Uptake of inorganic carbon and internal carbon cycling in symbiont-bearing benthonic foraminifera

Incorporation rates of inorganic carbon and its distribution between the organic matter and the skeleton have been measured using ¹⁴C tracer techniques on two species of symbiont-bearing benthonic foraminifera in the Gulf of Elat: Amphistegina lobifera (a perforate species) and Amphisorus hemprichii (an imperforate species). Under constant experimental conditions, incorporation rates of the radiotracer become linear with time after several hours in A. hemprichii and after one day in A. lobifera. A. lobifera showed a lag time of 24 h for skeletal incorporation, whereas in A. hemprichii uptake into the skeleton started within 2 h. Pulse-chase incubations in radioactive seawater, followed by unlabelled incubations, demonstrate transfer of photosynthetically acquired ¹⁴C into the skeleton of A. lobifera. No such transfer was found in A. hemprichii. The total ¹⁴C uptake by A. lobifera increased during the first 24 h of cold chase incubation. This increase suggests the existence of an internal inorganic carbon pool that was lost (probably evaporated) during the analysis of pulse incubations. However, during the following chase incubations, the ¹⁴C in this pool was incorporated mainly into the skeleton and retained during analysis, causing the increase in the total uptake. No such increase was found in A. hemprichii. Additional ¹⁴C uptake experiments on other species of the genera Operculina, Heterostegina and Borelis suggest that the differences in pathways for incorporation of carbon between A. lobifera and A. hemprichii can be generalized to the perforate and imperforate foraminiferal groups. In perforate species, respired carbon originally taken up through photosynthesis is partly recycled into the skeleton. In imperforate species such a transfer has not been demonstrated. Perforate species seem to have a large internal inorganic carbon pool which serves mainly for calcification and possibly also for photosynthesis, while imperforate species may take up carbon for calcification directly from seawater or have a very small inorganic carbon pool.     

Calcification in the staghorn coral Acropora acuminata: Variations in apparent skeletal incorporation of radioisotopes due to different methods of processing

Pieces of branch from the staghorn coral Acropora acuminata were incubated with ⁴⁵CaCl₂ and NaH¹⁴CO₃ under identical conditions in the light or in the dark. Specimens were then processed in different ways. All specimens were placed in N KOH to digest tissues. Some were placed in KOH immediately after incubation; others were placed in KOH after 2 h washing, or after 2 h extraction with methanol-chloroformwater. Specimens were washed in running fresh water or running seawater; some were killed in liquid N₂ before washing. Radioactivity associated with skeleton and tissues was determined. The method of processing profoundly affected the results. In dark incubations, there was up to a four-fold difference in apparent skeletal incorporation of ⁴⁵Ca⁺⁺ between average values obtained for the different treatments. For ¹⁴C incorporation, there was a difference of up to 2.5 times. In light incubations, skeletal incorporation of both radioisotopes showed a two-fold difference between high and low average values obtained for the different treatments.     

Zooxanthellae providing assimilatory power for the incorporation of exogenous acetate in Heteroxenia fuscescens (Cnidaria: Alcyonaria)

The soft coral Heteroxenia fuscescens (Ehrb.) and its isolated zooxanthellae (endosymbiotic dinoflagellates) were investigated with particular regard to uptake and utilization of exogenously supplied ¹⁴C-acetate in the light and in the dark. The incorporation of ¹⁴C from ¹⁴C-acetate into the host tissue and into the zooxanthellae was consistently much higher in the light than in the dark. The incorporated ¹⁴C-acetate was rapidly metabolized by the host and algae and was recovered from different assimilate fractions. The major proportion of radiocarbon from metabolized ¹⁴C-acetate was located in host tissue. The CHCl₃-soluble fraction composed of diverse lipids showed the strongest ¹⁴C-labelling. Zooxanthellae isolated prior to incubation accounted for about 80% of the acetate incorporation recorded for zooxanthellae in situ (in vivo). It is concluded from a comparison of acetate incorporation and conversion under light and dark conditions that most of the lipid reserve of the host tissue originates from fatty acids, which are synthesized within the algal symbionts and are then translocated to the heterotrophic partner via extrusion. The acetate units needed for lipid synthesis are obtained by absorption of free acetate from dissolved organic matter (DOM) in the seawater as well as by photosynthetic assimilation of inorganic carbon. Thus, in H. fuscescens, lipogenesis is operated as a light-driven process to which the zooxanthellae considerably contribute assimilatory power by performing fatty acid synthesis and translocation of lipid compounds to their intracellular environment (host cell). A metabolic scheme is proposed to account for the different pathways of carbon conversion observed in H. fuscescens. The incubations took place in August 1980 and the analytical part from October 1980 to January 1984.     

