Organic nitrogen uptake and growth by the chrysophyte Aureococcus anophagefferens during a brown tide event

The quantitative importance of light-mediated, dissolved organic nitrogen (DON) utilization in relation to overall nitrogen-assimilation in Aureococcusanophagefferens Hargraves et Sieburth was assessed during a brown tide event in Shinnecock Bay, Long Island, 24 through 26 July 1995. The growth response of A. anophagefferens was maximal in organic-rich Bay water and decreased proportional to the organic:inorganic nutrient ratio of the water. Short-term uptake measurements with six nitrogenous substrates revealed that reduced nitrogen could potentially represent 95% of overall nitrogen uptake of which 70% was due to organic nitrogen alone. Potential uptake of urea by the A. anophagefferens-dominated bloom was substan tially greater than uptake of the other substrates tested during the study, contributing the largest percentage of total nitrogen uptake (58 to 64%; ρ^′ _max(urea) 4.4 μg  atom N l⁻¹ h⁻¹), followed by NH₄ ⁺ (18 to 26%; ρ′_max(NH4+) 2 μg atom N l⁻¹ h⁻¹). The combined rates of uptake of algal extract, lysine and glutamic acid contributed between 11 and 16% of total uptake, whereas NO₃ ⁻contributed 5 to 8%. Based on the kinetic determinations from this study we suggest an ecological framework for the events leading to the dominance and abundance of A. anophagefferens in coastal bays. Received: 29 March 1997 / Accepted: 24 April 1997     

Interactions between NH+4 and NO−3 uptake and assimilation: comparison of diatoms and dinoflagellates at several growth temperatures

Ammonium concentrations of ∼1 M are commonly cited as being the threshold for inhibition of NO₃ ⁻ uptake, but the applicability of this threshold to phytoplankton from different taxonomic classes has rarely been examined. Additionally, little is known about the influence of environmental variables (e.g. growth temperature) on the interaction between ambient NH₄ ⁺ and NO₃ ⁻ uptake. Four species of estuarine phytoplankton, two diatom [Chaetoceros sp., and Thalassiosira weissflogii (Grunow) Fryxell et Hasle] and two dinoflagellate [Prorocentrum minimum (Pavillard) Schiller, and Gyrodinium uncatenum Hulburt], were grown on NO₃ ⁻ at several different temperatures (4, 10, 15, or 20 °C), and the impact of NH₄ ⁺ additions on NO₃ ⁻ uptake/assimilation (non-TCA-extracted) and assimilation (TCA-extracted) was assessed. For all species at all temperatures, NO₃ ⁻ uptake/assimilation and assimilation rates decreased in a roughly exponential manner with increasing NH₄ ⁺ concentrations but were not completely inhibited even at elevated NH₄ ⁺ concentrations of 200 μM. Estimated half-inhibition concentrations (K _i) were significantly greater in the diatom species (mean ± SE; 2.70 ± 0.67 μM) than in the dinoflagellate species (1.26 ± 0.55 μM). Half-inhibition constants were positively related to temperature-limited relative growth rate although not significantly. The observed inhibition of NO₃ ⁻ uptake and assimilation, as a percentage of NO₃ ⁻ uptake in the absence of NH₄ ⁺, averaged about 80% and ranged from 49 to 100%. For all species, a significant (P < 0.001) positive correlation was found between percent inhibition of NO₃ ⁻ assimilation and temperature-limited relative growth rate. Two experiments on Chesapeake Bay phytoplankton during an April 1998 diatom bloom showed that in short-term (∼1 h) temperature manipulation experiments, percent inhibition of NO₃ ⁻ uptake/assimilation was also positively related (P = 0.05) to experimental temperature. The observed relationships between temperature-limited relative growth rate and percent inhibition of NO₃ ⁻ assimilation rates for the species tested suggest that at the enzyme level, the inhibitory mechanism of NO₃ ⁻ assimilation is similar among species, but at the whole cell level may be regulated by species-specific differences in the accumulation of internal metabolites. These findings add not only to our understanding of species-specific variability and the role of growth temperature, but also provide additional data with which to evaluate current models of NH₄ ⁺ and NO₃ ⁻ interactions. Received: 31 August 1998 / Accepted: 7 December 1998     

Effects of ambient ammonia levels on blood ammonia, ammonia excretion and heart and scaphognathite rates of Nephrops norvegicus

