Response of an estuarine ecosystem to reduced organic waste discharge

Since the mid-19^th century large amounts of organic wastewere sluiced out into the Ems Estuary on the border between Germany and TheNetherlands. This waste originated from the regional potato flour and cardboardindustries making the inland waterways completely anoxic. In the estuaryseriousoxygen depletion occurred, especially during autumn. Most of the organic wasteentered the estuary in the Dollard, a brackish embayment of the Ems Estuary. Anintensive sanitation scheme was started in the 1970s, leading to a stepwisereduction of the organic waste load on the estuary. In this paper, a review isgiven of the response of the benthos living at intertidal mudflats,viz. microphytobenthos (diatoms), meiofauna (nematodes)andmacrozoobenthos. The benthos response is described mainly on the basis of dataobtained under conditions of high (ca. 1980), intermediate (1987) and largelyreduced (1993) organic waste loading.Reduction of organic loading caused significant changes in abundance, speciescomposition and standing stock of diatoms and nematodes. Macrobenthicpopulations recovered from being severely reduced regularly during the autumnalwaste discharges towards a more stable situation. Altogether, the intertidalmudflat benthos changed from organic waste loading stress towards a normal,estuarine environmental stress. This was especially the case at high and muddyflats in the southeast of the Dollard. At mixed sand/mud flats in the centre ofthe Dollard, a response of macrozoobenthos may have been obscured by theinvasion of the polychaete Marenzelleria cf.wireni.     

Immunocytochemical identification of prolactin cells in the pituitary gland of tilapia larvae (Oreochromis mossambicus: Teleostei)

Summary Using an antiserum to highly purified chum salmon prolactin, prolactin cells were identified in the putative rostral pars distalis of newly hatched tilapia larvae (Oreochromis mossambicus) by the immunogold method for the electron microscope. In the putative rostral pars distalis, some cells had another kind of secretory granule which was much less numerous, much smaller in size, and without immunoreactivity to salmon prolactin antiserum. Controls incubated with salmon prolactin-preabsorbed antiserum or normal serum showed no immunoreactive cells, confirming the specificity of the antiserum. The possible role of prolactin in the osmoregulation of tilapia larvae is discussed.     

The composition,nutritional status and digestibility of the diets of Sarotherodon mossambicus from nine man-made lakes in Sri Lanka

Synopsis The dietary composition and the nutritional status and the digestibility of the diets of Sarotherodon mossambicus from nine reservoirs in Sri Lanka were evaluated. The feeding habits of S. mossambicus were variable from reservoir to reservoir; they ranged from herbivory to total carnivory. The protein, total lipid, carbohydrate and total organic matter content of the ingested material were related to the dietary composition and ranged from 18.53% to 35.15% (x−24.18%), 5.94% to 9.84% (x−7.91%), 11.6% to 34.7% (x−22.34%) and 34.4% to 64.4% (x−45.71%), respectively. Irrespective of the feeding habits, the diet contained a significant proportion of organic material which cannot be accounted for by protein, total lipid and carbohydrate. As much as the ingested material was related to the feeding habit, the digestibility of the nutrient components was related to the food material devoured. For example, the mean digestibility of the total organic matter in S. mossambicus feeding on detritus, plants and animal were 36.85, 33.5 and 29.5 respectively, and compared well with observations from elsewhere. It is hypothesised that the favourable nutrient quality of the available dietary material in the reservoirs of Sri Lanka, which could be and is effectively utilized by S. mossambicus, may have been, at least partially, responsible for its almost unprecedented success in Sri Lanka.     

