Comparison of effectiveness of different ashing auxiliaries for determination of phosphorus in natural waters, aquatic organisms and sediments by ignition method

An improved method for the determination of phosphorus in natural waters, aquatic organisms and sediments by ignition method is proposed. The recoveries of phosphorus (P) from selected inorganic and organic P-containing compound standards after ignition with different auxiliaries, such as MgSO4, Mg(NO3)(2), MgO2, Mg(Ac-2) and CaCl2, were compared. We found that the phosphorus from most compound standards could not be completely recovered when these compounds were ignited (450-500degreesC) with the MgSO4 as auxiliary and the baked residue was extracted with 0.2 mol l(-1) HCl for 30min at 80degreesC or at room temperature. P recoveries, for example, were poor, less than 85%, if pyrophosphate and metaphosphate were ignited with the addition Of MgSO4 prior to the extraction of the baked residue with 0.2 mol l(-1) HCl at 80degreesC for 30 min. In contrast, MgO2, Mg(Ac)(2) and CaCl2, as well as Mg(NO3)(2), could all yield complete P recoveries at routine ashing temperatures (450-500degreesC). The results demonstrate that MgC12 is a more effective auxiliary agent for the determination of phosphorus in natural waters, aquatic organisms and sediments by ignition method than MgSO4 which is commonly used. (C) 2003 Elsevier Science Ltd. All rights reserved.

Construction of habitat suitability models (HSMs) for benthic macroinvertebrate and their applications to instream environmental flows: A case study in Xiangxi River of Three Gorges Reservior region, China

Based on a long-term ecological monitoring, the present study chose the most dominant benthic macroinvertebrate (Baetis spp.) as target organisms in Xiangxi River, built the habitat suitability models (HSMs) for water depth, current velocity and substrate, respectively, which is the first aquatic organisms model for habitat suitability in the Chinese Mainland with a long-term consecutive in situ measurement. In order to protect the biointegrity and function of the river ecosystem, the theory system of instream environmental flow should be categorized into three hierarchies, namely minimum required instream flow (hydrological level), minimum instream environmental flow (biospecies level), and optimum instream environmental flow (ecosystem level). These three hierarchies of instream environmental flow models were then constructed with the hydrological and weighted usable area (WUA) method. The results show that the minimum required instream flow of Xiangxi River calculated by the Tennant method (10% of the mean annual flow) was 0.615 m(3) s(-1); the minimum instream environmental flow accounted for 19.22% of the mean annual flow (namely 1.182 m(3) s(-1)), which was the damaged river channel. ow in the dry season; and 42.91% of the mean annual flow (namely 2.639 m(3) s(-1)) should be viewed as the optimum instream environmental flow in order to protect the health of the river ecosystem, maintain the instream biodiversity, and reduce the impact of small hydropower stations nearby the Xiangxi River. We recommend that the hydrological and biological methods can help establish better instream environmental. ow models and design best management practices for use in the small hydropower station project. (C) 2008 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Identification of differentially expressed immune-relevant genes in Chinese soft-shelled turtle (Trionyx sinensis) infected with Aeromonas hydrophila

Expressed sequence tag (EST) analysis is an efficient tool for gene discovery and profiling gene expression. Aeromonas hydrophila, a ubiquitous waterborne bacterium, is one of the most frequent pathogens isolated from diseased aquatic organisms. In order to understand the molecular mechanism of anti-bacteria immune response in reptile, we have investigated the differentially expressed genes in Chinese soft-shelled turtle (Trionyx sinensis) experimentally infected with A. hydrophila by suppression subtractive hybridization (SSH). Forty-two genes were identified from more than 200 clones, of which 25 genes are found for the first time in reptiles, and classified into 6 categories: 18 in defense/immunity. 4 in catalysis, 2 in retrotransposon; 2 in cell signal transduction, 5 in cell metabolism, 10 in protein expression, and 1 in cell structure. Of the 42 differentially expressed genes, 6 genes, IL-8, serum amyloid A (SAA), CD9, CD59, activating transcription factor 4 (ATF4) and cathepsin L genes, were further observed to be up-regulated in the infected turtles by virtual Northern hybridization and RT-PCR assays. (C) 2008 Elsevier B.V. All rights reserved.