During commercial handling of Nephropsnorvegicus (L.) there are a number of situations when the prawns may be exposed to very high ambient ammonia levels. These experiments evaluated the effects of increased levels of ambient total ammonia (TA = NH₃ + NH₄ ⁺) on␣blood ammonia, ammonia efflux rates and on the cardio-ventilatory performance of N. norvegicus. When prawns were taken from <1 to 2000 μmol TA l⁻¹ medium, blood TA concentrations increased rapidly for the first 2 h but tended to drop thereafter. Original blood TA levels were restored 6 h after the prawns were transferred back from seawater containing 2000 to <1 μmol TA l⁻¹. Sudden exposure to 500, 1000, 2000 or 4000 μmol TA l⁻¹ medium induced blood TA concentrations to increase respectively to 50, 30, 33 and 36% of external concentrations (normally, internal TA values are much higher than external levels). Immediately after transfer back to seawater with low ammonia concentration ( <1 μmol TA l⁻¹), excretion rates were higher than those of control prawns, and the absolute amounts of TA excreted were considerably higher than those calculated to have accumulated in the haemolymph. Heart rate (HR) and scaphognathite rate (SR) were not altered when prawns were subjected to sudden alterations in ambient ammonia ( <1 to 2000 to <1 μmol TA l⁻¹). When water ammonia concentrations were altered more gradually, both rates increased, but only at 4000 μmol TA l⁻¹. These results show that N. norvegicus is able to remove ammonia from the haemolymph and/or transform ammonia into some other substance when subjected to increased levels of ambient ammonia. Possible mechanisms involved (e.g. active transport across the gills; storage in some other tissue; glutamate synthe sis) are discussed. Received: 20 May 1996 / Accepted: 1 July 1996     

Effect of temperature fluctuations and food supply on the growth and metabolism of juvenile sea scallops (Placopecten magellanicus)

On the eastern shore of Nova Scotia late summer atmospheric systems cause upwelling of shelf water; the associated temperature variations of 10 °C with a 6 to 8 d period are comparable in magnitude to the seasonal variation. A laboratory study was undertaken to assess the effects of these temperature fluctuations on sea scallop (Placopecten magellanicus) growth and metabolism. In a factorial design, scallops were subjected to constant (10 °C) or a variable (6 to 15 °C) 8 d temperature cycle, and either a low (seston in filtered seawater) or high (seston supplemented with cultured phytoplankton) food diet. During the 48 d experiment scallop mortality was low and growth positive in all treatments. Shell and total tissue growth rate did not differ between temperature treatments, but growth in the high food treatments was 40 to 50% higher than in the low food treatments. However, soft tissue (excluding adductor) growth did show a temperature treatment effect; growth rates were significantly higher in the fluctuating temperature treatment, due in part to greater gonad development. Weight-standardized rates of scallop oxygen consumption (V _sO₂ , μmol O₂ g⁻¹ h⁻¹) were 20 to 25% higher in high food than in low food treatments, consistent with the expected increase in respiration due to the higher growth rates. Scallop metabolism did not acclimate to the fluctuating temperature cycle; V _sO₂ and ammonium excretion (V _sNH⁺ ₄, μmol O₂ g⁻¹ h⁻¹) remained dependent on ambient temperature throughout the experiment. V _sNH⁺ ₄ Q₁₀ (2.77) was higher than V _sO₂ Q₁₀ (2.01) which was reflected in a decrease in the O:N ratio at 15 °C, indicating a shift toward increased protein catabolism and a stressed state. At 10 °C, V _sO₂ and V _sNH⁺ ₄ in the variable temperature treatments were 15 to 18% lower than in the constant temperature treatments, a difference that was not detected in growth measurements. Results demonstrate that the metabolism of Placopecten magellanicus, unlike some bivalve species, is tightly coupled to fluctuations in ambient temperature. Although an absence of compensatory acclimation had a minimal effect on growth in this study, if high temperatures were combined with low food conditions a reduction in scallop production could result. Received: 23 June 1998 / Accepted: 8 February 1999     

Urea as a nitrogen source for the phytoplankton in the Oslofjord

Ambient concentrations of urea in the inner Oslofjord, Norway, showed a pronounced yearly cycle in 1980, with values in the range 0.1 to 10.0 μg-at N l^-1; this cycle resemble that of ammonia although urea concentrations were usually lower. The uptake of urea by phytoplankton was investigated using ¹⁵N. Urea was usually a less important N source than NH ₄ ⁺ , and accounted for 0 to 53% (mean 19%) of summed NH ₄ ⁺ +NO ₃ ^- + urea uptake rates from April to October. Absolute as well as relative (specific) uptake rates of urea were higher in the summer (June–August) than at other times. Uptake of urea was inhibited by NH ₄ ⁺ concentrations higher than 1 to 2 μg-at N l^-1. The summed NH ₄ ⁺ +NO ₃ ^- + urea uptake rate was exponentially related to temperature.     

