soil organic C mineralization under different water conditions in the Minjiang River estuary wetland, Southeast China. The results showed that the organic C mineralization in the wetland soils was significantly affected by human disturbance and water conditions(P 0.001), and the interaction between human disturbance activities and water conditions was also significant(P 0.01). The C mineralization rate and the cumulative mineralized carbon dioxide-carbon(CO_2-C)(at the 49th day) ranked from highest to lowest as follows: Phragmites australis wetland soil aquaculture pond sediment soil near the discharge outlet rice paddy soil. This indicated that human disturbance inhibited the mineralization of C in soils of the Minjiang River estuary wetland, and the inhibition increased with the intensity of human disturbance. The data for cumulative mineralized CO_2-C showed a good fit(R~2 0.91) to the first-order kinetic model C_t = C_0(1 – exp(–kt)). The kinetic parameters C_0, k and C_0 k were significantly affected by human disturbance and water conditions. In addition, the total amount of mineralized C(in 49 d) was positively related to C_0, C_0 k and electrical conductivity of soils. These findings indicated that anthropogenic disturbance suppressed the organic C mineralization potential in subtropical coastal wetland soils, and changes of water pattern as affected by human activities in the future would have a strong influence on C cycling in the subtropical estuarine wetlands.     

Soil Organic Carbon Contents and Stocks in Coastal Salt Marshes with <Emphasis Type="Italic">Spartina alterniflora</Emphasis> Following an Invasion Chronosequence in the Yellow River Delta,China

Plant invasion alters the fundamental structure and function of native ecosystems by affecting the biogeochemical pools and fluxes of materials and energy. Native (Suaeda salsa) and invasive (Spartina alterniflora) salt marshes were selected to study the effects of Spartina alterniflora invasion on soil organic carbon (SOC) contents and stocks in the Yellow River Delta. Results showed that the SOC contents (g/kg) and stocks (kg/m²) were significantly increased (P < 0.05) after Spartina alterniflora invasion of seven years, especially for the surface soil layer (0–20 cm). The SOC contents exhibited an even distribution along the soil profiles in native salt marshes, while the SOC contents were gradually decreased with depth after Spartina alterniflora invasion of seven years. The natural ln response ratios (LnRR) were applied to identify the effects of short-term Spartina alterniflora invasion on the SOC stocks. We also found that Spartina alterniflora invasion might cause soil organic carbon losses in a short-term phase (2–4 years in this study) due to the negative LnRR values, especially for 20–60 cm depth. And the SOCD in surface layer (0–20 cm) do not increase linearly with the invasive age. Spearman correlation analysis revealed that silt + clay content was exponentially related with SOC in surface layer (Adjusted R2 = 0.43, P < 0.001), suggesting that soil texture could play a key role in SOC sequestration of coastal salt marshes.     

Leaf stable carbon isotope composition in <Emphasis Type="Italic">Picea schrenkiana</Emphasis> var. <Emphasis Type="Italic">tianschanica</Emphasis> in relation to leaf physiological and morphological characteristics along an altitudinal gradient

To understand the effects of leaf physiological and morphological characteristics on δ ¹³C of alpine trees, we examined leaf δ ¹³C value, LA, SD, LNC, LPC, LKC, Chla+b, LDMC, LMA and N_area in one-year-old needles of Picea schrenkiana var. tianschanica at ten points along an altitudinal gradient from 1420 m to 2300 m a.s.l. on the northern slopes of the Tianshan Mountains in northwest China. Our results indicated that all the leaf traits differed significantly among sampling sites along the altitudinal gradient (P<0.001). LA, SD, LPC, LKC increased linearly with increasing elevation, whereas leaf δ ¹³C, LNC, Chla+b, LDMC, LMA and N_area varied non-linearly with changes in altitude. Stepwise multiple regression analyses showed that four controlled physiological and morphological characteristics influenced the variation of δ ¹³C. Among these four controlled factors, LKC was the most profound physiological factor that affected δ ¹³C values, LA was the secondary morphological factor, SD was the third morphological factor, LNC was the last physiological factor. This suggested that leaf δ ¹³C was directly controlled by physiological and morphological adjustments with changing environmental conditions due to the elevation.     

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.     

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.     

