Improved methodology for ATP determination in marine environments

A simple technique is described for de-salting and concentrating adenosine triphosphate (ATP) extracted from seawater or marine sediment samples prior to assays with the standard luciferin-luciferase procedure. The technique involves chromatography of H₂SO₄ extracts on columns of activated carbon. The efficiency of ATP recovery from marine sediments using this pre-treatment was superior to that attained with either boiling Tris extraction or with H₂SO₄ extraction without subsequent purification. All ions which interfere with the luciferase reaction or precipitate ATP upon neutralization of acid extracts are removed with this procedure, thus eliminating the 50- to 100-fold dilutions required with other acid-extraction procedures. In addition, the purified ATP extracts may be concentrated up to 100-fold, thus greatly improving the sensitivity of ATP measurement in samples containing very low biomass. This procedure has been applied to ATP determination in marine sediments in the upwelling area off northwest Africa. The average ATP concentrations in the upper 6 cm of sediment from 1000 and 200 m were 195 and 545 ng per ml wet sediment, respectively.     

Nutrient cycling between the water column and a marine sediment. I. Organic carbon

The carbon flow through the sediments at a station located in 18.3 m of water off the Scripps Institution of Oceanography, San Diego, California (USA) was determined. The parameters studied [and their mean rates of input (+) or output (-) to the benthos] were macro-detritus (+0.028 gC m^-2day^-1), fallout of particulate debris (+3.3 gC m^-2day^-1), benthic net photosynthesis during the day (-0.06 gC m^-2 daylight period^-1), burial (0 gC m^-2day^-1), benthic respiration at night (-0.28 gC m^-2 night period^-1), and resuspension (-3.0 gC m^-2day^-1). Resuspension of sediment at this station was found to have a controlling effect on the sediment organic carbon content. Benthic photosynthesis was able to provide 79% of the organic carbon required by the benthos for respiration during the daylight hours. A carbon-flow diagram linking together all of the above measurements is presented.     

Marine diatoms grown in chemostats under silicate or ammonium limitation. II. Transient response of Skeletonema costatum to a single addition of the limiting nutrient

Skeletonema costatum was grown at different steady-state growth rates in ammonium or silicate-limited chemostats. The culture was perturbed from its steady-state condition by a single addition of the limiting nutrients ammonium or silicate. The transient response was followed by measuring nutrient disappearance of the liliting perturbation experiment indicate that three distinct modes of uptake of the limiting nutrient can be distinguished; surge uptake (V _s ), internally controlled uptake (V _i ), and externally controlled uptake (V _e ). An interpretation of these three modes of uptake is given and their relation to control of uptake of the limiting nutrient is discussed. The uptake rates of the non-limiting nutrients were shown to be depressed during the surge of the uptake of the limiting nutrient. Kinetic uptake parameters, K _s and V _max, were obtained from data acquired during the externally controlled uptake segment, V _e . The same V _max value of 0. 12 h^-1, was obtained under either silicate or ammonium limitation. Estimates of K _s were 0.4 g-at NH₄-N l^-1 and 0.7 g-at Si l^-1. Short-term ¹⁵N uptake-rate measurements conducted on nitrogen-limited cultures appear to be a combination of V _s or V _i , or at lower substrate concentrations V _s and V _e . It is difficult to separate these different uptake modes in batch or tracer experiments, and ensuing problems in interpretation are discussed.Contribution No. 882 from the Department of Oceanography, University of Washington, Seattle, Washington 98195, USA. This work represents portion of three dissertations submitted to the Department of Oceanography, University of Washington, Seattle, in partial fulfillment of the requirements for the Ph.D. degree.     

Decomposition of urea associated with photosynthesis of phytoplankton in coastal waters

Decomposition of urea in seawater was studied in Mikawa Bay, a shallow eutrophic bay on the southern coast of central Japan. The urea concentration in seawater ranged from 1.3 to 5.9 μg-at. N/1 and comprised 12 to 40% of the dissolved organic nitrogen. Using ¹⁴C labelled urea, the rate of CO₂ liberation from urea and the incorporation rate of urea carbon into the particulate organic matter were determined. For the surface samples, high rates of CO₂ liberation from urea as well as the incorporation of urea carbon into the particulate organic matter were observed in the light, while much lower rates were obtained in the dark. Incubation experiments with exposure to different light intensities revealed that the rate of CO₂ liberation from urea and the incorporation of urea carbon into particulate organic matter changed with light intensity, showing a pattern similar to that of photosynthesis. The highest liberation and incorporation rates were observed at 12,000 lux. Incubation in light and in dark produced marked decreases and increases, respectively, in urea and ammonia, while no appreciable changes were observed for nitrate and nitrite. It is suggested that urea decomposition associated with photosynthetic activity of phytoplankton is one of the major processes of urea decomposition, and that it plays a significant role in the nitrogen supply for phytoplankton in coastal waters.     