Amino acid synthesis in the symbiotic sea anemone Aiptasia pulchella

Symbiotic Aiptasia pulchella and freshly isolated zooxanthellae were incubated in NaH¹⁴CO₃ and NH₄Cl for 1 to 240 min, and samples were analysed by reverse-phase high-performance liquid chromatography (HPLC) and an online radiochemical detector. NH₄ ⁺ was first assimilated into ¹⁴C-glutamate and ¹⁴C-glutamine in the zooxanthellae residing in A. pulchella. The specific activities (dpm nmol⁻¹) of ¹⁴C-glutamate and ¹⁴C-glutamine in vivo, were far greater in the zooxanthellae than in the host tissue, indicating that NH₄ ⁺ was principally incorporated into the glutamate and glutamine pools of the zooxanthellae. ¹⁴C-α-ketoglutarate was taken up from the medium by intact A. pulchella and assimilated into a small amount of ¹⁴C-glutamate in the host tissue, but no ¹⁴C-glutamine was detected in the host fraction. The ¹⁴C-glutamate that was synthesized was most likely produced from transamination reactions as opposed to the direct assimilation of NH₄ ⁺. The free amino acid composition of the host tissue and zooxanthellae of A. pulchella was also measured. The results presented here demonstrate that NH₄ ⁺ was initially assimilated by the zooxanthellae of A. pulchella. Received: 3 February 1997 / Accepted: 24 October 1997     

Effect of nutrient enrichment in the field on the biomass, growth and calcification of the giant clam Tridacna maxima

Nutrients were added separately and combined to an initial concentration of 10 μM (ammonium) and/or 2 μM (phosphate) in a series of experiments carried out with the giant clam Tridacna maxima at 12 microatolls in One Tree Island lagoon, Great Barrier Reef, Australia (ENCORE Project). These nutrient concentrations remained for 2 to 3 h before returning to natural levels. The additions were made every low tide (twice per day) over 13 and 12 mo periods for the first and second phase of the experiment, respectively. The nutrients did not change the wet tissue weight of the clams, host C:N ratio, protein content of the mantle, calcification rates or growth rates. However, ammonium (N) enrichment alone significantly increased the total population density of the algal symbiont (Symbiodinium sp.: C = 3.6 · 10⁸ cell clam⁻¹, N = 6.6 · 10⁸ cell clam⁻¹, P = 5.7 · 10⁸ cell clam⁻¹, N + P = 5.7 · 10⁸ cell clam⁻¹; and C = 4.1 · 10⁸ cell clam⁻¹, N = 5.1 · 10⁸ cell clam⁻¹, P = 4.7 · 10⁸ cell clam⁻¹, N + P = 4.5 · 10⁸ cell clam⁻¹, at the end of the first and second phases of the experiment, respectively), although no differences in the mitotic index of these populations were detected. The total chlorophyll a (chl a) content per clam but not chlorophyll a per cell also increased with ammonium addition (C = 7.0 mg chl a clam⁻¹, N = 13.1 mg chl a clam⁻¹, P = 12.9 mg chl a clam⁻¹, N + P = 11.8 mg chl a clam⁻¹; and C = 8.8 mg chl a clam⁻¹, N = 12.8 mg chl a clam⁻¹; P = 11.2 mg chl a clam⁻¹, N + P = 11.3 mg chl a clam⁻¹, at the end of the first and second phases of the experiment, respectively). The response of clams to nutrient enrichment was quantitatively small, but indicated that small changes in inorganic nutrient levels affect the clam–zooxanthellae association. Received: 2 June 1997 / Accepted: 9 June 1997     

Effects of dissolved ammonium addition and host feeding with Artemia salina on photoacclimation of the hermatypic coral Stylophora pistillata

