The high content of polyunsaturated fatty acids in Nannochloropsis limnetica (Eustigmatophyceae) and its implication for food web interactions, freshwater aquaculture and biotechnology

In the eustigmatophycean Nannochloropsis limnetica the content of polyunsaturated fatty acids (PUFA) is extremely high in comparison to different planktonic green algal taxa in freshwater ecosystems. The sums of n-6 and n-3 fatty acids in N. limnetica were ten-fold higher than in the other picoplankton Choricystis minor and Pseudodictyosphaerium jurisii, and higher than in the nanoplanktonic green algae Chlorella vulgaris, Monoraphidium neglectum and Scenedesmus obtusiusculus. The content of fatty acids in N. limnetica was highly variable under different culture conditions. The highest concentrations of PUFA in N. limnetica were found in non-aerated suspension cultures, with a high content of phosphate (40 mg l⁻¹ K₂HPO₄) in the culture medium: linoleic acid 22.19 mg g⁻¹ DW, arachidonic acid 10.52 mg g⁻¹ DW, and eicosapentaenoic acid 55.56 mg g⁻¹ DW. N. limnetica represent a high-quality food resource in freshwater food chains. Furthermore, cultures of this eustigmatophycean alga have a high potential for use in biotechnology and aquaculture.     

The Accumulation of Crimidin in Some Fish Organs

The accumulation of crimidin in total samples and in some organs of five fish species was studied under experimental conditions. The species were Cyprinus carpio, Carassius carassius, Tinca tinca, Scardinus erythrophthalmus and Leucaspius delineatus. Fish were exposed to concentrations of 10 mg · l^?l and 50 mg · l^?1. During short-term experiments water and fish samples were taken at intervals of t = 0, 6, 12, 24, 48 and 72 hours, during the long-term experiment sampling was performed weekly for six weeks. Total samples were analysed for C. carassius, T. tinca, S. erythrophthalmus and L. delineatus. Samples of individual organs and tissues were taken as follows: C. carpio – gills, digestive tract, muscular tissue, kidneys, gonads; C. carassius – gills, digestive tract, muscular tissue, ovaries, testes. Crimidin was determined by gas chromatography. In samples from the short-term experiments at a concentration of 10 mg · l^?1 in the water the amount was roughly 10¹–10 mg · kg^?1, at a concentration of 50 mg · l^?1 in the water roughly 10¹ mg · kg^?1. In most cases the accumulation coefficient was lower than 1.0. The accumulation capacity of individual fish species did not differ greatly. Of the internal organs only the kidneys had a high accumulation capacity, otherwise the highest values were found in muscular tissue and the gills. After one – three weeks the amount of crimidin in most organs falls, and after transfer to clean water there is a general sharp decline. Thus crimidin is not firmly bound in the body.     

Effects of 4-nonylphenol exposure in mussels (Mytilus galloprovincialis) and crabs (Carcinus aestuarii) with particular emphasis on vitellogenin induction

Since it is often difficult to estimate possible adverse effects due to contamination in selected ecosystems, multi-species biomonitoring may provide more information, taking into account different routes of exposure, ecological roles and metabolic capabilities of animals. In this context, we exposed for 7 days the mussel Mytilus galloprovincialis and the crab Carcinus aestuarii to 4-nonylphenol (NP), a well-known xenoestrogen. In mussels (0–0.2 mg NP l⁻¹), we measured NP bioaccumulation in soft tissues and vitellogenin (Vg)-like protein levels in digestive glands from both males and females by the alkali-labile phosphate assay (ALP). As no reference data were available for crab exposure, the NP 96-h LC₅₀ value was previously determined. Then, in sublethally exposed (0–1.0 mg NP l⁻¹) male crabs, NP bioaccumulation and Vg levels were measured in hemolymph, gonads and digestive gland. Bioaccumulation of NP increased from 43 to 371 μg g⁻¹ d.w. in mussels, and from 3.6 to 37 μg g⁻¹ d.w. in crabs, depending on the NP concentration in water. Dose-dependent Vg-like protein induction was observed in both species, appearing to be related to NP bioaccumulation, although a partial decrease was recorded at the highest concentration tested. A similar trend was observed in both digestive gland and gonad of exposed crabs; Vg increased to a lesser extent, although significantly, in hemolymph. Results demonstrated that NP induces Vg synthesis both in male and female mussels, as well as in male crabs. On the basis of the responsiveness of both species investigated, a multi-species approach is indicated in biomonitoring programmes.     