Impact of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and white spot syndrome virus (WSSV) co-infection on survival of penaeid shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

White spot syndrome virus (WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect Litopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus (WSSV+Vp), or we first infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection (Vp+WSSV). The eff ect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of Vibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction (PCR) method. LvMyD88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the diff erent mortality rates. Our results show that (1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection; (2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed significant increases in the numbers of Vibrio (P<0.05); (3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10⁵ to 10⁷ /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.     

Changes of Biogenic Elements in <Emphasis Type="Italic">Phragmites australis</Emphasis> and <Emphasis Type="Italic">Suaeda salsa</Emphasis> from Salt Marshes in Yellow River Delta,China

Little information is available on biogenic elements (carbon, nitrogen, phosphorus and sulfur) and the ecological stoichiometric characteristics of plants in coastal wetlands. To investigate the contents of carbon, nitrogen, phosphorus and sulfur of plants, and their ecological stoichiometric characteristics in the Yellow (Huanghe) River Delta, plant samples were collected from two typical salt marshes (Suaeda salsa and Phragmites australis wetlands) during the period of from August to October in 2007, and the ratios of C/N, C/P, N/P, C/N/P and C/N/P/S were calculated. Results showed that during the studying period, plant C, N and P were lower than the global average values, and plant N and P were lower than the China’s average values. Leaf C and S in Suaeda salsa were significantly lower than those in Phragmites australis (P < 0.05), and leaf N and P in Suaeda salsa and Phragmites australis showed no significant differences (P > 0.05). Average C/N ratios were 23.75 in leaf, 73.36 in stem, 65.67 in root of Suaeda salsa, and 33.77 in leaf, 121.68 in stem, 97.13 in root of Phragmites australis. Average C/N ratios of Suaeda salsa and Phragmites australis were all great than 25, indicating the salt marsh in the Yellow River Delta is an N limitation system. Average C/P ratios were 276.78 in leaf, 709.28 in stem and 1031.32 in root of Suaeda salsa, and 536.94 in leaf, 768.13 in stem and 875.22 in root of Phragmites australis. The average N/P ratios of Suaeda salsa were 12.92 in leaf, 10.77 in stem and 10.91 in root, and the average N/P ratios of Phragmites australis were 16.40 in leaf, 7.40 in stem and 6.92 in root, indicating the Suaeda salsa wetlands were N limited and Phragmites australis wetlands were N limited in August and P limited in October in 2007. The average C/N, C/P and C/N/P ratios in Suaeda salsa and Pragmites australis were higher than the global average values, indicating the lower quality of organic matter provided by wetland plants in the Yellow River delta.     

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.     

Karyotype analysis of four nemertean species

We performed a karyotype analysis on four species from Phylum Nemertea using regenerating somatic cells. Two palaeonemertean species, Cephalothrix hongkongiensis and Cephalothrix sp., had the same chromosome number (2n=28), but different karyotypes (16 m+10 sm+2 st, NF=54 and 22 m+4 sm+2 st, NF=54, respectively). The karyotypes of the two heteronemerteans, Notospermus geniculatus and Ramphogordius sanguineus were 2n=38, 30 m+6 sm+2 sm/st (NF=76/74) and 2n=56, 48 m+4 sm+4 sm/st (NF=112/108), respectively.     

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.     

Distribution and quantitative zonation of unloading cracks at a proposed large hydropower station dam Site

Rock mass unloading is an important rock engineering problem because unloading may impact the stability of a rock mass slope. Based on hydroelectric engineering principles, this study focuses on the classification of unloading zones to reflect the rock mass structure characteristics. Geological background and slope structure of the study region were considered to investigate the distribution and deformation of the unloading process. Quantitative indices were classified according to the formation mechanisms and the geological exhibition of unloading zones. The P-wave velocity (V _P ), the ratio of the wave velocity (K _V , the ratio of the test P-wave velocity along the adit depth to the P-wave velocity of intact rock), the sum of joint openings every 2 meters (S _t ), and the density of open joints (D _t ) were calculated as quantitative indices for the rock mass unloading zone. The characteristics of the unloading zone of rock mass slopes at the dam site were successfully determined. The method of combining qualitative data with quantitative indices was found to be effective for the classification of slope unloading zones.     

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.     

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.     

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.