 Effects of nutrient treatments on photoacclimation of the hermatypic coral Stylophora pistillata (Esper) were studied. Studies on photoacclimation of colonies from different light regimes in the field were evaluated and used to design laboratory experiments. Coral colonies were collected in the Gulf of Eilat (Israel) from January to March 1993. Exterior branches of colonies from different depths (1 to 40 m) displayed different trends in production characteristics at reduced and very low levels of illumination. From 24 ± 3% to 12 ± 2% of incident surface photosynthetic active radiation (PAR_o), zooxanthella population density and chlorophyll a+c per 10⁶ zooxanthellae increased, a trend seen in the range of light levels optimal for coral growth (90 to 30% PAR_o). The P _max of CO₂ per 10⁶ zooxanthellae decreased, while P _max of CO₂ per 10³ polyps increased, indicating an increase in zooxanthella population density at low light levels. Proliferous zooxanthella frequency (PZF, a measure of zooxanthella division) declined significantly at light levels <18 ± 3% PAR_o. At the lowest levels of illumination (<5% PAR_o), zooxanthella population density decreased, as did the PZF; chl a+c per 10⁶ zooxanthellae was unchanged. In 28-d experiments, exterior coral branches from the upper surfaces of colonies from 3 m depth (65 ± 4% PAR_o) were incubated in aquaria under bright (80 to 90% PAR_o), reduced (20 to 30% PAR_o), and extremely low (2 to 4% PAR_o) light intensities. At each light intensity, the corals were maintained in three feeding treatments: sea water (SW); ammonium enriched SW (SW + N); SW with Artemia salina nauplii (SW + A). An increase in P _max of CO₂ per 10³ polyps was found in corals acclimated to reduced light (20 to 30% PAR_o) in nutrient-enriched SW, while in SW, where the increase in zooxanthella population density was smaller, it did not occur. Nutrient enrichments (SW + N at 2 to 4% PAR_o and SW + A at 20 to 30% PAR_o) increased zooxanthella population density, but had no effect on chl a+c per 10⁶ zooxanthellae. Acclimation for 14 d to reduced (10 to 20% PAR_o) and extremely low (1 to 3% PAR_o) light intensities shifted ¹⁴C photoassimilation into glycerol and other compounds (probably glycerides), rather than sugars. Both ammonium addition and feeding with Artemia salina nauplii resulted in an increase in photosynthetic assimilation of ¹⁴C into amino acids. We conclude that acclimation to reduced light consists of two processes: an increase in photosynthetic pigments and in zooxanthella population density. Both processes require nitrogen, the increase in zooxanthella population density needing more; this adaptation is therefore limited in nitrogen-poor sea water. Received: 19 June 1998 / Accepted: 13 June 2000     

Intraspecific variation in the red alga Portieria hornemannii : monoterpene concentrations are not influenced by nitrogen or phosphorus enrichment

The red alga Portieria hornemannii (Lyngbye) Silva was selected to test the effects of enhanced nutrient availability on the production of carbon-based secondary metabolites, because of its notable site-to-site variation in monoterpene production. On Guam, the major secondary metabolite produced by this alga is ochtodene, a cyclic monoterpene. Quantitative high-performance liquid-chromatography analysis of the extracts of P. hornemannii collected from six sites on Guam showed that both ochtodene and triglyceride concentrations differed significantly among sites. Internal nitrogen and phosphorus content of the algae did not correlate with the observed variation in chemistry. Experimental enhancement of N-alone, P-alone or N + P in the field for 5 wk failed to induce a significant change in ochtodene concentrations in the alga, while triglyceride concentrations increased significantly in the N + P treatment. Ochtodene and triglyceride concentrations did not change among similar treatments in shaded (18 d) and unshaded (11 d) fertilization experiments conducted in the laboratory. Variation in ochtodene concentrations in P. hornemannii cannot be attributed to N and P availability; however, the decrease in ochtodene and triglyceride concentrations during the shaded laboratory experiment suggests that light may be a factor influencing monoterpene biosynthesis. The difference in ochtodene concentration between the initial and final sets of field controls collected for the unshaded laboratory experiment suggests that temporal variation might also contribute to differences observed among the algae at the different sites. Received 11 June 1996 / Accepted 20 July 1996     

Nitrate reductase and urease enzyme activity in the marine diatom<Emphasis Type="Italic"> Thalassiosira weissflogii</Emphasis> (Bacillariophyceae): interactions among nitrogen substrates

The dissolved nitrogen pool in aquatic systems is comprised of many different nitrogen forms, both inorganic and organic. Interaction among these nitrogen forms at the level of uptake and enzyme activity is, with the exception of NH₄⁺ and NO₃^–, not completely understood. Nitrate reductase (NR) and urease (UA) activities in the marine diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle were measured in NO₃^–, NH₄⁺, and urea-sufficient cultures before and after challenge additions of NH₄⁺, NO₃^–, and urea in a factorial design. NR and UA were constitutively expressed during growth on NO₃^–, NH₄⁺, and urea. Growth on NH₄⁺ or urea resulted in NR activities that were <10% of the activity observed in the NO₃^–-grown culture, while growth on NO₃^– resulted in UA values that were ~35% of the activities during growth on either NH₄⁺ or urea. The addition of NH₄⁺ or urea to NO₃^–-grown cultures resulted in an immediate decrease in cellular NO₃^– uptake rate, which was not mirrored by an immediate repression of in vitro NR activity; however, the diel peak in NR was suppressed in these challenge experiments. The addition of NO₃^– or NH₄⁺ to urea-grown cultures resulted in non-significant decreases in the urea uptake rate. UA was not impacted by NO₃^– addition, but NH₄⁺ addition significantly decreased UA throughout the experiment. These studies demonstrate that the uptake and assimilation of NO₃^– and urea may not be subject to the same internal feedback mechanism when challenged with other nitrogen substrates.Communicated by J.P. Grassle, New Brunswick     

Comparison between chemical and isotopic measurements of biological nitrate utilization: further evidence of low new-production levels in the equatorial Pacific

