Changes in carbohydrates and lipids during embryonic development ofAntheraea mylitta (Lepidoptera)

Changes in carbohydrate and lipid metabolism during embryonic development inAntheraea mylitta were studied. While carbohydrates were metabolized during early embryogenesis, lipids were catabolised at the later stages. A significant increase in both total carbohydrates and glycogen on days 5 and 6 suggested the concurrent occurrence of both gluconeogenesis and glycogenesis. As the development of the embryo proceeds, both lipids and carbohydrates were utilised, resulting in the increase in the concentration of citrate, pyruvate and lactate.     

Recovery of adenine-nucleotide pools in terrestrial blue-green algae after prolonged drought periods

Summary The response of Nostoc commune and Nostoc flagelliforme (terrestrial blue-green algae), grown in their natural habitat, towards rewetting after prolonged drought periods (2 weeds up to five years) has been investigated. In Nostoc flagelliforme, the energy charge (EC) about 0.18 in dry condition increases rapidly (EC=0.7 after 1 h) and more slowly in a second phase (EC=0.8 after 6 h). The total content of AXP (=ATP+ADP+AMP) apparently increases due to de novo synthesis of adenine nucleotides. ATP-build-up after a drought period is probably provided by oxidative phosphorylation. It has been found to be about the same, regardless of whether the foregoing drought period had been extended over 6 months or 5 years.Dry samples of colony mats of N. commune exhibit very low ATP-, but high ADP-contents. Within 6 h after rewetting, the final level of extractable ATP (60–100 nmol/mg chlorophyll) is recovered.     

Glycogen metabolism in resting and growing cells of Saccharomyces carlsbergensis

Saccharomyces carlsbergensis cells, growing under carbohydrate or nitrogen limitation, initially deplete their glycogen, which is resynthesized only during the late exponential phase. Cells, harvested in the carly exponential phase, are even unable to synthesize glycogen in glucose-containing phosphate buffer. This is in contrast to cells from the stationary phase which rapidly synthesize glycogen under the same conditions. Lack of O₂ slows down glycogen synthesis.Contrary to cells suspended in complete medium, addition of ammonia alone to nitrogen free-media induced neither breakdown of glycogen, nor complete cessation of glycogen synthesis. Ammonia slowed down glycogen synthesis (both aerobic and anaerobic), only, in cells grown either under carbohydrate or under nitrogen limitation.Glycogen synthesis was observed 1 min after addition of glucose to a starved cell suspension in phosphate buffer. Removal of the sugar from the buffer resulted in an instantanous decrease of the glycogen level in the cells. The results indicate that glycogen-metabolism is regulated by a variety of endogenous and environmental factors.     

Desiccation and recovery of antarctic cyanobacterial mats

Summary The ability of cyanobacterial mats from Antarctic ponds and streams to recover from desiccation is described. Mats dominated by Nostoc dehydrated rapidly and were dry within 5 h of exposure. Nostoc mats recovered to pre-desiccation rates of photosynthesis and respiration within as little as 10 min of rewetting. Recovery of acetylene reduction activity was slower (>24 h). Phormidium dominated mats were less tolerant of desiccation, and recovery on rewetting from air-drying was not complete after 10 days. Viable diaspores were, however, found in Phormidium mats which had been exposed for 3 years. Partial hydration during aerial exposure improved the survival of Phormidium mats, but appeared to slow the recovery of Nostoc mats on subsequent rewetting.     

Carbohydrate Composition during Germination and Outgrowth of Ascospores of Saccharomyces cerevisiae

Single spores of Saccharomyces cerevisiae were examined to distinguish changes in the synthesis and degradation of intracellular and wall carbohydrates during germination and outgrowth. Intracellular carbohydrate was fractionated into trehalose and glycogen. Trehalose degradation occurred during germination and outgrowth. The intracellular glycogen was degraded during germination and then synthesized during outgrowth. Wall carbohydrate was fractionated into glycogen, glucan and mannan. The wall glycogen and the KOH-soluble glucan were degraded during germination and then synthesized prior to and during outgrowth, respectively. The major component of the KOH-insoluble glucan in the wall is β-1,3-glucan. The glucan and mannan were synthesized during outgrowth.