The genetic basis of morphological differentiation in some Laminaria populations

Clinal differentiation of stipe traits in Laminaria (simplices) populations occurs on a gradient of exposure to wave action. Crossability tests and the production of fertile hybrids established interfertility between populations from the extremes of the morphological range. Reciprocal transplantation experiments between exposed and sheltered sites did not produce significant changes in stipe morphology. Quantitative genetic analysis of the degree of resemblance between relatives indicated a relatively high genetic component in the interpopulation differentiation. This is consistent with the results of the transplantation experiments. Within-population heritability estimates at the exposed site were low, perhaps because of intensive local stabilising selection which acts to reduce genetic variability. The question of conspecificity of L. saccharina and L. longicruris in view of these results is discussed.     

Background levels of microelements in soils of Italy

A systematic survey on the distribution of geochemically and texicologically relevant trace elements in the soils of Italy was carried out on 32 soil profiles, developed in different environmental and pedological conditions. The aim of this study was to collect basic information on the background levels of trace elements, their geochemical behaviour and the relationships between weathering and pedogenetic processes. The statistically processed analytical results led to the following conclusions: (i) Trace element distribution in the profiles studied is consistent with pedochemical behaviour and rock lithology. A comparison with the world soil means shows that there are no significant differences, except for those elements largely utilised in agriculture (Cu, Zn) or, because of pollution, (Pd, Cd), or natural accumulation (Ni, Cr); (ii) data processing by cluster analysis shows marked correlations between some elements, for example, Al-V, Ni-Cr, Mn-Co. Groups of geochemically homogeneous samples, which correspond to the great soil families characteristic of each pedological landscape, are also indicated; (iii) evidence of soil contamination is provided by the high levels of some elements, especially in the A horizon. Cu and Zn seem to be concentrated in the Ap horizon of cultivated soils, while Pd and Cd may be derived from the atmosphere.     

Male mating speed promote hybridization in the Rana lessonae–Rana esculenta waterfrog system

Hybridization is a widespread phenomenon in many vertebrate groups. Prezygotic isolating mechanisms, probably caused by selection against hybrids with reduced fitness, reduce the likelihood of such events. Although hybrid-reduced fitness relatively to parental species is common, hybridization can also be beneficial, and hybrids sometimes outperform the pure species type. In this study, we examined two potential processes, Hubbs’s principle and male–male competition, which could enhance hybridization in the waterfrog complex and thus explain the proportion of heterospecific pairs collected in a natural pond. Firstly, by collecting 791 frogs in the field to study pair and chorus composition, we showed that in a mixed Rana lessonae–Rana esculenta population, the scarcity of hybrid R. esculenta males did not account for the proportion of heterospecific pairs: indeed, when examining pairing composition in six different choruses, we found that hybrid males were always under-represented and that R. esculenta females were found paired with R. lessonae males. Secondly, we investigated experimentally whether or nor male–male competition mechanism could explain pair formation in waterfrogs. Our mating speed experiment highlights mechanisms that could explain heterospecific pairs in a context of promiscuous mating where scramble competition was intense. To measure the rapidity with which a male grasps a female, we placed males in a grid cage with a female, and the dynamics of pair formation was monitored. R. esculenta males showed a lower pairing success than R. lessonae males as a smaller proportion of them amplexed females, and more time was needed for them to get amplexed. Thus, a less adaptative mechanism than female mate choice may also explain the mating pattern observed in waterfrog species.     

Dynamics of soil organic matter in primary and secondary forest succession on sandy soils in The Netherlands: An application of the ROMUL model