New-production (nitrate uptake) rates in the equatorial Pacific were estimated by parallel measurements of nitrate disappearance from sea water using a colorimetric method and of ¹⁵N-labelled nitrate (¹⁵NO₃ ⁻) incorporation into particulate organic nitrogen (PON) collected on GF/F filters (net nitrate uptake, conventional ¹⁵N-tracer method) and Anopore (0.2 μm) membranes. Regression analyses of 74 sample pairs gathered during 12 and 24 h productivity experiments revealed a significant positive relationship between decreasing nitrate level and ¹⁵NO₃ ⁻ accumulation into PON retained on GF/F filters, but the slopes of Model I and Model II regression lines were 1.18 and 1.29, respectively, suggesting that 15 to 22% of ¹⁵NO₃ ⁻ removed from the dissolved fraction were lost to another N-pool. Two possible avenues for the missing ¹⁵NO₃ ⁻ have been examined: uptake by submicron particles passed through the GF/F filters, and loss as dissolved organic nitrogen (DON). Nitrate uptake by small cells not recovered on GF/F filters, could be safely eliminated as a cause of loss, since ¹⁵NO₃ ⁻ uptake rates obtained from ¹⁵N entering PON collected on GF/F filters agreed well with those obtained from ¹⁵N entering PON collected on Anopore membranes (32 sample pairs). Inspection of the DON pool of 0.2 μm filtrates for excess-¹⁵N enrichment (20 samples) revealed that in nitrate-rich waters (equatorial upwelling between 1°N and 10°S), loss of ¹⁵NO₃ ⁻ as DO¹⁵N accounted for <5% of net nitrate uptake. In samples from subtropical oligotrophic waters (from 11°S southward), however, ¹⁵NO₃ loss as DO¹⁵N represented up to 20% of net NO₃ ⁻ uptake. These results, as well as experimental considerations concerning the use of colorimetric and isotopic methods to measure new production show that: (1) earlier reported high discrepancies between nitrate decreases (ΔNO₃ ⁻) and ¹⁵NO₃ ⁻ incorporation into filterable particles (ΔNO₃ ⁻/¹⁵NO₃ ⁻ incorporation >2) were probably erroneous; (2) the use of GF/F filters does not result in an underestimation of new production, although it was found to underestimate PON concentrations by up to 60%; (3) in the equatorial upwelling area (1°N to 10°S), which has high ambient nitrate levels (>2000 nmol l⁻¹) but only slight changes in concentration (0 to 80 nmol l⁻¹ d⁻¹), new production is more accurately estimated by the isotopic method than by the chemical method; (4) in subtropical oligotrophic waters (from 11°S southward) with low ambient nitrate levels (0 to 100 nmol l⁻¹), both procedures are appropriate as long as nitrate removal per incubation period is >3 nmol l⁻¹ (lower rates are only detectable with the isotopic method); (5) the traditional ¹⁵N-tracer technique does not substantially underestimate net new-production in the equatorial Pacific, and failure to account for the loss of ¹⁵NO₃ ⁻ as DON, i.e. to estimate gross nitrate uptake (gross uptake = net uptake + ¹⁵N loss) tends to underestimate new production on an average by only 10%. Overall, the apparent low level of new production in the nitrate-rich area of the central equatorial Pacific seems to be a fact, and may be ascribable to other nutrient (macro and micro) deficiencies and/or to intense in situ recycling of ammonium and nitrate (regenerated production) rather than to inaccurate nitrate uptake rates measured with the classical ¹⁵N-tracer technique. Received: 24 November 1998 / Accepted 10 March 2000     

Feeding, metabolism and growth in the Antarctic limpet, Nacella concinna (Strebel 1908)

Post-prandial increases in metabolism, the specific dynamic action of feeding (SDA), were evaluated in the Antarctic limpet Nacella concinna. O₂ consumption rose to a peak value 2.3 times higher than pre-feeding standard metabolic rates. This peak rise is low for marine ectotherms, but is typical of polar species. There were three peaks in the SDA, the first lasted only for the 1st day, was caused by handling, and was minor. The second was the major peak. It lasted from post-prandial days 4–9 inclusive, and accounted for around 70% of the SDA response. The third peak lasted from day 11 to day 15 and accounted for 30% of the total SDA. A 15-day SDA is much longer than values for temperate species, but is again typical for polar marine ectotherms. NH₃ excretion declined post-prandially from around 0.4 μmol animal⁻¹ h⁻¹ to values between 0.025 and 0.223 μmol animal⁻¹ h⁻¹ throughout the SDA. The total O₂ consumed in the SDA was 90.2 μmol O₂, which converts to 44.7 J of energy. This was 45–50% of the energy consumed in the meal (93.5 J). Pre-feeding O:N ratios, after 26 days without food, were around 1, indicating protein as the sole metabolic substrate prior to initiating the SDA. After feeding, O:N ratios rose to between 2.5 and 19, indicating significant use of lipid or carbohydrate from the food. Experiments were conducted in ambient seawater with enhanced levels of Sr (SrCl added at 800 mg kg⁻¹), and limpets were fed microalgal films also grown in enhanced Sr media. Sr incorporated in the shells during the experiment allowed the measurement of shell increments deposited during the SDA. Between five and eight microgrowth bands were present in the Sr-enhanced increments, which was similar to the number of days in the second SDA peak. The mean shell increment laid down was 17.6 μm. Estimating tissue deposition from measured growth increments and published ash-free dry mass (AFDM) to length relationships produced a value of 0.81 mg AFDM, which converted to 26.4 J of energy, or 25–30% of the energy ingested in the meal. Estimates of growth increments associated with a single SDA have not previously been possible. Overall energy used in the SDA and tissue deposition accounted for 75–80% of the energy ingested; the remainder was probably accounted for by unmeasured costs such as mucus production. Received: 6 June 2000 / Accepted: 20 September 2000     