The study revealed that the development of a vegetative cell from a spore follows rapid decreases in the amounts of trehalose, glycogen and KOH-soluble glucan during germination, and great increases in the amounts of glycogen, β-1,3-glucan and mannan during outgrowth.     

Rewetting of drought-resistant blue-green algae: Time course of water uptake and reappearance of respiration,photosynthesis, and nitrogen fixation

Summary The response of the terrestrial blue-green algae Nostoc flagelliforme, Nostoc commune, and Nostoc spec. to water uptake has been investigated after a drought period of approximately 2 years. Rapid half-times of rewetting (0.6, 3.3, and 15.5 min, respectively) are found. The surfaceto-mass ratio of the three species is inversely correlated to the speed of water uptake and loss. The ecological relevance of these different time courses is discussed.Respiration starts immediately after a 30-min rewetting period, whereas photosynthetic oxygen evolution reaches its maximum activity after 6 and 8 h with N. commune and N. flagelliforme, respectively. In the dark, recovery of oxygen uptake by N. commune is somewhat impaired, while slightly stimulated with N. flagelliforme. With both species, recovery of photosynthesis is inhibited by darkness.Using colonies kept dry for two years, nitrogenase activity of N. commune attains its maximum 120 to 150 h after rewetting, while only 50 h were needed with algal mats kept dry for two days.Thus, after a 2-year drought period, the physiological sequence of reactivation is respiration—photosynthesis—nitrogen fixation. Respiration and photosynthesis precede growth and are exhibited by existing vegetative cells, whereas recovery of nitrogen fixation is dependent on newly differentiated heterocysts.     

Compartmentation and fluxes of carbon in leaf blades and leaf sheaths of Poa annua L. and Poa x jemtlandica (Almq.) Richt

Abstract The allocation of photosynthetically fixed carbon in the leaf blades and sheaths of Poa annua (a ruderal grass) and Poa x jemtlandica (a sub-arctic grass) was followed over a light-dark cycle. Labelling with ¹⁴Carbon and gas exchange measurements provide data for an eight-compartment model describing the partitioning of carbon between spatially and chemically separated pools and their rates of turnover. Soluble sugars and fructans were turned over rapidly in the leaf blades of both species. The flux of carbon through pools of storage carbohydrates was higher in the leaves of P. x jemtlandica than in P. annua. The exchange of carbon between pools was slower in the sheath than the blade. Carbohydrates stored in the sheath appeared to have no significant role in metabolism over the light-dark cycle studied here.     

Detection of protein and carbohydrate in the extracellular matrix released byCochliobolus heterostrophus during germination

The extracellular matrix (ECM) ofCochliobolus heterostrophus (anamorph:Bipolaris maydis) was made visible by gold/silver and FITC-lectin staining at different stages of germ tube development. A proteinaceous material was released from conidia as germ tubes began to emerge and continued to be released from the germ tube tip throughout elongation. A material that did not stain for protein was observed to surround germ tubes upon their elongation. At later stages of maturation, germ tubes were surrounded by a sheath of proteinaceous material. After 15 h of incubation, staining with the FITC-labeled Concanavalin A revealed that a carbohydrate material surrounded and extended between hyphae. The ECM extract was separated into two fractions which were shown by SDS-PAGE and HPLC analyses to consist of proteins and carbohydrates. The results demonstrate that the composition and physical structure of the ECM change over time. Thus, the ECM is not a static material. Rather, the components of the ECM appear to be laid down at different stages of fungal morphogenesis, possibly related to germ tube emergence, elongation, and maturation.     

Carbohydrate as Fuel for Foraging,Resource Defense and Colony Growth – a Long‐term Experiment with the Plant‐ant Crematogaster nigriceps