Hemolytic activity and fatty acids composition in the ichthyotoxic dinoflagellate Cochlodinium polykrikoides isolated from Bahía de La Paz, Gulf of California

The hemolytic activity of the dinoflagellate Cochlodinium polykrikoides from Bahía de La Paz, Gulf of California was investigated as part of the ichthyotoxic mechanism of this microalga. Two different kinds of erythrocytes, fish and human, were tested for the hemolytic assay. Since fatty acids have been associated with hemolytic activity in C. polykrikoides, the composition of fatty acids of this dinoflagellate was also analyzed. The concentration of C. polykrikoides causing 50% hemolysis (HE₅₀) was 4.88 and 5.27 × 10⁶ cells L⁻¹, for fish and human erythrocytes, respectively. According to the standard curve of saponin, an equivalence between the hemolytic activity of saponin and the dinoflagellate concentration was found with 1 μg saponin mL⁻¹ equivalent to 1 × 10⁶ cells L⁻¹ of C. polykrikoides. The polyunsaturated fatty acids: hexadecaenoic (16:0), docosahexaenoic (22:6 n3) and octadecapentaenoic (18:5 n3) were found in an abundance of 62% of total fatty acids.     

Hypoxia does not promote naphthalene bioaccumulation in the brown shrimp, Penaeus aztecus

Since increased ventilation is known to be a common strategy used by aquatic animals to cope with hypoxia, we tested in present study the hypothesis that hypoxia can promote the bioaccumulation of naphthalene, a representative polycyclic aromatic hydrocarbon (PAH), in Penaeus aztecus, a penaeid shrimp subject to hypoxia and PAH contamination in the northern Gulf of Mexico. For each of the two naphthalene concentrations, five groups of shrimps were, respectively, subjected to five different conditions, namely, clean seawater under normoxia, seawater containing acetone under normoxia and hypoxia, and seawater containing 10 or 250 μg/L naphthalene under normoxia and hypoxia. Our results suggest that hypoxia does not significantly alter naphthalene bioaccumulation in either the gills or the hepatopancreas of P. aztecus. The absence of a promoting effect of hypoxia on naphthalene bioaccumulation is attributed to the increased disposition of naphthalene when the shrimps are subjected to hypoxia.     

Effects of some environmental parameters on the reproduction and development of a tropical marine harpacticoid copepod Nitocra affinis f. californica Lang

The effects of salinity, temperature, and light conditions on the reproduction and development of harpacticoid copepod, Nitocra affinis f. californica under controlled laboratory conditions were determined. Seven different salinity levels (5, 10, 15, 20, 25, 30, 35 ppt), four temperatures (20, 25, 30, 35 °C), three different light intensities (25, 56, 130 μmol m⁻² s⁻¹) and photoperiods (24 h:0 h, 1 h:23 h, 12 h:12 h LD cycle) were employed in this study. The highest (p < 0.05) overall reproduction and fastest development time were achieved by copepods reared under 30–35 ppt salinity. The optimum temperature required for the maximum reproduction was 30 °C while under 30 °C and 35 °C the copepod development time was shortest (p < 0.05) compared to other temperature levels. The overall reproduction was highest (p < 0.05) and development rate of N. affinis was shortest (p < 0.05) under lowest light intensity (25 μmol m⁻² s⁻¹). Continuous light (24 h:0 h LD) inhibited the egg production while, continuous darkness (1 h:23 h LD) and 12 h:12 h LD significantly favoured the overall reproductive activity of the female. Photoperiods 1 h:23 h and 12 h:12 h LD yielded highest total (p < 0.05) offspring female⁻¹ coupled with highest (p < 0.05) survival percentage. This study illustrated that although N. affinis can tolerate wide range of environmental conditions, prolonged exposure to subnormal environments affect its reproduction and development. This study showed that this species can be mass cultured for commercial purposes and has a potential to be used for toxicity studies due to its high reproductive performance fast development and a wide range of tolerance to environmental conditions.     

Cloning and developmental expression of kinesin superfamily7 (kif7) in the brackish medaka (Oryzias melastigma), a close relative of the Japanese medaka (Oryzias latipes)

kif7 is a member of the kinesin superfamily members which are molecular motor proteins that move along microtubules in a highly regulated manner through ATP hydrolysis. In this paper, we report on the cloning of the Oryzias melastigma kif7 (omkif7) using primers designed according to the Japanese medaka (Oryzias latipes) database. The cloned omkif7 has an open reading frame of 3762bp and is deduced to encode a polypeptide of 1254 amino acids that possesses the putative ATP-binding and microtubule-binding motifs in its motor domain at the N-terminal region. We characterized the cloned omkif7 by comparison with the zebrafish kif7. Both omkif7 and zebrafish kif7 are shown to be expressed in all embryonic stages and adult tissues examined with higher expression level in the testis and ovary. Whole-mount in situ hybridization revealed that the expression of omkif7 is ubiquitous during the early stages of embryonic development, but became more restrictive and localized to the brain, fin bud and eye at later development. This study suggested that the brackish O.melastigma can serve as a good seawater model organism for developmental studies by utilizing the resources developed from its close relative of the Japanese medaka.     