Growth of zooxanthellae in culture with two nitrogen sources

Physiological characteristics of zooxanthellae were examined under nutrient-saturated conditions created by mixing ammonium (¹⁵NH₄) with nitrate (¹⁵NO₃) to give 0.88 mM total nitrogen. Growth rate varied with the form of nitrogen provided. Ammonium alone resulted in the lowest C:N and C:chl-a ratios. Although zooxanthellae took up nitrate in the absence of ammonium, ammonium assimilation was 1.3 times higher than nitrate assimilation. Ammonium strongly inhibited nitrate assimilation. While high-ammonium treatments resulted in the highest ¹⁴C incorporation into intermediate compounds, high nitrate levels resulted in the highest ¹⁴C incorporation into protein, suggesting that the intermediate compounds are produced prior to the subsequent production of protein when ammonium is the dominant N source. The enhanced production of intermediate compounds at the expense of carbon directed to protein synthesis in the presence of ammonium might be analogous to the “host factor” observed in zooxanthellae–host symbioses, since growth rate is depressed due to low production of protein. Received: 16 March 2000 / Accepted: 26 August 2000     

Ammonium regeneration and carbon utilization by marine bacteria grown on mixed substrates

We examined the impact of exposing natural populations of marine bacteria (from seawater collected near Woods Hole, Massachusetts, USA) to multiple nitrogen and carbon sources in a series of batch growth experiments conducted from 1989 through 1990. The substrate C:N ratio (C:N_s) was varied from 1.5:1 to 10:1 either with equal amounts of NH ₄ ⁺ and different amino acids or an amino acid mixture, all supplemented with glucose to maintain the C:N_s ratio equal to that of the respective amino acid, or with combinations of glucose and NH ₄ ⁺ alone. A common feature of the experiments involving amino acids was the concurrent uptake of NH ₄ ⁺ and amino acids that persisted as long as a readily assimilable carbon source (glucose in our case) was taken up. There was no net regeneration of NH ₄ ⁺ , even though catabolism of amino acids occurred. Regeneration of NH ₄ ⁺ was evident only after glucose was completely utilized, which usually occurred at the end of exponential growth. The contribution of¹⁵NH ₄ ⁺ to total nitrogen uptake by the end of exponential growth varied from ~60 to 80% when individual amino acids were present and down to ~24% when the amino acid mixture was added. These estimates are conservative because we did not account for possible isotope dilution effects resulting from amino acid catabolism. When NH ₄ ⁺ and glucose were the sole nitrogen and carbon sources, there was a stoichiometric balance between glucose and NH ₄ ⁺ uptake over a wide range of C:N_s ratios, leading to a constant bacterial biomass C:N ratio (C:N_B) of ~4.5:1. As a result NH ₄ ⁺ usage varied from 50% when the C:N_s ratio was 3.6:1, to 100% when the C:N_s ratio was 10:1. Gross growth efficiency varied from ~60% when NH ₄ ⁺ plus glucose were added alone or with the amino acid mixture, to 47% when the individual amino acids were used in place of the mixture. It is thus evident that actively growing bacteria will act as sinks for nitrogen when a carbon source that can be assimilated easily is available to balance NH ₄ ⁺ uptake, even when amino acids are available and are being co-metabolized.     