A reevaluation of the role of glycerol in carbon translocation in zooxanthellae-coelenterate symbiosis

Glycerol has been traditionally viewed as the main form of carbon translocated from zooxanthellae to the coelenterate host. Most of this glycerol was postulated to be used by the coelenterate host for lipid synthesis. Recent work suggests that large amounts of photosynthetically fixed carbon is synthesized into lipid in the algae, and then translocated as lipid droplets to the host. These two hypotheses of carbon translocation are not mutually exclusive, but to reconcile them the role of glycerol must be reevaluated. In this study the short term metabolic fate of uniformly labelled ¹⁴C-glycerol, ¹⁴C-bicarbonate, and ¹⁴C-acetate was examined in zooxanthellae and coelenterate host tissue isolated from Condylactis gigantea tentacles. When host and algal triglycerides, synthesized during 90-min light and dark incubations in ¹⁴C-bicarbonate and ¹⁴C-acetate, were deacylated, more than 80% of the activity was found in the fatty acid moiety. In contrast, triglycerides isolated from zooxanthellae and coelenterate host tissue incubated in ¹⁴C-glycerol in the dark for 90 min were found to have more than 95% of their radioactivity in the glycerol moiety. During the 90-min ¹⁴C-glycerol incubations in the light, the percentage of radioactivity in the fatty acid moiety of zooxanthellae triglycerides increased to 37%. The percentage of radioactivity in the host tissue triglycerides fatty acid moiety stayed below 5% during the 90-min ¹⁴C-glycerol incubations in the light. These results show that neither the zooxanthellae nor the host can rapidly convert glycerol to fatty acid. Radioactivity from ¹⁴C-glycerol, which does eventually appear in host lipid, may have been respired to ¹⁴CO₂, then photosynthetically fixed by the zooxanthellae and synthesized into lipid fatty acid.     

The pattern of carbon fixation in the marine unicellular alga Phaeodactylum tricornutum

The short- and long-term fixation of ¹⁴CO₂ by Phaeodactylum tricornutum was studied using methods of fractionation that allowed examination of all the products labelled with ¹⁴C. There was no doubt that the major pathway of CO₂ fixation was into 3-phosphoglycerate, but there was also significant incorporation by -carboxylation by means of phosphoenol-pyruvate carboxylase and transamination into aspartate. At short time intervals (10 sec), 90% of the radioactive products found were accounted for by 3-phosphoglycerate and aspartate. The lipids associated with the photosynthetic apparatus contained a high proportion of the ¹⁴C fixed, which at 60 sec was located mainly in the carbohydrate portion of the lipids. At 30 and 300 min, the chlorophylls, carotenoids and the long-chain fatty acids were heavily labelled with ¹⁴C. The monogalacto-diglycerides, the digalacto-diglycerides and the sulpholipids each had a characteristic long-chain fatty acid composition. The cell proteins and a reserve polysaccharide were also labelled with ¹⁴C at short time intervals and increased their radioactivity in a linear fashion up to the longest period studied, 300 min. The activity of the enzymes ribulose diphosphate carboxylase and phosphoenolpyruvate carboxylase was sufficient to account for the pattern of fixation found.     

Extent and nature of extracellular organic production by the marine coccolithophorid Hymenomonas carterae

Production of extracellular dissolved organic substances by cultures of the marine coccolithophorid Hymenomonas carterae SMBA 254 was investigated by the ¹⁴C-tracer technique. In 4 h incubations, extracellular ¹⁴C production represented 20 to 64% of ¹⁴C incorporation into cell material for cultures nearing, or during stationary growth; for rapidly growing cultures with relatively low cell densities the ¹⁴C-production was only 4 to 10% of incorporation. Intra-and extra-cellular material was fractionated by ion-exchange membrane electrodialysis. Ionic compounds accounted for 40 to 60% of extracellular material; in contrast only 16 to 27% of intracelular metabolites were in this category. Of a range of methods which were investigated, separation of compounds by gel-filtration in conjunction with two-dimensional thin-layer chromatography proved most effective. Up to 80% of extracellular material was of less than 1 800 mol. wt and included carbohydrates and amino acids, with glutamic acid predominating. The intracellular material was more heterogeneous, with a substantially greater proportion of high molecular weight material. The quite distinct compositions show clearly that extracellular production observed in these experiments arose through excretion and was not attributable to cell lysis.     