We applied the simulation model ROMUL of soil organic matter dynamics in order to analyse and predict forest soil organic matter (SOM) changes following stand growth and also to identify gaps of data and modelling problems. SOM build-up was analysed (a) from bare sand to forest soil during a primary succession in Scots pine forest and (b) on mature forest soil under Douglas fir plantations as an example of secondary succession in The Netherlands. As some of the experimental data were unreliable we compiled a set of various scenarios with different soil moisture regime, initial SOM pools and amount and quality of above and below ground litter input. This allowed us to find the scenarios that reflect the SOM dynamics more realistically. In the Scots pine forest, total litter input was estimated as 0.50 kg m⁻² year⁻¹. Two scenarios were defined for the test runs: (a) forest floor moisture regimes—‘dry, mesic and hydric’ and (b) augmenting a root litter pool with three ratios of needles and branches to roots: 1:1, 1:1.5 and 1:2.0. The scenario finally compiled had the following characteristics: (a) climate for dry site with summer drought and high winter moisture of forest floor; (b) a litter input of 0.25 kg m⁻² year⁻¹ above ground and 0.50 kg m⁻² year⁻¹ below ground; (c) a low nitrogen and ash content in all litter fall fractions. The test runs for the estimation of the initial SOM pools and the amount and proportion of above and below ground litter fall were also performed in the Douglas fir plantation. The inputs of above ground litter tested in various combinations were 0.30 and 0.60 kg m⁻² year⁻¹, and below ground litter 0.30, 0.60 and 0.90 kg m⁻² year⁻¹. The scenario that fitted the experimental data had an SOM pool of 20–25 kg m⁻², an aboveground litter input of 0.6 kg m⁻² year⁻¹and a below ground litter input of 0.9 kg m⁻² year⁻¹. The long-term simulation corresponded well with the observed patterns of soil organic matter accumulation associated with the forest soil development in primary and secondary succession. During primary succession in Scots pine forest on dry sand there is a consistent accumulation of a raw humus forest floor. The soil dynamics in the Douglas fir plantation also coincide with the observed patterns of SOM changes during the secondary succession, with SOM decreasing significantly under young forest, and SOM being restored in the older stands.     

Metabolic specialization of mitochondria from scallop phasic muscles

In fast, glycolytic muscles, oxidative phosphorylation presumably facilitates recuperation from exhaustive exercise and supports growth and maintenance metabolism. Given the shifts in pH with extensive glycolytic activity, the pH optima of mitochondrial processes should indicate whether mitochondria are adapted for recuperation from exercise or for growth and maintenance. We examined this question using mitochondria from the phasic adductor muscle of the scallop, Euvola (Pecten) ziczac, collected from the Golfo de Cariaco, Venezuela in 1992 and 1993. Scallop muscle mitochondria showed well coupled oxidation of glutamate and pyruvate at pH 7.0 and 6.4. The preferred substrates (glutamate, pyruvate and succinate) were oxidized at approximately 40 nmol O₂ min^-1 mg^-1 mitochondrial protein at 25°C, while malate and glutamine were oxidized at 75% and proline at 30% of these rates. Neither palmitoyl carnitine nor aspartate were oxidized. Succinate oxidation was not coupled to ADP utilization at pH 7.0 but was somewhat coupled at pH 6.4. Generally, State 3 rates of oxygen uptake were similar at pH 7.0 and 6.4. Maximal rates of oxidation of glutamate and pyruvate showed broad pH optima. For both glutamate and pyruvate, the highest respiratory control ratio (RCR) values were found at pH 6.5. The saturation curves of scallop muscle mitochondria for pyruvate, glutamate and ADP were well described by the Michaelis-Menten equation. The affinity for pyruvate was greater at pH 6.4 (apparent K _{m, app}=0.013 mM) than at pH 7.0 (K _{m, app}=0.026 mM) while the affinity for ADP (K _{m, app}=0.015 mM) and that for glutamate (K _{m, app}=0.55 mM) changed little with pH. The ADP affinity was the same whether pyruvate or glutamate was the carbon substrate. The combination of maintenance of sensitivity to ADP with an enhanced affinity for pyruvate at acidic pH values should facilitate recuperation from bouts of glycolytic activity. Scallops harvested in September and those harvested in January differed in the maximal rates of glutamate and pyruvate oxidation.     

Cadmium bioaccumulation in organs of the scallopMizuhopecten yessoensis

Cadmium and Zinc content was determined in organs of the Japanese scallopMizuhopecten yessoensis, collected in 1984 from the Sea of Japan and aged between 1 and 8 yr. Under conditions of background Cd, Cd concentration in hepatopancreas and kidney increased linearly with age from 39 to 400µg g^?1 dry wt in hepatopancreas, and from 100 to 640µg g^?1 dry wt in kidney. Such a pattern was not determined for Zn. Cd concentration in muscle, mantle and gill did not exceed 6µg g^?1 dry wt in the oldest scallops. In subcellular fractions of the hepatopancreas, cytosolic Cd accounted for 71.7% in 1-yr-old scallops and 98.8% in 8-yr-olds. A similar ratio was established for gills, although gill Cd content was an order of magnitude lower. Analysis of Cd distribution in cytoplasmic proteins (of different molecular weight) in the hepatopancreas, showed that the amount of Cd bound to metallothionein-like proteins increases with scallop growth. A considerable amount of Cd also was detected in high molecular weight proteins. Thus, Cd accumulation, with age, in organs of the Japanese scallop is due to metal binding by cytoplasmic proteins.