Distribution and bioaccumulation of microcystins in water columns: A systematic investigation into the environmental fate and the risks associated with microcystins in Meiliang Bay, Lake Taihu

For the purpose of understanding the environmental fate of microcystins (MCs) and the potential health risks caused by toxic cyanobacterial blooms in Lake Taihu, a systematic investigation was carried out from February 2005 to January 2006. The distribution of MCs in the water column, and toxin bioaccumulations in aquatic organisms were surveyed. The results suggested that Lake Taihu is heavily polluted during summer months by toxic cyanobacterial blooms (with a maximum biovolume of 6.7 x 10(8) cells/L) and MCs. The maximum concentration of cell-bound toxins was 1.81 mg/g (DW) and the dissolved MCs reached a maximum level of 6.69 mu g/L. Dissolved MCs were always found in the entire water column at all sampling sites throughout the year. Our results emphasized the need for tracking MCs not only in the entire water column but also at the interface between water and sediment. Seasonal changes of MC concentrations in four species of hydrophytes (Eichhornic crassipes, Potamogeton maackianus, Alternanthera philoxeroides and Myriophyllum spicatum) ranged from 129 to 1317, 147 to 1534, 169 to 3945 and 124 to 956 ng/g (DW), respectively. Toxin accumulations in four aquatic species (Carassius auratus auratu, Macrobrachium nipponensis, Bellamya aeruginosa and Cristaria plicata) were also analyzed. Maximum toxin concentrations in the edible organs and non-edible visceral organs ranged from 378 to 730 and 754 to 3629 ng/g (DW), respectively. Based on field studies in Lake Taihu, risk assessments were carried out, taking into account the WHO guidelines and the tolerable daily intake (TDI) for MCs. Our findings suggest that the third largest lake in China poses serious health threats when serving as a source of drinking water and for recreational use. In addition, it is likely to be unsafe to consume aquatic species harvested in Lake Taihu due to the high-concentrations of accumulated MCs. (C) 2007 Elsevier Ltd. All rights reserved.

Isolation and characterization of Scophthalmus maximus rhabdovirus

A rhabdovirus associated with a lethal hemorrhagic disease in cultured turbot Scophthalm us maximus Linnaeus was isolated. The virus induced typical cytopathogenic effects (CPE) in 9 of 15 fish cell lines examined and was then propagated and isolated from infected carp leucocyte cells (CLC). Electron microscopy observations revealed that the negatively stained virions had a typical bullet-shaped morphology with one rounded end and one flat base end. The bullet-shaped morphology was more obvious and clear in ultrathin sections of infected cells. Experimental infections also indicated that the S. maximus rhabdovirus (SMRV) was not only a viral pathogen for cultured turbot, but also had the ability to infect other fish species, such as freshwater grass carp. A partial nucleotide sequence of the SMRV polymerase gene was determined by RT-PCR using 2 pairs of degenerate primers designed according to the conserved sequences of rhabdovirus polymerase genes. Homology analysis, amino acid sequence alignment, and phylogenetic relationship analysis of the partial SMRV polymerase sequence indicated that SMRV was genetically distinct from other rhabdoviruses. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified SMRV revealed 5 major structural proteins, and their molecular masses were estimated to be about 250, 58, 47, 42, and 28 kDa. Significant serological reactivity differences were also observed between SMRV and its nearest neighbor, spring viremia of carp virus (SVCV). The data suggest that SMRV is likely a novel fish rhabdovirus, although it is closely related to rhabdoviruses in the genus Vesiculovirus.

Nonylphenol and octylphenol in urban eutrophic lakes of the subtropical China

The paper studied two estrogenic pollutants, 4-nonylphenol (NP) and 4-tert-octylphenol (OP) in water, suspended particle (SP) and sediments in urban eutrophic lakes. The concentrations of NP ranged from 1.94 to 32.85 mu g/l, 0.876 to 31.13 mu g/l and 3.54 to 32.43 mu g/g dry weight (dw) in water, suspended particle (SP) and sediments, respectively, and that of OP from 0.027 to 1.44 mu g/l, 0.008 to 1.777 mu g/l and 0.058 to 1.245 mu g/g dw in water, suspended particle (SP) and sediments, respectively. An increasing trend in the concentration was noticed in all matrices close to the sewage inlets, which was found to be the major factor affecting the spatial distribution of alkylphenols (APs) in the lakes. Due to restoration of submerged macrophytes, which might accumulate APs, the contaminations of APs in the Little Moon Lake (LML) and the Little Lotus Lake (LLL) were lower than those in the Big, Moon Lake (BML) and the Bier Lotus Lake (BLL). A reasonable correlation of NP and OP was obtained among water, suspended particle and sediment. The possible environmental stress of APs concentration on aquatic organisms in Wuhan urban lakes was also discussed.