Flagellate Cryptobia branchialis (Bodonida: Kinetoplastida), ectoparasite of tilapia from the Salton Sea

An infestation of young tilapia, Oreochromis mossambicus Peters, by the flagellate Cryptobia branchialiswas observed at the Salton Sea, California, in September, 1997. This is the first report of C. branchialis in a highly saline water-body (43 g l^–1). Ultrastructure of C. branchialis as well as its effect on the gills of tilapia were studied using the scanning and transmission electron microscopy. No direct effect of C. branchialis on the epithelial cells of fish gills was observed. However, alterations of gill general structure, such as deposition of copious mucus on the gill surface, swelling of filaments, reduction of respiratory lamellae and their transformation into short club-shaped structures were found in infected fish. This suggests mortality of young tilapia may arise from decreased gill function in response to Cryptobia infestation.     

In vitro stimulation and inhibition of steroid hormone release from postovulatory follicles of the tilapia,Oreochromis mossambicus

Summary Postovulatory follicles of the tilapia, Oreochromis mossambicus, were incubated with graded doses of salmon gonadotropin to identify the steroid hormones released by this tissue. In addition, the effects of either cytochalasin B or colchicine on steroid hormone release were studied. After the incubation, the tissue was examined by electron microscopy. Postovulatory follicles released testosterone and estradiol-17B in a dose-dependent manner with gonadotropin. There was no detectable release of progesterone or 17a-OH-progesterone. When stimulated with high doses of gonadotropin, the steroidogenic cells showed an increase in smooth endoplasmic reticulum, Golgi complexes, and lipid droplets. Also, microfilaments became arranged in orderly bundles and were found close to the numerous secretory vesicles and lipid droplets. Upon incubation with gonadotropin and either colchicine or cytochalasin B, the cells still appeared steroidogenic, but the filaments were not organized nor associated with vesicles or lipid droplets. Release of steroid hormone decreased significantly. Also in these tissues, vesicles were no longer numerous in the apical region of the granulosa cells, but were located primarily near smooth endoplasmic reticulum and Golgi complexes. This suggests that disruption of the cytoskeleton results in reduced steroid hormone synthesis or release.     

The diet of the mud clam Geloina coaxans (Mollusca, Bivalvia) as indicated by fatty acid markers in a subtropical mangrove forest of Okinawa, Japan

Fatty acid compositions in the tissues of the clam Geloina coaxans collected from Oura mangal, Okinawa, Japan, during the cold and warm seasons (January and July 2001, respectively) were compared with those in suspended materials (SM) in order to assess the clams' diet. In both seasons, the suspended mangrove detritus at the sediment-water interface was high as indicated by the mean percentage of even-numbered long-chain fatty acids in SM (12.8-18.4%). The contribution of this marker in the clam tissues, especially during the cold season (3.9%), indicates the consumption of mangrove detritus in considerable amounts by the clams. The occurrence of the fatty acids 16:1ω7, 18:1ω9, 18:2ω6 and 18:3ω3 in SM was most likely due to the mangrove detritus sources, whereas in the SM they together constituted 12.9% and 23.9% of total fatty acid contents during the cold and warm seasons, respectively. As a result, their contribution in the clam tissues was high in the cold (15.4%) and warm seasons (19.0%). These results indicate that mangrove detritus play a significant role in the clams' diet. The mean percentages of bacterial markers (odd-numbered branched fatty acids and vaccenic acid, 18:1ω7) in the SM and tissues during both seasons ranged from 8.1% to 9.5%. This indicates that the clam diet is also dependent on the attached bacteria on the partially decomposed leaf detritus suspended at the sediment-water interface. The relative contribution by microalgae markers (18:4ω3, 20:5ω3 and 22:6ω3) in clam tissues ranged from 4.3% to 7.6%, suggesting considerable microalgae sources in the diets.     