Attenuation characteristics of coda waves and estimation of Q c values in eastern China

For short-period near-earthquake records in eastern China, from the empirical attenuation formula of coda ground motion amplitudeA with timeτ: lgA=G−2. 235 lgτ, using the single scattering theory modified with epicentral distance, we obtain the curve family of corrected coda amplitudeA _c(r,t), andω/2Q _c values for each time interval of coda. From this,Q _c(f,h) values, which correspond to each observational average frequency and sampling depth, are calculated. The results substantially agree with those observationalQ _c values in Yunnan, Beijing and central Asia.     

Molecular cloning and beta-naphthoflavone-induced expression of a cytochrome P450 1A (CYP1A) gene from an anadromous river pufferfish, Takifugu obscurus

In recent years, there has been a decline in the wild populations of river pufferfish, Takifugu obscurus. Besides overexploitation for commercial purposes, environmental pollution is believed to have contributed to its decline. However, almost no information exists about genes involved in metabolism of xenobiotics by this species. Nevertheless, there is interest in fugu fishes, since they possess the smallest genome among vertebrates. We cloned and characterized the full-length cDNA sequence of a cytochrome P450 1A (CYP1A) gene from T. obscurus. Phylogenic relationship of T. obscurus CYP1A was also compared to other fish species. The tissue distribution and time-dependant induction of CYP1A mRNA were studied by real-time PCR in T. obscurus exposed to an aryl hydrocarbon receptor (Ahr) agonist, beta-naphthoflavone (BNF). The greatest basal expression in livers of control as well as BNF-treated individuals. However, brain, gill, gonad, intestine, and kidney also expressed CYP1A. Muscles expressed the least CYP1A. The results of the time-course study revealed induction in brain and gills after 6h and at 12h in most tissues. Except for gills, all other organs retained induced expression of CYP1A mRNA up to 96h.     

Differential developmental toxicity of naphthoic acid isomers in medaka (Oryzias latipes) embryos

Polycyclic aromatic hydrocarbons (PAHs) are widespread persistent pollutants that readily undergo biotic and abiotic conversion to numerous transformation products in rivers, lakes and estuarine sediments. Here we characterize the developmental toxicity of four PAH transformation products each structural isomers of hydroxynaphthoic acid: 1H2NA, 2H1NA, 2H3NA, and 6H2NA. Medaka fish (Oryzias latipes) embryos and eleutheroembryos were used to determine toxicity. A 96-well micro-plate format was used to establish a robust, statistically significant platform for assessment of early life stages. Individual naphthoic acid isomers demonstrated a rank order of toxicity with 1H2NA > 2H1NA > 2H3NA > 6H2NA being more toxic. Abnormalities of circulatory system were most pronounced including pericardial edema and tube heart. To determine if HNA isomers were AhR ligands, spatial-temporal expression and activity of CYP1A was measured via in vivo EROD assessments. qPCR measurement of CYP1A induction proved different between isomers dosed at respective concentrations affecting 50% of exposed individuals (EC50s). In vitro, all ANH isomers transactivated mouse AhR using a medaka CYP1A promoter specific reporter assay. Circulatory abnormalities followed P450 induction and response was consistent with PAH toxicity. A 96-well micro-plates proved suitable as exposure chambers and provided statistically sound evaluations as well as efficient toxicity screens. Our results demonstrate the use of medaka embryos for toxicity analysis thereby achieving REACH objectives for the reduction of adult animal testing in toxicity evaluations.     