Extra- and intracellular acid-base balance and ionic regulation in cod (Gadus morhua ) during combined and isolated exposures to hypercapnia and copper

Cod (Gadus morhua) were exposed to hypercapnia (water Pco₂ = 7.5 mmHg), elevated copper level (0.4 ppm) or a combination of both in order to study extra- and intracellular acid-base regulation and the influence hereupon of copper. During pure hypercapnia, the extracellular respiratory acidosis was completely compensated within 12 to 24 h via a chloride-mediated increase in extracellular [HCO₃ ⁻]. Exposure to copper in normocapnic seawater caused a large and progressive increase in plasma [Na⁺] and [Cl⁻] and a metabolic acidosis. Exposure to copper in hypercapnic seawater was associated with smaller elevations of plasma [Na⁺] and [Cl⁻] than in normocapnic seawater, showing that hypercapnia had a protective effect on the copper-induced osmoregulatory disturbances. The compensation of the hypercapnic acidosis was, however, slow and incomplete in fish exposed to both copper and hypercapnia. Extracellular pH remained depressed by 0.3 pH units after 72 h. The data reveal that acid-base regulation was immediately and persistently inhibited by copper. The limited acid-base regulation during combined copper and hypercapnia exposure was chloride-mediated as during hypercapnia alone. Intracellular pH recovery was complete and very rapid in ventricular and skeletal muscle tissues during environmental hypercapnia, whereas acid-base compensation in liver tissue was slower, the kinetics being similar to that in the extracellular compartment. Intracellular pH compensation was significantly slowed down by copper. Copper concentration increased drastically in gill tissue already at 3 h, while copper concentrations in liver, muscle and plasma were significantly elevated only after 48 h, with liver showing the largest elevation. Received: 15 November 1996 / Accepted: 2 December 1996     

Effect of an organophosphorus insecticide, fenitrothion, on survival and osmoregulation of various developmental stages of the shrimp Penaeus japonicus (Crustacea: Decapoda)

The toxicity of fenitrothion was determined in larvae (nauplii, Zoeae 1 to 3, Mysis 1 to 3), postlarvae (PL stages) and juvenile shrimp (Penaeus japonicus Bate), in two media, seawater (SW) and diluted seawater (DSW) (1100 and 550 mosM kg⁻¹, ≃ 37 and 19‰ S). The effects of fenitrothion on the osmoregulatory capacities (OC) of juveniles were recorded. A gill and epipodite histopathological study was also conducted. For larvae in seawater, 24 and 48 h LC₅₀s ranged from 32.9 μg l⁻¹ (Zoeae 2) to 10.7 μg l⁻¹ (Mysis 3), and from 3.9 μg l⁻¹ (Zoeae 3) to 2.0 μg l⁻¹ (Mysis 3), respectively; 48 and 96 h  LC₅₀s in postlarvae (PL) at the same salinity ranged from 1.8 μg l⁻¹ (PL1) to 0.6 μg l⁻¹ (PL5), and from 0.3 μg l⁻¹ (PL7) to 0.4 μg l⁻¹ (PL15). In juveniles, 96 h LC₅₀s were 0.8 μg l⁻¹ in seawater and 1.5 μg l⁻¹ in diluted seawater. From hatching to juvenile stages, the overall trend was a rapid decrease (from nauplii to PL5–PL7) followed by a slight increase (from PL7 to PL15 and juveniles) in the shrimp's ability to tolerate the insecticide. In juveniles kept in seawater and in diluted seawater, fenitrothion decreased the osmoregulatory capacity (OC = difference between the hemolymph osmotic pressure and the osmotic pressure of the medium) at both lethal and sublethal concentrations. This effect was time- and dose-dependent. In SW, the decrease in hypo-OC was ˜ 25% at sublethal concentrations and ˜ 35% at the 96 h LC₅₀. In DSW, the decrease in hyper-OC was ˜ 10 to 15% at sublethal concentrations. In SW, shrimp were able to recover their OC in less than 48 h when transferred to water free of pesticide. In DSW, recovery at 48 h was only possible after exposure to the lowest tested sublethal concentration. Haemocytic congestions (thrombosis) of the gills, lamellae necrosis and other alterations of gills and epipodites (breakage of the cuticle, reduction of the hemolymph lacunae) were noted in juveniles exposed to lethal and sublethal concentrations of fenitrothion. Received: 7 October 1996 / Accepted: 13 November 1996     

Allozyme electrophoresis indicates high gene flow between populations of Holothuria (Microthele) nobilis (Holothuroidea: Aspidochirotida) on the Great Barrier Reef

 Genetic variation in 15 Holothuria (Microthele) nobilis (Selenka, 1867) populations on the Great Barrier Reef was studied at seven polymorphic allozyme loci. Although populations were separated by distances up to 1300 km, there were no apparent restrictions to gene flow (F _ST values were not significantly different from 0) and the maximum Nei's unbiased genetic distance was 0.003. Populations were in Hardy–Weinberg equilibrium at all loci. The estimated maximum sexual input [this is the ratio of the number of sexually produced individuals (N*) to the sample size (N _i )] and the minimum sexual input [this is the ratio of the number of genotypes (N _go ) over the sample size (N _i )] were used as estimators for the amount of asexual reproduction. Both parameters suggested that H. nobilis reproduces solely by sexual means (N*:N _i : = 1; N _go :N _i  = 0.74 to 1). The allozyme data indicated high gene flow between populations, but the possibility that allozyme frequencies may not be at equilibrium means that it was not possible to distinguish whether the patterns reflect present-day dispersal or dispersal that occurred in the past. Received: 27 March 2000 / Accepted: 29 June 2000     