The immune response of tilapia Oreochromis mossambicus and its susceptibility to Streptococcus iniae under stress in low and high temperatures

Mozambique tilapia Oreochromis mossambicus acclimated to 27 degrees C were then held at 19, 23, 27 (control), 31 and 35 degrees C, and were examined for non-specific cellular and humoral responses after 12-96 h. Total leucocyte count decreased significantly when fish were transferred to 19 and 23 degrees C after 48 and 96 h, and when transferred to 35 degrees C over 12-96 h, respectively. Respiratory burst decreased significantly when fish were transferred to 19, 31 and 35 degrees C over 24-96 h, whereas phagocytic activity and phagocytic index decreased significantly when fish were transferred to low temperatures (19 and 23 degrees C) and high temperatures (31 and 35 degrees C) over 12-96 h. Lysozyme activity decreased significantly when fish were transferred to 19 degrees C after 12-96 h, but increased significantly when transferred to 31 and 35 degrees C over 48-96 h. Alternative complement pathway (ACH(50)) also decreased significantly when transferred to 19 and 23 degrees C after 12h, but increased significantly when transferred to 31 and 35 degrees C after 24h. In another experiment, tilapia reared at 27 degrees C were injected intraperitoneally with Streptococcus iniae at a dose of 1 x 10(7)colony-forming units (cfu)fish(-1), and then reared onward at water temperatures of 19, 23, 27 (control), 31 and 35 degrees C. Over 48-168 h, the cumulative mortality of S. iniae-injected fish held in 19 and 35 degrees C was significantly higher than that of injected-fish held in 23, 27 and 31 degrees C. It is concluded that transfer of tilapia O. mossambicus from 27 degrees C to low temperatures (19 and 23 degrees C) after 12h, and transfer of fish from 27 degrees C to high temperatures (31 and 35 degrees C) reduced their immune capability. Moreover, tilapia under temperature stress at 19 and 35 degrees C from 27 degrees C decreased its resistance against S. iniae.     

Characterization of fatty acid composition in healthy and bleached corals from Okinawa, Japan

Under bleaching conditions, corals lose their symbiotic zooxanthellae, and thus, the ability to synthesize fatty acids (FAs) from photosynthetically derived carbon. This study investigated the lipid content and FA composition in healthy and bleached corals from the Odo reef flat in Okinawa, southern Japan, following a bleaching event. It was hypothesized that the FA composition and abundance would change as algae are lost or die, and possibly microbial abundance would increase in corals as a consequence of bleaching. The lipid content and FA composition of three healthy coral species (Pavona frondifera, Acropora pulchra, and Goniastrea aspera) and of partially bleached and completely bleached colonies of P. frondifera were examined. The FA composition did not differ among healthy corals, but differed significantly among healthy, partially bleached, and completely bleached specimens of P. frondifera. Completely bleached corals contained significantly lower lipid and total FA content, as well as lower relative amounts of polyunsaturated FAs and higher relative amounts of saturated FAs, than healthy and partially bleached corals. Furthermore, there was a significantly higher relative concentration of monounsaturated FAs and odd-numbered branched FAs in completely bleached corals, indicating an increase in bacterial colonization in the bleached corals.     

The effects of predation on the survival and size-distribution of estuarine fishes: an experimental approach

Synopsis The effect of southern flounder,Paralichthys lethostigma, predation on the survival and size-distribution of spot,Leiostomus xanthurus, was investigated using feeding experiments in outdoor tanks and estuarine ponds. In the outdoor tank experiments, southern flounder, 143 mm average standard length (SL), fed for 48 h on spot (25–30 mm SL), over a range of densities (4–20 m^–2). In these trials, flounder showed a Type II functional response to spot density. In the estuarine pond experiment, flounder density (2 or 4 flounders 70 m^–2) contributed significantly to spot mortality relative to predator-free controls. The effect of flounder density on spot mortality was non-linear; in the 4-flounder treatment the daily instantaneous mortality due to flounder (0.0177) was 3.5 times that in the 2-flounder treatment (0.0051). The total spot mortality rate in the 4-flounder treatment (0.028) was similar to that observed from published field observations. Flounder also altered the size distribution of spot relative to predator-free controls. Predation by flounder resulted in fewer intermediate-sized spot at the end of the experiment when compared to predator-free controls. Flounder also significantly reduced survival and mean length of two killifishes,Cyprinodon variegatus andFundulus heteroclitus, that invaded the pond. Flounder predation was a significant size-structuring force on the prey fish assemblage in the pond. Results from both the outdoor tank experiment and the pond experiment indicate that flounder may represent a significant source of mortality for juvenile estuarine fishes.     