Incorporation of 32PO4 directly taken-up into acid-soluble phosphates of Artemia salina

A study of the direct uptake by Artemia salina of phosphate ion from the medium and its incorporation into acid-soluble organic phosphorous compounds over a range of exposure time from 2 to 30 min, using ³²PO₄ ion, indicated that the phosphate ion was directly taken up and was rapidly incorporated into the energy-rich compounds, such as adenosine triphosphate (ATP), guanosine triphosphate (GTP), and adenosine diphosphate (ADP), which were separated by ion-exchange chromatography using Dowex-1, X2. Even after an exposure of 2 min, the sum of the radioactivity of nucleotide-fractions was 37.4% of that of the whole acid-soluble extract. The most rapid incorporation of ³²P occurred into ATP, followed by GTP and ADP. The amount of ³²P incorporated into each fraction increased with increased exposure, giving straight lines when the radioactivity of each fraction was plotted against the exposure time on a logarithmic scale. Almost no difference, however, was observed in the distribution rate of ³²P into each fraction at 2, 5, 10 and 30 min. These results show that inorganic phosphate absorbed by A. salina is rapidly incorporated into the energy-rich nucleotides, and that a dynamic equilibrium is established among various acid-soluble phosphorous compounds even after very short periods of time.     

Temperature and salinity adaptation in the purple shore crab Hemigrapsus nudus: An in vitro metabolic flux study with excised gills

The effects of temperature and salinity acclimation on intermediary metabolism in excised gill homogenates from Hemigrapsus nudus were examined. In general, a decrease in salinity was followed by an increase in the oxidation of the substrates glucose-1-¹⁴C, acetate-1-¹⁴C and glycine-¹⁴C to ¹⁴CO₂. Also, there was an increase in amino acid incorporation into the protein fraction. Both of these metabolic parameters were differentially temperature-sensitive. An enzymic model which may explain the increase in respiration rate observed in several intertidal invertebrates meeting an hypo-osmotic stress is proposed, using data from these experiments and also from the literature.     

Compartmentation and turnover of organic carbon in the Staghorn coral Acropora formosa

Four colonies of Acropora formosa were incubate with Na₂ ¹⁴CO₃ for separate 2 h periods within a 24 h period, and then returned to the reef from which they were collected. Terminal branches were collected at intervals over the following 5 d and analysed for radioactivity associated with the skeleton and certain organic pools. Colonies incubated at night showed little or no loss of fixed radioactivity during the 5 d on the reef. However, 50–60% of photosynthetically-fixed ¹⁴C was lost from the terminal branches during the first 40 h on the reef. This loss of radioactivity probably resulted from release of mucus and dissolved organic carbon from the coral tissues. Most of the loss of photosynthetically-fixed ¹⁴C was due to decrease in the radioactivity of lipids (80% of the total ¹⁴C loss) and methanol-water soluble compounds. Determination of any sequencing in metabolic compartments was made difficult by the rapidity with which ¹⁴C dissappeared from most of the metabolic pools measured. ¹⁴C was incorporated into the skeleton throughout the 5 d on the reef, although the rate of incorporation was very low in colonies which had been incubated with Na₂ ¹⁴CO₃ at night.     

A method to determine in situ zooplankton grazing rates on natural particle assemblages

In situ zooplankton grazing rates on natural particle assemblages were stimated by measuring zooplankton uptake of labelled autotrophic (with Na¹⁴CO₃) and heterotrophic (with [methyl-³H]-thymidine) particulate matter in 1-h incubations in clear, Plexiglas, Haney chambers. The in situ grazing rates are in the same range as those measured for zooplankton in the laboratory using standard particle counting techniques. A negative selection coefficient for ³H-labelled particles indicated a lower filtration efficiency or avoidance of these particles by zooplankton.     