Molecular cloning and mRNA expression of cytochrome P4501A1 and 1A2 in the liver of common minke whales (Balaenoptera acutorostrata)

This study presents full-length cDNA sequences of CYP1A1 and 1A2, in common minke whale (Balaenoptera acutorostrata) from the North Pacific. Both CYP1A1 and CYP1A2 cDNAs had an open reading frame of 516 amino acid residues, and predicted molecular masses were 58.3 kDa and 58.1 kDa, respectively. The deduced full-length amino acid sequence of CYP1A1 revealed higher identities with those of sheep (86%) and pig (87%), and that of CYP1A2 was most closely related to human (82%) and monkey CYP1A2 (82%) among species from which CYP1A2 has been isolated so far. Differences in certain conserved and functional amino acid residues of CYP1A1 and 1A2 between common minke whale and other mammalian species indicate the possibility of their specific metabolic function. Concentrations of organochlorine compounds (OCs) including PCBs and DDTs analyzed in common minke whale liver showed no significant correlation with hepatic mRNA expression levels of CYP1A1 and CYP1A2, indicating no induction of these enzymes by such OCs.     

Cloning and expression of alpha class glutathione S-transferase gene from the small hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae)

In order to assess its potential as a biomarker of aquatic pollution, an alpha class glutathione S-transferase gene (GSTα gene) was cloned from the small hermaphroditic fish Rivulus marmoratus. The R. marmoratus GSTα gene spanned 1.3 kb, consisting of 6 exons encoding 221 amino acid residues. It showed high similarity to zebrafish GST. We named this R. marmoratus GSTα gene as rm-GSTα. The cDNA of the rm-GSTα gene was also investigated for its phylogeny, tissue-specific and chemical-induced expression. Rm-GSTα was subcloned into a 6 × His-tagged pCR^®T7 TOPO TA expression vector to produce the recombinant 6 × His-tagged rm-GST protein. This will be used in future to raise an rm-GSTα antibody for use in the study of phase II metabolism involved in detoxification. We also exposed R. marmoratus to 300 μg/l of 4-nonylphenol in water, and found approximately 4-fold induction of R. marmoratus GSTα mRNA in the treated animals. In this paper, we discuss the characteristics of the R. marmoratus GSTα gene as well as its potential use in relation to environmental pollution.     

Copper toxicity in the marine copepod Tigropus japonicus: Low variability and high reproducibility of repeated acute and life-cycle tests

The intertidal copeopod Tigriopus japonicus, which is abundant and widely distributed along the coasts of Western Pacific, has been suggested to be a good marine ecotoxicity testing organism. In this study, a series of experiments were conducted to investigate the reproducibility and variability of copper (Cu) sensitivity of T. japonicus so as to evaluate its potential to serve as an appropriate test species. To understand the seasonal variation of Cu sensitivity, individuals of T. japonicus were collected from the field in summer and winter, and subjected to standard 96 h acute (static renewal) toxicity tests. 96 h-LC50 values of T. japonicus collected from the two seasons were marginally different (p = 0.05), with an overall coefficient of variation (CV) of 33%. Most importantly, our results indicated that chronic Cu sensitivity of T. japonicus was highly reproducible. The CVs of intrinsic rates of increase in the population of the control and Cu treatment (10 μg Cu l⁻¹) groups were only 10–11% between 10 runs of a standardised complete life-cycle test. Moreover, different Cu(II) salts generally resulted in a similar 96 h-LC50 value while Cu(I) chloride was consistently slightly less toxic than Cu(II) salts. Given such a high reproducibility of toxic responses, it is advocated to use T. japonicus as a routine testing organism.     

Q and high-frequency strong motion spectra

Empirical scaling equations for Fourier amplitude spectra of strong ground motion are used to describe A₀ and τ in the assumed (high-frequency) shape of strong motion amplitudes: FS(_φ) = A₀e-^πτφ. The res of computed A₀ and τ with other related estimates of spectral amplitudes; (2) smooth decay of strong motion spectral amplitudes up to φ = 25 Hz, without an abrupt low-pass filtering of high frequecies; and (3) good agreement with other estimates of the regionally specific attenuation of high-frequncy seismic waves.As the recorded strong earthquake shaking in the western United States typically samples only the shallow (10 km) and local (100km) characteristics of wave attenuation, the processed strong motion accelerograms can be used as the most direct means of describing the nature of the high-frequency attenuation of the entire strong motion signal for use in earthquake engineering applications. Seismological body wave, Lg and coda wave estimates of Q sample different volumes of the crust surrounding the station, and involve different paths of the waves. These differences must be carefully documented and understood before the results can be used in earthquake engineering characterization of strong motion amplitudes.     