Mismatches in nutrient composition (e.g., protein, carbohydrates, lipids, etc.) between consumers and the resources they depend on can have ecological consequences, affecting traits from individual behavior to community structure. In many terrestrial ecosystems, ants depend on plant and insect mutualist partners for carbohydrate‐rich rewards that are nutritionally unbalanced (especially in protein) relative to colony needs. Despite imbalances, many carbohydrate‐feeding ant mutualists dominate communities—both competitively and numerically—raising the question of whether excess carbohydrates ‘fuel’ colony acquisition of limiting resources and growth. In a 10‐month field study, we manipulated carbohydrate access for the obligate plant‐ant Crematogaster nigriceps to test whether carbohydrate availability could be mechanistically linked to ecological dominance via heightened territory defense, increased protein foraging, and colony growth. Supplementation increased aggressive defense of hosts after only two weeks, but was also strongly linked to variation in rainfall. Contrary to predictions, we did not find that supplemented colonies increased protein foraging. Instead, colonies with reduced carbohydrate access discovered a greater proportion of protein baits, suggesting that carbohydrate deprivation increases foraging intensity. We found no significant effect of carbohydrate manipulation on brood or alate production. These results contrast with findings from several recent short‐term and lab‐based nutrient supplementation studies and highlight the role of seasonality and biotic context in colony‐foraging and reproductive decisions. These factors may be essential to understanding the consequences of carbohydrate access in natural plant‐ant systems.     

Seasonal patterns of carbohydrate translocation and synthesis of structural carbon components in <Emphasis Type="Italic">Typha angustifolia</Emphasis>

Quantitative studies of material budgets and resource allocation patterns of emergent plants are needed to fully understand nutrient and carbon cycling in wetlands. Whole-plant translocation patterns of nonstructural carbohydrates and synthesis of structural carbon were documented using two (shallow and deepwater) populations of Typha angustifolia in floodplain habitats of the Arakawa River, Japan. Monthly and bimonthly measurements of the concentrations of total carbon, non-structural carbohydrates, and water-soluble carbohydrates, as well as, estimates of standing stocks of aboveground (AG) and belowground (BG) biomass for both populations from 2002 to 2004 are described here. Annual patterns of carbon gain, rates of carbohydrate translocation between AG and BG organs, and rates of synthesis of structural carbon were estimated. Upward translocation supported all AG production for approximately 30 days. Afterward, the fraction of AG production supported by upward translocation decreased linearly with time, and completely diminished by Day 80 (counted from first day of growth; March 15). At Day 80, material translocation was directed downward and the percentage of downward translocation relative to AG net production increased until Day 170; there was a vigorous increase in downward translocation prior to senescence. In early summer (Day 80–110), more than half of the translocated materials were transformed into structural components, but by Day 125 only 30% was synthesized into total structural carbohydrates. Prior to senescence, there was a greater percentage of nonstructural carbohydrates in the rhizomes. Across the growing season (Day 60–153), the total amount of downward translocation was found to be proportional to AG production and the quantitative evaluation of the carbon budget shed further insight into the translocation process of this rhizomatous aquatic plants. In addition, insights on the differences between shallow and deepwater populations were gained. Especially, the deepwater population supported a higher fraction of vertical rhizomes compared to individuals sampled from the shallow water population. Handling editor: S. M. Thomaz.     

Effect of glucose starvation on germ-tube production byCandida albicans

By incubating starved and unstarved yeast cells in synthetic media with a pH of 4.5 or 6.7 at 37°C the effect of a 3 hours' glucose starvation on germ-tube production byCandida albicans was evaluated. In addition the endocellular content of total carbohydrates, glycogen, trehalose and proteins after and before the starvation were dosed. The most interesting result was the overcoming of the pH-regulated dimorphism, thanks to the starvation treatment. Infact the starved cultures produced germtubes indifferently in neutral or acid media, whereas the filamentation of the unstarved cultures was more copious in pH 6.7 medium. The endocellular content of trehalose and protein was unchanged, whereas total carbohydrates and glycogen showed a shortage after the 3 hours' glucose starvation. The possible involvements of these metabolic changes in the regulation of dimorphic transition are discussed.     

D2O effects on association behavior and binding properties of phospho- and dephospho-cytoplasmic malic dehydrogenase