Respiratory quotient and calcification of Nautilus macromphalus (Cephalopoda: Nautiloidea)

Respiration and calcification were investigated in the ectocochleate cephalopod Nautilus macromphalus Sowerby. Specimens were collected off New Caledonia, in October 1991, and kept at the Nouméa Aquarium until December 1991. The respiratory quotient and calcification rate of 5 individuals were measured during 14 short term incubations (63 to 363 min). Oxygen uptake was recorded with a polarographic oxygen sensor. CO₂ flux and calcification were calculated from changes in pH and alkalinity (alkalinity-anomaly technique). Several methods were used to compute the respiratory quotient (RQ); a functional regression indicated an RQ of 0.74. CaCO₃ exchange rates were linearly related to respiratory quotient, calcification occurring in individuals with a low RQ. CaCO₃ uptake from the surrounding water was noncontinuous. From the highest CaCO₃ uptake, maximum growth rate was estimated as 7.1 mg shell wt h^- (=61 g yr^-1).     

Studies on the mechanism of biosynthesis of wax esters in Euchaeta norvegica

Preparations of Euchaeta norvegica catalyse the incorporation of (1-¹⁴C) hexadecanol, (9,10-³H₂) oleic acid, (U-¹⁴C) glucose, (U-¹⁴C) alanine and (U-¹⁴C) aspartic acid into wax esters. Both the fatty alcohol and fatty acid moieties of the wax esters are labelled, indicating de novo biosynthesis. A limited interconversion of fatty acid and fatty alcohol has been demonstrated. Incorporation of glucose into wax esters is diminished by prior starving of E. norvegica; it is also reduced in the presence of ecdysterone. Formation of wax esters from glucose is inhibited by increased oxygen tensions, rotenone and dinitrophenol, and is relatively insensitive to decreased oxygen tensions and cyanide. The data are discussed with particular reference to the reasons why wax esters are biosynthesised by calanoids.     

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     

Incorporation of pesticides by soil micro-organisms as a way of bound residues formation

We studied the ¹⁴C-tagged biochemical compartments such as proteins, lipids and polysaccharides from bacterial and fungal cultures supplied with ¹⁴C-pesticides and ¹⁴C-glucose. We showed that ¹⁴C incorporation depends both on the substrate structure and on the nature of the micro-organism. Both bacterial and fungal cells incorporated glucose ¹⁴C mainly into cell wall proteins. By contrast, glyphosate ¹⁴C was mainly incorporated into cytoplasm carbohydrates by fungi. On the other hand, 2,4-dichlorophenoxyacetic acid ¹⁴C was mainly found in the carbohydrate fractions of the bacteria walls.     

Direct measurement of dissolved organic carbon release by phytoplankton and incorporation by microheterotrophs

It is now possible to divide particulate primary production into algal and heterotrophic components without physical separation. This depends on two innovations, the introduction of isotope in the form of labelled dissolved product(s) of primary production and the employment of a data analysis specifically designed for tracer kinetic incorporation experiments. The ¹⁴C technique described by Steemann Nielsen (1952) is inapplicable in the analyses of certain classes of systems and kinetic tracer incorporation experiments must be employed instead. We show that measurement of PDOC production rate requires such kinetic tracer analyses. Measurements made in the laboratory on water taken from 2 m depth in South West Arm of the Port Hacking estuary showed that: (1) the steady-state rate of PDOC production was 0.10 to 0.13 mg C.m^-3.h^-1; (2) the rate of PDOC incorporation into microheterotroph particulate organic carbon was 0.10 to 0.12 mg C.m^-3.h^-1; (3) the rate at which PDOC was respired to CO₂ was 0.001 to 0.003 mg C.m^-3.h^-1. (4) the PDOC makes up only about 0.1% of the total dissolved organic carbon. The size class of particles associated with PDOC production differed from the size class responsible for uptake of PDOC. More than 50% of the PDOC production was associated with particles having a nominal diameter range of 20 to 63 m, while this fraction was responsible for <10% of the incorporation.     

Significance of active and passive depuration in the clearance of naphthalene from the tissues of Hemigrapsus nudus (Crustacea: Decapoda)

Photosynthate incorporation into lipids, low molecular weight compounds, polysaccharides and proteins by individual phytoplankton species isolated from natural populations is described. This sensitive method uses serial solvent extraction and liquid scintillation counting and gives results indentical to those obtained from filtering large numbers of cells from suspension. The time course of incorporation of ¹⁴C into these polymers for algae isolated from estuarine and coastal populations shows (i) considerable differences in the carbon metabolism among species, (ii) the pattern of incorporation by any individual species may not reflect that of the phytoplankton community and (iii) significant reallocation of cell carbon among carbon pools and net protein synthesis at night. This technique permits the in-situ carbon metabolism of individual species to be examined.     