Biomarker responses in caged rockfish (Sebastes schlegeli) from Masan Bay and Haegeumgang, South Korea

The purpose of this study was to compare enzymatic biomarker activities in fish caged at two sites, Masan Bay (contaminated) and Haeguemgang (reference). In the present study, ethoxyresorufin O-deethylase (EROD), brain acetyl cholinesterase (bAChE), muscle acetyl cholinesterase (mAChE) and butyryl cholinesterase (mBChE) in caged rockfish (Sebastes schlegeli) were measured 0, 1, 3, 7, 14, 21 and 30 days after caging. The level of CYP1A mRNA and Protein expression was induced higher in Masan Bay at 1, 3, 7, 14 and 30 days after caging. EROD activity in the caged fish was significantly higher in Masan Bay than in Haeguemgang 3 and 7 days after caging, but not at 14 and 30 days after caging. bAChE activity was significantly inhibited at 7 and 14 days after caging in Masan Bay. However, mBChE activity was not significantly inhibited during the experiment. Taken together, the data suggest that the caged fish were exposed, at least transiently, to CYP1A inducers and ChE inhibitors, which is consistent with our previous observations.     

Changes in the Level of Proteins,Free Amino Acids and Protease Activities of Clarias batrachus in Response to Sodium meta‐arsenite Intoxication

Increased hazard of arsenic toxicity at many provinces of India causing extensive contamination of their wetlands. To illustrate the consequences of the arsenic menace on the aquatic fauna, a nutritionally important freshwater catfish Clarias batrachus (Linn.) was subjected to the stress of sublethal concentration (1 mg/L; 5% of 96‐h LC₅₀ value) of sodium meta‐arsenite. The alterations induced by the arsenic salt on certain biomolecules (proteins, free amino acids (FAA), and protease activities) in five tissue components (muscle, liver, brain skin, and gills) of the fish using biochemical techniques were estimated. Significant (p < 0.01) decrease in the concentration of proteins in all the tissues (19.74–44.84% in muscles; 3.17–49.39% in liver; 10.05–36.16% in brain; 12.62–52.01% in skin and 7.34–61.69% in gills), and significant (p < 0.01) increase in FAA (6.84–269.89% in muscles; 125.89–358.03% in liver; 1.07–152.90% in brain; 29.31–401.62% in skin and 38.53–165.07% in gills), and protease activities (43.59–183.33% in muscles; 19.52–131.90% in liver; 8.85–46.72% in brain; 33.33–261.53% in skin and 25.39–50.79% in gills) of the exposed fish were observed. This increase in amino acids and enzyme activity point out toward breakdown of protein leading to their depletion.     

Modern and Pleistocene boron isotope composition of the benthic foraminifer Cibicidoides wuellerstorfi

Here we present the first species-specific study of boron isotopes in the epibenthic foraminifer species Cibicidoides wuellerstorfi. Coretop samples from a water depth profile from 1000 to 4500 m on the northern flank of the Walvis Ridge are 4.4‰ lower than the values expected, based on calculations of the δ¹¹B_borate of ambient seawater. Similar values for this foraminifer species are presented from ODP site 668B at the Sierra Leone Rise, in the equatorial Atlantic. The consistency between data of the same species suggests the offsets are primary, rather than diagenetic. Glacial C. wuellerstorfi from ODP 668B and Walvis Ridge have boron isotope compositions only slightly different to interglacial samples, that is no larger than + 0.10 pH units, or + 23 µmol kg^− 1 in [CO₃²⁻] above the reconstructed glacial lysocline, and − 0.07 pH units, or − 14 µmol kg^− 1 in [CO₃²⁻] below. We use these results to suggest that glacial deep water pH in the Atlantic was similar to interglacial pH. The new data resolve the inconsistency between the previously reported high bottom water pH and the lack of significant carbonate preservation of the glacial deep ocean.     

Controls on shell Mg/Ca and Sr/Ca in cultured planktonic foraminiferan, Globigerinoides ruber (white)

Mg/Ca and Sr/Ca ratios were determined on a single species of planktonic foraminiferan, Globigerinoides ruber (white), collected from the Gulf of Eilat and cultured in seawater at five different salinities (32 to 44), five temperatures (18 to 30 °C) and four pH values (7.9 to 8.4). The Mg/Ca-temperature calibration of cultured G. ruber (with an exponential slope of 8 ± 3%/°C) agrees well with previously published calibrations from core-tops and sediment traps. However, the dependence of Mg/Ca on salinity (with an exponential slope of 5 ± 3%/psu) is also significant and should be included in the calibration equation. With this purpose, we calculated a calibration equation for G. ruber dependent on both temperature and salinity within the 95% confidence limits:

Mg/Ca(mmol/mol)=exp[0.06(±0.02)*S(psu)+0.08(±0.02)*T(°C)−2.8(±1.0)],R²=0.95