Changes in the concentration of three major carbohydrates, e.g., glycogen, trehalose, and cellulose, were determined during differentiation of Dictyostelium discoideum in a stage study. These three carbohydrates consituted 50–63% of the total carbohydrates. Total carbohydrate content per cell aliquot did not change between the aggregation and sorocarp stages of differentiation. The isolation, purification, and characterization of cellulose is described. Cellulose consisted of an alkali-insoluble (alpha) and an alkali-soluble (beta) fraction. Total cellulose accumulated from very low amounts in late pseudoplasmodium cells to about 35% of carbohydrates in mature sorocarps at a rate of 0.07 μmole glucose equiv/min/ml packed cell volume. Purified alkali-insoluble cellulose constituted about 19% of total carbohydrates in mature sorocarps and accumulated at a rate of 0.035 μmole glucose equiv/min/ml packed cell volume. Trehalose constituted 10–11% of the carbohydrates in sorocarps and accumulated at a rate of 0.035 μmole glucose equiv/min/ml packed cell volume. Glycogen, comparing several methods of determination, was rapidly degraded between the culmination and sorocarp stages of differentiation at an average rate of 0.05 μmole glucose equiv/min/ml packed cell volume. The major portion of glycogen was soluble in TCA and constituted 35% of total carbohydrates in aggregated cells and about 11% in mature sorocarps. A minor fraction of glycogen, about 15% of total carbohydrates in aggregated cells, was solubilized by KOH from a TCA precipitate. A mild acidic treatment of solubilized cell constituents increased the glycogen content by 55%, as judged by an enzymatic assay.     

Changes in the fungus-specific,soluble-carbohydrate pool during rapid and synchronous ectomycorrhiza formation of Picea abies with Pisolithus tinctorius

A simple and convenient culture system has been developed for the analysis of ectomycorrhiza formation under controlled conditions. Rapid and synchronous mycorrhiza synthesis was observed when thin and even layers of Pisolithus tinctorius (Pers.) hyphae were brought at once into contact with the entire root system of 3-month-old Picea abies (L. Karst) plants. Suitable fungal layers were grown on cardboard with limiting glucose supply in the medium to maximize radial growth. The glucose was almost consumed by the time the fungus had spread over the whole cardboard and was ready for inoculation of the roots. At this stage, the fungus contained trehalose and arabitol as the main soluble carbohydrates. A few hours after the assembly of the culture system, contacts between roots and aerial hyphae were observed and a sheath was formed 3 days later, suggesting very rapid ectomycorrhiza formation under these conditions. The pool of soluble carbohydrates of the inoculum, i.e. the extramatrical mycelium, declined after inoculation of the roots and was almost zero after 2 weeks. The supply of carbon by the plant was then sufficient for the fungus to expand the soluble pool efficiently in both the mycorrhizas and the extramatrical mycelium. The kinetics of the carbohydrate pool and the observed differentiation of the short roots to mycorrhizas imply that in our culture system fully functional symbiosis was established no later than 14 days after the plants were inoculated with the fungus.     

Studies on Photosynthesis,Respiration and Morphology of Nostoc flagelliforme

Nostoc flagelliforme Born. et Flah. is one of the terrestrial cyanobacteria naturally distributed in arid and semi-arid areas in the Northern and the North-western parts of China. The cyanobacterium is an edible delicacy with special medical value. However, commercial N. flagelliforme has nov been collected from the field only. For cultivation of this valuable cyanobacterium, it is necessary to understand how it grows and how it adapts to the environment.The experimental material was collected in Siziwangqi of Nei Monggol. The effects of light intensity, temperature, pH, salinity, length of thallus and rewetting on photosynthesis and respiration of N. fiagelli[orme were measured using an oxygen electrode. The results were as follows: The photosynthetic light compensation point was around 40–90μmol photons·m-2·s-1, the light saturation point was 1200μmol photons·m-2·s-1, and no photoinhibition appeared when the light intensity was increased to 1800μmol photons· m-2·s-1. N. fiagelliforme exhibited its photosynthetic and respiratory activities in the temperature range of 5–45℃. The optimum temperature for its photosynthesis was 25℃ and that for respiration was 35--40℃. Between pH range of 4.5–9.5 N. flagelliforme had photosynthetic activity and respiratory activity at pH range of 4-10, with optimum pH for photosynthesis at 7.5 and for respiration at 7.5–8.0. N. flagelliforme exhibited maximum net photosynthesis in 0.15mol/L of NaC1 in BG-11 medium. When the salinity was increased to 0.9 mol/L the net photosynthesis dropped down to zero. Respiration decreased concordantly with the increasing salinity as well. Maxima photosynthesis and respiration was also detected when the thallus of N. flagelliforme reached a length of 0.5cm and aftewords the more the length the less the activities. The recovery time attaining to the maximum photosynthesis and respiration activities after rewetting was dependent on storage time in dryness. The cyanobacterial mats after being reserved for 3 months, attained its maximum photosynthesis by 0.5h after rewetting, and that being reserved for 18 months needed 3.5h after rewetting. For respiration, the mats reserved for 3 months and 18 months required 5 minutes and lh after rewetting, respectively to attain its maximum. Under scanning electron microscope, cells of N. flagelliforme were wrapped up within a gluey sheath, and usually attached closely to each other in pairs and the filaments were uni-trichome with branches in some cases. The surface of thallus tip was rougher than other parts which meant that the tip portion had greater surface area beneficial to water absorption and cell growth.     