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.     

Isolation,characterization and glucose metabolism of glycogen cells (=vesicular connective-tissue cells) from the labial palps of the marine mussel Mytilus edulis

A method has been developed to isolate glycogen cells (=vesicular connective tissue cells) from the labial palps of the marine mussel Mytilus edulis L. These cells have a modal density of 1.14 g ml^-1 and this property has been exploited to separate them from pronase-dissociated cell dispersions by density-gradient centrifugation. The isolated cells were mainly spherical, with a small peripheral nucleus. The quasitotality of the cell was filled with -like glycogen particles with cellular organelles being limited to the cell periphery. The major recipients of [U-¹⁴C] glucose-derived carbon were glycogen and amino acids. Only small quantities of ¹⁴C radiolabel were recovered from lipids, protein and CO₂. Glucose incorporation into glycogen increased linearly over 6 h and showed saturation kinetics with respect to exogenous glucose concentration.     

Coral illumination through an optic glass-fiber: incorporation of 14C photosynthates

The pathways of ¹⁴C incorporation into the three major compartments of the coral body were analysed in colonies of Stylophora pistillata. We used the optic glass-fiber method to carry out two sets of experiments: in the first, 11 different colonies were sampled immediately after incubation; in the second, 3 colonies were returned to the reef at the termination of incubation for a further period of 29 h. Within the tissue compartment, significantly more ¹⁴C labeled products were incorporated into illuminated tips or bases than into unilluminated sections. Tips located above illuminated bases contained amounts of ¹⁴C products similar to unilluminated tips. Within the organic matrix compartment, illumination of tip or base segments again resulted in increased amounts of ¹⁴C fixation, and again unilluminated tips located above the illuminated bases did not accumulate more ¹⁴C photosynthates than other tips on the same branches. The absence of detectable translocation was also confirmed after a post-incubation period of 29 h, and raises questions as to the validity of the widely accepted theory of upward translocation. Within the skeletal carbonate compartment, although the results were associated with a high coefficient of variation, significantly more ¹⁴C accumulated in the tips than in the bases. Illumination of tips or bases did not enhance ¹⁴C uptake. A light-independent carbon assimilation (dark fixation) is significant in S. pistillata within the three tested compartments (the tissue, the organic matrix of the skeleton, and the skeletal carbonate). It is suggested that the dark fixation process in corals in a result of accumulation of respiratory CO₂ and CO₂ from sea water as malate or other titratable acids during the night. During the day these acids are broken down, releasing free CO₂ for C₃ pathway photosynthesis.     

RQ of benthic marine invertebrates

This study investigated an incubation method which employed simultaneous measurement of CO₂ production and O₂ consumption rates to calculate the RQ (respiratory quotient; CO₂ production rate: O₂ consumption rate) of individual benthic marine invertebrates. Carbon dioxide production rates were calculated from changes in CO₂ concentration determined using seawater pH. O₂ consumption rates were calculated from changes in O₂ concentration with a correction applied for O₂ flux across the air/water interface due to gaseous exchange. Species examined were Triphyllozoon sp. cf. moniliferum (MacGillivray 1860), a bryozoan; Herdmania momus (Savigny), a solitary ascidian; Poneroplax albida (Blainville 1825), a chiton; and Haliotis roei (Gray 1826), an abalone. Six individuals of each were collected on 14 November 1985 from the limestone walls of a cave in a nearshore reef off Marmion, Western Australia. After acclimation for 6 h in experimental conditions, rates of CO₂ production and O₂ consumption were measured. A minimum period of 4 h was required to obtain consistent RQ values for each species. The standard error (SE) of the (calculated) RQ ratio was 14 to 33% of the mean in incubations of 4 h, and less than 14% in incubations of 4 to 12 h. The RQ is commonly used as an indicator of unknown catabolic substrates by comparing it with biochemically determined limits for known substrates. This study provides a strong argument against using the RQ of individual animals to draw any conclusions about catabolic substrates. Unexplained variation in the components of the RQ of an individual, measured over short time periods, and the potential involvement of stored reserves in catabolism, over longer time periods, obscure the relationship between the RQ of individual animals and the ratio's biochemically determined limits.