Phospholipid composition and fatty acid desaturation in the roots of rye during acclimatization of low temperature

When the roots of rye plants grown at 20°C were cooled to 8°C the concentration of phospholipid in them more than doubled over a 7 d period in comparison with that in roots remaining at 20°C. The relative abundance of lecithin (PC) declined while that of phosphatidyl ethanolamine (PE) increased; this change was completed after 2 d cooling. Labelling with ³²P suggested that turnover of phospholipids may be inhibited by low temperature. Acyl lipids contained an increased proportion of linolenic acid (18:3) and reduced proportion of linoleic acid (18:2) when roots were cooled at 8°C for 7 d. The ratio of these acids is a relatively more sensitive indicator of desaturation than is the double bond index. Cooling brought about no change in the abundance of the principal saturated acid, palmitic (16:0). In the first 3 days of cooling PC and PE desaturated markedly while there was no change in galactosyl and neutral lipids. Desaturation did not appear to be greatly sensitive to the concentration of dissolved O₂ and was only partly inhibited in 8°C solutions where the oxygen concentration was lowered to 0.5–2.0%. Positional analysis of acyl chains in PC and PE showed that more than 90% of all 16:0 is associated with position I while 65% of the 18:2+18:3 is associated with position II. When roots are cooled the abundance of 18:3 increases in both chains but the relative distribution of saturated and unsaturated fatty acids remains constant in positions I and II. At both 20°C and 8°C there is a high probability that a saturated chain in position I will be paired with the polyunsaturated one in position II.Abbreviations PC Lecithin - PE phosphatidyl ethanolamine - TLC thin layerchromatography - BHT butylatedhydroxytoluene     

Comparison of two fatty acid analysis protocols to characterize and differentiate Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici

The utility of fatty acid methyl ester (FAME) profiles for characterization and differentiation of isolates of Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici was investigated. Two fatty acid analysis protocols of the normal (MIDI) and a modified MIDI method were used for their utility. Only the modified MIDI method allowed a clear differentiation between F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicislycopersici. FAME profiles using the modified MIDI method gave the most consistent and reproducible analyzed fatty acid data. Evaluation of the FAME profiles based on cluster analysis and principal-component analysis revealed that FAME profiles from tested isolates were correlated with the same vegetative compatibility groups (VCGs) compared to the same races in F. oxysporum f. sp. lycopersici. Results indicated that FAME profiles could be an additional tool useful for characterizing isolates and forma species of F. oxysporum obtained from tomato.     

Comparative study on the fatty acid composition of two marine vertebrates: striped dolphins and loggerhead turtles

The fatty acid composition of total lipids extracted from seven different tissues (fat, liver, cerebrum, cerebellum, lung, kidney and muscle) of 10 striped dolphins (Stenella coeruleoalba) (weight 75.7±16.2 kg) found dead or moribund during a morbillivirus epizootic, and from two tissues (fat and liver) of 54 loggerhead turtles (Caretta caretta) (18.5±10.5 kg) seized dead after illegal capture, both from the Mediterranean Sea, have been determined by high resolution chromatography techniques. When comparing both species, fatty acid composition is surprisingly similar, with a predominance of the monoenoic followed by the saturated group of fatty acids, and a very close ratio of polyunsaturates n-3/n-6. The relatively high arachidonic acid content in the liver of the two marine species is remarkable. The similar diet can play an important role in these findings, but it is suggested that probably metabolic pathways and essential fatty acid requirements between both marine vertebrates are similar, more than is expected from their earlier filogenetic evolutionary divergence.     

Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared toward the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically expressed enzymes. Here, we show that the addition of an N-terminal epitope tag sequence (either Myc or hemagglutinin) to oleate desaturase (FAD2) or omega-3 linoleate desaturase (FAD3) enzymes from plants, which catalyze consecutive reactions in the production of long chain omega-3 fatty acids, significantly increases their activity up to fourfold when expressed in yeast cells. Quantitative protein blotting using an antibody specific for native FAD2 revealed that the steady-state amount of the epitope-tagged FAD2 protein was also approximately fourfold higher than that of its untagged counterpart, demonstrating a direct relationship between the epitope tag-induced increase in enzyme amount and fatty acid product formation. Protein half-life and RNA blotting experiments indicated that the half-lives and mRNA content of the tagged and untagged FAD2 proteins were essentially the same, suggesting that the epitope tags increased protein abundance by improving translational efficiency. Taken together, these results indicate that the addition of an epitope tag sequence to a plant fatty acid desaturase (FAD) not only provides a useful means for protein immunodetection using highly specific, commercially available antibodies, but that it also significantly increases FAD activity and the production of polyunsaturated fatty acids in yeast cells.     

Functional analysis of a fatty acid elongase from arachidonic acid-producing <Emphasis Type="Italic">Mortierella alpina</Emphasis> 1S-4

We describe the isolation and characterization of a gene (MAELO) that encodes a fatty acid elongase from arachidonic acid-producing fungus Mortierella alpina 1S-4. Although the homologous MAELO gene had already been isolated from M. alpina ATCC 32221, its function had not yet been identified. The MAELO gene from M. alpina 1S-4 was confirmed to encode a fatty acid elongase by its expression in yeast Saccharomyces cerevisiae. Analysis of the fatty acid composition of the yeast transformant revealed the accumulation of 22-, 24-, and 26-carbon saturated fatty acids. On the other hand, RNA interference of the MAELO gene in M. alpina 1S-4 was carried out. The gene-silenced strain obtained on RNA interference exhibited low contents of 20-, 22-, and 24-carbon saturated fatty acids and a high content of stearic acid (18 carbons), compared with those in the wild strain. The enzyme encoded by the MAELO gene was demonstrated to be involved in the biosynthesis of 20-, 22-, and 24-carbon saturated fatty acids in M. alpina 1S-4.     

Variation of lipid and fatty acid compositions of the marine microalga Pavlova viridis (Prymnesiophyceae) under laboratory and outdoor culture conditions

The marine microalga Pavlova viridis (Prymnesiophyceae) is widely used in marine aquaculture industries of China for feeding bivalves and has been proposed as an alternative source of eicosapentaenoic acid (EPA). To investigate variation of its lipid and fatty acid compositions during laboratory and outdoor cultivation, a 60-1 photobioreactor was established in Nanjing, China. Outdoor cultivation, paralleled with laboratory cultures in mid-October, was performed from autumn through midwinter. The results showed that the total lipid and EPA contents of outdoor cultures were both lower than those of indoor cultures. When the outdoor temperature and illumination decreased, total lipid experienced no significant change. Although the level of saturated fatty acids decreased, polyunsaturated fatty acids, especially EPA, increased.     

The relative importance of sediment and water column supplies of nutrients to the growth and tissue nutrient content of the green macroalga Enteromorpha intestinalis along an estuarine resource gradient