An evaluation of methods for quantifying the enzymatic degradation of red seaweed Grateloupia turuturu

For better exploitation of the red seaweed Grateloupia, enzymatic digestion of the thallus may be a way to increase access to metabolites of industrial interest. With this aim, we have tried to find a method to quantify the efficiency of enzymatic digestion. Vegetative algal material was treated with polysaccharidases (Onozuka R-10 cellulase, agarase, and Ultraflo L mixture). The proportion of degraded surface area was determined by microscopic measurement of the residue surface using imaging software and compared with the analysis of carbohydrates and R-phycoerythrin released in the incubation solution. Both the reducing carbohydrate concentration and percentage of degraded surface area appeared the most reliable methods to study enzymatic efficiency. The amount of solubilized total carbohydrates, and particularly that of R-phycoerythrin, showed non-specific variations, so no conclusions could be drawn. The application of this procedure to the screening of the efficient digestion of Grateloupia material demonstrated that cell walls were only partially digested by polysaccharidase enzymes alone. The Ultraflo L mixture and Onozuka R-10 cellulase produced a greater degradation of Grateloupia tissues and a higher release of reducing carbohydrates, whereas agarase did not display any specific action. Thus, the proposed procedure based on the quantification of residue surface area seems to be an accurate method to analyze enzymatic digestion. Other tests using different concentrations and combinations of enzymes are now required.     

Honeydew feeding in the solitary bee Osmia bicornis as affected by aphid species and nectar availability

Like honey bees (Apis mellifera), non-Apis bees could exploit honeydew as a carbohydrate source. In addition to providing carbohydrates, this may expose them to potentially harmful plant products secreted in honeydew. However, knowledge on honeydew feeding by solitary bees is very scarce. Here we determine whether the polylectic solitary bee Osmia bicornis (=O. rufa) collects honeydew under semi-field conditions, and whether this is affected by aphid species and presence of floral nectar. Bees were provided with oilseed rape plants containing flowers and/or colonies of either Myzus persicae or Brevicoryne brassicae. We used the total sugar level of the bee crop as a measure of the individual's nutritional state and the oligosaccharide erlose as indicator for honeydew consumption. Erlose was present in honeydews from both aphid species, while absent in oilseed rape nectar, nor being synthesized by O. bicornis. When bees were confined to a single honeydew type as the only carbohydrate source, consumption of M. persicae honeydew was confirmed for 47% of the bees and consumption of B. brassicae honeydew for only 3%. Increased mortality in the latter treatment provided further evidence that B. brassicae honeydew is an unsuitable food source for O. bicornis. All bees that were given the choice between honeydew and floral nectar showed significantly increased total sugar levels. However, the fact that no erlose was detected in these bees indicates that honeydew was not consumed when suitable floral nectar was available. This study demonstrates that honeydew exploitation by O. bicornis is dependent on honeydew type and the presence of floral nectar.     

An autolysin dissolves the inner cell wall layer of the algaPediastrum (Hydrodictyaceae)

Summary An autolysin produced by young colonies ofPediastrum frees them from the vesicle in which they are formed within 12 hours of release of zoospores from the parent cell. The polysaccharide vesicle is derived from the inner wall layer of the parent cell. Refrigeration delays vesicle disintegration; boiling stops it completely. A purified, lyophilized extract of the vesicle fluid added to boiled vesicled colonies removes the vesicle in 2 hours with the release of reducing sugars and polysaccharides.Biogel P2 and P10 chromatography of the products following incubation of the enzyme preparation and wall showed no more than 1% oligosaccharides; the remaining carbohydrates had a molecular weight of several thousand daltons. Analyses of isolated vesicle wall material (70–85% of the dry weight) showed mannose accounting for approximately 50% of the dry weight, with none of the other neutral sugars present (fucose, xylose, galactose and glucose) representing more than 3%. Uronic acids account for 20–25% of the wall weight, and proteins less than 2%. Pediastrum colonies are thus freed from the vesicles in which they are formed by the action of an autolysin they produce. The autolysin acts on the vesicle wall material to generate reducing sugars and cause it to disintegrate into its constituent polysaccharides.     