Carbon and nitrogen isotopic composition of the fauna from the Broken Spur hydrothermal vent field

 The carbon and nitrogen isotopic composition of 18 faunistic groups collected during the 39th cruise of the R.V. “Akademik Mstislav Keldysh” in September 1996 at the Broken Spur vent field, MAR, was studied. The trophic structure of the Broken Spur vent community is considered. All age stages of the shrimp Rimicaris exoculata living 5 m below the main aggregations at black smokers show higher δ¹⁵N and more depleted δ¹³C values than the same stages inhabiting the black smokers themselves. The shrimps R. exoculata and Chorocaris chacei demonstrate ontogenetic changes in δ¹³C (the former also in δ¹⁵N), with smaller individuals showing higher δ¹⁵N and more depleted δ¹³C values than larger shrimps. Benthopelagic and benthic components of the vent community differ significantly in δ¹³C and δ¹⁵N, the benthic fauna being less dependent upon chemosynthetic production. Received: 30 March 1999 / Accepted: 28 September 1999     

Availability of two forms of dissolved nitrogen to the coral Pocillopora damicornis and its symbiotic zooxanthellae

The relative contribution of dissolved nitrogen (ammonium and dissolved free amino acids DFAAs) to the nitrogen budget of the reef-building coral Pocillopora damicornis was assessed for colonies growing on control and ammonium-enriched reefs at One Tree Island (southern Great Barrier Reef) during the ENCORE (Enrichment of Nutrient on Coral Reef; 1993 to 1996) project. P. damicornis acquired ammonium at rates of between 5.1 and 91.8 nmol N cm⁻² h⁻¹ which were not affected by nutrient treatment except in the case of one morph. In this case, uptake rates decreased from 80.5 to 42.8 nmol cm⁻² h⁻¹ (P < 0.05) on exposure to elevated ammonium over 12 mo. The presence or absence of light during measurement did not influence the uptake of ammonium ions. Nitrogen budgets revealed that the uptake of ammonium from concentrations of 0.11 to 0.13 μM could completely satisfy the demand of growing P. damicornis for new nitrogen. P. damicornis also took up DFAAs at rates ranging from 4.9 to 9.8 nmol N cm⁻² h⁻¹. These rates were higher in the dark than in the light (9.0 vs 5.1 nmol m⁻² h⁻¹, P < 0.001). Uptake rates were highest for the amino acids serine, arginine and alanine, and lowest for tyrosine. DFAA concentrations within the ENCORE microatolls that received ammonium were undetectable, whereas they ranged up to 100 nM within the control microatolls. The contribution of DFAAs to the nitrogen budget of P. damicornis constituted only a small fraction of the nitrogen potentially contributed by ammonium under field conditions. Even at the highest field concentrations measured during this study, DFAAs could contribute only ≃11.3% of the nitrogen demand of P.␣damicornis. This contribution, however, may be an important source of nitrogen when other sources such as ammonium are scarce or during periods when high concentrations of DFAAs become sporadically available (e.g. cell breakage during fish-grazing). Received: 22 April 1998 / Accepted: 3 November 1998     

Die stickstoffhaltigen stoffwechselendprodukte und ihre exkretion bei Paracentrotus lividus

The perivisceral fluid of Paracentrotus lividus (Lamarck) contains, as main end-products of the nitrogenous metabolism, ammonia, urea, creatine, creatinine and traces of uric acid. In the organs analysed, the distribution and abundance of ammonia and urea fluctuate considerably. the intestine was found to have the highest NH₄ ⁺?N and the lowest urea-N contents. The axial organ contained the highest amount of urea-N and the lowest quantity of NH₄ ⁺?N; the perivisceral fluid, including the coelomocytes, contained intermediate amounts. The special relations to hemal system and coelothel, the presence of excretory material, and the analogous conditions to other invertebrates, suggest that the coelomocytes and parts of the intestine and axial organ are excretophoric and able to synthesize urea. From the known distribution of ammonia and urea in echinoid species, it is concluded that the ability to synthesize urea must have developed and improved in efficiency during echinoid evolution. P. lividus is predominantly ureotelic. It excretes approximately 91% of the nitrogenous wastes released into the surrounding sea water as urea-N, and only 9% as ammonia-N. Ammonia, and most of the urea, are excreted via the body surface — probably through respiratory surfaces; however, one third of the urea is excreted through the intestine, since urea excretion decreases by this amount when the anus is sealed artificially.