Identification of a WSSV neutralizing scFv antibody by phage display technology and in vitro screening

White spot syndrome virus (WSSV) is one of the most significant viral pathogens causing high mortality and economic damage in shrimp aquaculture. Although intensive efforts were undertaken to detect and characterize WSSV infection in shrimp during the last decade, we still lack methods either to prevent or cure white spot disease. Most of the studies on neutralizing antibodies from sera have been performed using in vivo assays. For the first time, we report use of an in vitro screening method to obtain a neutralizing scFv antibody against WSSV from a previously constructed anti-WSSV single chain fragment variable region (scFv) antibody phage display library. From clones that were positive for WSSV by ELISA, 1 neutralizing scFv antibody was identified using an in vitro screening method based on shrimp primary lymphoid cell cultures. The availability of a neutralizing antibody against the virus should accelerate identification of infection-related genes and the host cell receptor, and may also enable new approaches to the prevention and cure of white spot disease.

Population dynamics of the water mite Unionicola arcuata (Unionicolidae) in the freshwater bivalve Cristaria plicata (Unionidae) in Poyang Lake, eastern China

Population dynamics of the water mite Unionicola arcuata were investigated in the freshwater bivalve Cristaria plicata during the period from January to December 2002 in Poyang Lake, East China. A pattern of seasonal variation was observed, with prevalence and abundance peaking in early spring and autumn. The number of mites in individual hosts was significantly correlated with the size, but not with the sex, of bivalves. The change in infection level of mites on different infection sites in C. plicata was significant, with > 58% of the mites found on the outer and inner gills, indicating that U. arcuata shows site preference.

Exposure of spermatozoa to duroquinone may impair reproduction of the common carp (Cyprinus carpio) through oxidative stress

Toxicity of many waterborne organic contaminants to aquatic organisms is mediated through oxidative damages resulting from the production of reactive oxygen species (ROS). Using duroquinone as a model ROS inducer, we carried out in vitro and in vivo experiments to test the hypothesis that reproduction in common carp (Cyprinus carpio) can be impaired through oxidative damage of their spermatozoa. In vitro exposure of fish spermatozoa to 0, 12.5, 25, 50, 100 and 200 mu M duroquinone for 2 h showed a significant increase in the level of ROS in a dose-dependant manner. Sperm motility was significantly reduced in all exposure groups, but lipid peroxidation (LPO) and DNA strand break (measured by comet assay) were only enhanced at 50 mu M and above. A significant decrease in subsequent hatching rate was recorded in all the exposure groups, despite fertilization rate was not affected. In the in vivo experiment, spermatozoa were collected 24 and 72 h after fish received intra-peritoneal injections of 1.0 and 10 mg kg(-1) body weight duroquinone. DNA damage was clearly evident in spermatozoa of all treatment groups after 72 h exposure, and ROS was significantly enhanced in the high concentration group. LPO however, remained unchanged in both treatment groups. The overall results of both our in vitro and in vivo experiments demonstrated that duroquinone can induce ROS production in spermatozoa, which may impair sperm quality and subsequently reproductive success through oxidative stress. (c) 2006 Elsevier B.V. All rights reserved.

Environmental signals: Synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis

According to outdated paradigms humic substances (HS) are considered to be refractory or inert that do not directly interact with aquatic organisms. However, they are taken up and induce biotransformation activities and may act as hormone-like substances. In the present study, we tested whether HS can interfere with endocrine regulation in the amphibian Xenopus laevis. In order to exclude contamination with phyto-hormones, which may occur in environmental isolates, the artificial HS 1500 was applied. The in vivo results showed that HS 1500 causes significant estrogenic effects on X. laevis during its larval development and results of semi-quantitative RT-PCR revealed a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, preliminary RT-PCR results showed that the thyroid-stimulating hormone (TSH beta-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Hence, HS may have estrogenic and anti-thyroidal effects on aquatic animals, and therefore may influence the structure of aquatic communities and they may be considered environmental signaling chemicals. (c) 2005 Elsevier Ltd. All rights reserved.