Large blooms of opportunistic green macroalgae such as Enteromorpha intestinalis are of ecological concern in estuaries worldwide. Macroalgae derive their nutrients from the water column but estuarine sediments may also be an important nutrient source. We hypothesized that the importance of these nutrient sources to E. intestinalis varies along a nutrient-resource gradient within an estuary. We tested this in experimental units constructed with water and sediments collected from 3 sites in Upper Newport Bay estuary, California, US, that varied greatly in water column nutrient concentrations. For each site there were three treatments: sediments + water; sediments + water + Enteromorpha intestinalis (algae); inert sand + water + algae. Water in units was exchanged weekly simulating low turnover characteristic of poorly flushed estuaries. The importance of the water column versus sediments as a source of nutrients to E. intestinalis varied with the magnitude of the different sources. When initial water column levels of dissolved inorganic nitrogen (DIN) and soluble reactive phosphorus (SRP) were low, estuarine sediments increased E. intestinalis growth and tissue nutrient content. In experimental units from sites where initial water column DIN was high, there was no effect of estuarine sediments on E. intestinalis growth or tissue N content. Salinity, however, was low in these units and may have inhibited growth. E. intestinalis growth and tissue P content were highest in units from the site with highest initial sediment nutrient content. Water column DIN was depleted each week of the experiment. Thus, the water column was a primary source of nutrients to the algae when water column nutrient supply was high, and the sediments supplemented nutrient supply to the algae when water column nutrient sources were low. Depletion of water column DIN in sediment + water units indicated that the sediments may have acted as a nutrient sink in the absence of macroalgae. Our data provide direct experimental evidence that macroalgae utilize and ecologically benefit from nutrients stored in estuarine sediments.     

First total synthesis and antileishmanial activity of (Z)-16-methyl-11-heptadecenoic acid, a new marine fatty acid from the sponge Dragmaxia undata

The first total synthesis for the (Z)-16-methyl-11-heptadecenoic acid, a novel fatty acid from the sponge Dragmaxia undata, was accomplished in seven steps and in a 44% overall yield. The use of (trimethylsilyl)acetylene was key in the synthesis. Based on a previous developed strategy in our laboratory the best synthetic route towards the title compound was first acetylide coupling of (trimethylsilyl)acetylene to the long-chain protected 10-bromo-1-decanol followed by a second acetylide coupling to the short-chain 1-bromo-4-methylpentane, which resulted in higher yields. Complete spectral data is also presented for the first time for this recently discovered fatty acid and the cis double bond stereochemistry of the natural acid was established. The title compound displayed antiprotozoal activity against Leishmania donovani (IC₅₀ = 165.5 ± 23.4 μM) and inhibited the leishmania DNA topoisomerase IB enzyme (LdTopIB) with an IC₅₀ = 62.3 ± 0.7 μM.     

Charge modification at conserved positively charged residues of fatty acid binding protein (FABP) from the giant liver fluke Fasciola gigantica: its effect on oligomerization and binding properties

Fatty acid binding proteins (FABPs) are capable of binding hydrophobic ligands with high affinity; thereby facilitating the cellular uptake and intracellular trafficking of fatty acids. In this study, functional characteristics of a cytoplasmic FABP from the giant liver fluke Fasciola gigantica (FgFABP) were determined. Binding of a fluorescent fatty acid analogue 11-[[5-dimethy aminonaphtalene-1-sulphonyl] amino] undecanoic acid (DAUDA) to FgFABP resulted in changes in the emission spectrum. The optimal excitation wavelength and maximum emission of fluorescence for binding activities with DAUDA were 350 nm and 550 nm, respectively. The binding activity for DAUDA was determined from titration experiments and revealed a K_d value of 2.95 ± 0.54 μM. Furthermore, we found that cross-linking profile of FgFABP with dithiobis-(succinimidylpropionate) (DSP) in the presence of DAUDA resulted in increased formation of higher-ordered oligomers compared to that in the absence of DAUDA. We also replaced five highly conserved positively charged residues (K9, K58, K91, R107 and K131) with alanine and studied their oligomerization and binding properties of the modified FgFABPs. The obtained data demonstrate that these residues do not appear to be involved in oligomerization. However, the K58A and R107A substitutions exhibited a reduction in binding affinities. K91A and R107A revealed an increase in maximal specific binding.