Carbohydrate synthesis by two Navicula strains isolated from benthic and pelagic mucilages in the Tyrrhenian Sea (Tuscan Archipelago)

Two exopolysaccharide (EPS)-producing strains of the diatom Navicula, were isolated from benthic and pelagic mucilaginous aggregates sampled in the Tyrrhenian Sea and cultured under laboratory conditions. The amount of carbohydrate formed over the growth period and on a per cell basis was quite similar. However, the benthic strain showed a preferential synthesis of the bound (i.e., cellular and capsular) carbohydrate fraction, whereas the pelagic strain preferentially synthesised soluble, polymeric carbohydrates. The polysaccharides released into the medium by the two strains showed the same qualitative monosaccharidic composition, being constituted by two acidic and six neutral sugars. It is suggested that the difference between the benthic and the pelagic strain in the synthesis of bound or soluble carbohydrates may be related to the different role of these compounds in the particular habitats of the strains.     

水体溶氧影响陆生植物喜旱莲子草(Alternanthera philoxeroides)和牛鞭草(Hemarthria altissima)对完全水淹的耐受力

溶氧是水环境中一个重要的环境因子,为了探讨水中的溶氧含量水平是否会对陆生植物的耐淹能力造成影响,研究了陆生植物喜旱莲子草(Alternanthera philoxeroides)和牛鞭草(Hemarthria altissima)在遭受不同溶氧含量水体完全淹没后的生长表现、存活情况和非结构碳水化合物的变化。实验结果表明:(1)水体中的溶氧含量显著影响了处于完全水淹环境中的喜旱莲子草和牛鞭草的存活。受高溶氧水体完全水淹的喜旱莲子草和牛鞭草主茎的完好程度和存活叶的数量均显著高于遭受低溶氧水体完全水淹的喜旱莲子草和牛鞭草,喜旱莲子草和牛鞭草在高溶氧水体完全水淹后的生物量比低溶氧水体完全水淹后要高;(2)水体中的溶氧含量显著影响了处于完全水淹环境中的喜旱莲子草和牛鞭草的生长,受高溶氧水体完全水淹的喜旱莲子草主茎伸长生长和不定根生长显著强于受低溶氧水体完全水淹的喜旱莲子草,在不定根的生长上牛鞭草也具有同样的表现。(3)高溶氧水环境有利于减小被完全淹没的喜旱莲子草和牛鞭草的碳水化合物消耗,两种植物在受高溶氧完全水淹后体内具有的非结构性碳水化合物含量均比受低溶氧完全水淹后高。(4)喜旱莲子草比牛鞭草能更好地耐受完全水淹,当处于低溶氧完全水淹时表现得更为明显,本研究表明入侵物种喜旱莲子草比本地物种牛鞭草具有更强的环境适应能力和水淹耐受能力。     

The effects of clipping and fertilization on nitrogen nutrition and allocation by mycorrhizal and nonmycorrhizal Panicum coloratum L., a C4 grass

Summary Mycorrhizal and nonmycorrhizal plants of Panicum coloratum L. were grown in a factorial treatment design under two nitrogen levels and two clipping heights with an unclipped control. The nitrogen concentration in different plant components was determined following 9 weeks of growth under experimental conditions. Mycorrhizal infection increased green leaf and sheath nitrogen concentration by a relatively small, but significant percentage and had no effect on nitrogen allocation to the various plant components. Clipping increased leaf nitrogen concentration but inhibited growth to the extent that, when compared with the unclipped controls, less nitrogen remained in residual plant biomass with up to half of the total nitrogen allocated to offtake (the material removed by clipping). Plants receiving the higher nitrogen fertilization had higher tissue concentration of N and more N allocated to above-ground living tissues. Mycorrhizal infection interacted with clipping height and also with N availability significantly. Infection was unable to ameliorate the negative effects of the most severe clipping regime and of the low nitrogen availability on leaf and sheath N content. This is possibly due to mycorrhizal demand for carbohydrates competing with the carbohydrate requirement of roots for nitrogen uptake.