Humoral immune responses of the grouper Eninephelus akaara against the microsporidium Glugea epinephelusis

The humoral immune responses of grouper Epinephelus akaara to a natural infection with Glugea epinephelusis was studied by ELISA utilizing intact mature spores as the coated antigen. Results showed that a specific humoral immune response was elicited, but the intensity of infection (in terms of the number of cysts) was not related to the antibody level in naturally infected hosts. The differences in the antigenicity of intact mature spores and soluble spore proteins derived from cracked mature spores were also analyzed. Results suggested that similar antigen epitopes existed between the 2 groups. Additionally, antigen component patterns and the distribution of antigen with immunogenicity were investigated by using the western blot and the immunofluorescent antibody technique (IFAT). The new parasitic microsporidium has specific polypeptide patterns comparable to the reported fish microsporidians. The main antigenic substances are concentrated on the surface of spores, and are mostly located on the anterior and posterior end of the spore bodies. Most surface components of the G. epinephelusis spores are soluble, The potential role of the surface components in initiating infection was also discussed.

Identification of two novel interferon-stimulated genes from cultured CAB cells induced by UV-inactivated grass carp hemorrhage virus

Interferon (IFN) exerts its antiviral effect by inducing the expression of a number of IFN-stimulated genes (ISGs) to establish a host antiviral state. Earlier studies identified some important fish IFN system genes from IFN-induced CAB cells (crucian carp Carassius auratus L. embryonic blastulae cells) after treatment with UV-inactivated GCHV (grass carp hemorrhage virus). Herein, the cloning of 2 novel IFN-stimulated genes, termed Gig1 and Gig2, is described for the same cell system. The complete cDNA sequences of Gig1 and Gig2 contain 1244 bp encoding for a 194-amino-acid protein and 693 bp for a 158-amino-acid protein, respectively. A search of public databases revealed that these are 2 novel IFN-stimulated genes, since neither significant homologous genes nor conserved motifs were identified. Active GCHV, UV-inactivated GCHV and CAB IFN-containing supernatant (ICS) induced transcription of these genes and distinct kinetics were observed. An analysis of differences in expression between the 2 genes and the IFN signal factors CaSTAT1 and CaIRF7 indicated that GCHV infection activated different signal pathways for their up-regulation. Upon virus infection, the transcription of Gig1 but not of Gig2 is strongly suppressed by cycloheximide (CHX). In contrast, following treatment with CAB IFN-containing supernatant, CHX does not inhibit either gene transcription. The results suggest that GCHV infection can induce expression of both Gig1 and Gig2 via newly synthesized CAB IFN, most probably through the JAK-STAT signal pathway, and can also directly activate Gig2 transcription without ongoing protein synthesis.

Infection and propagation of lymphocystis virus isolated from the cultured flounder Paralichthys olivaceus in grass carp cell lines

The causative agent of lymphocystis disease that frequently occurs in cultured flounder Paralichthys olivaceus in China is lymphocystis virus (LV). In this study, 13 fish cell lines were tested for their susceptibility to LV. Of these, 2 cell lines derived from the freshwater grass carp Ctenopharyngodon idellus proved susceptible to the LV, and 1 cell line, GCO (grass carp ovary), was therefore used to replicate and propagate the virus. An obvious cytopathic effect (CPE) was first observed in cell monolayers at 1 d post-inoculation, and at 3 d this had extended to about 75% of the cell monolayer. However, no further CPE extension was observed after 4 d. Cytopathic characteristics induced by the LV were detected by Giemsa staining and fluorescence microscopic observation with Hoechst 33258 staining. The propagated virus particles were also observed by electron microscopy. Ultrastructure analysis revealed several distinct cellular changes, such as chromatin compaction and margination, vesicle formation, cell-surface convolution, nuclear fragmentation and the occurrence of characteristic 'blebs' and cell fusion. This study provides a detailed report of LV infection and propagation in a freshwater fish cell line, and presents direct electron microscopy evidence for propagation of the virus in infected cells. A possible process by which the CPEs are controlled is suggested.

Detection of Myxobolus rotundus (Myxozoa : Myxosporea) in skin mucus of crucian carp Carassius auratus auratus using a monoclonal antibody

Diagnosis of myxosporean Myxobolus rotundus infection was conducted by examining skin mucus from the infected crucian carp Carassius auratus auratus with a monoclonal antibody, MAb 2D12, raised previously against the parasite. A positive reaction was observed in skin mucus collected from infected fish, and spores and pre-spore stages of the parasite were identified by the MAb 2D12. It was also demonstrated that M. rotundus infection can be successfully detected by a simple method, enzyme-linked immunosorbent assay (ELISA), and that skin mucus collected from infected fish skin had a significantly higher optical density (OD) value than that from uninfected fish.