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The behavioral effects of short periods (2, 4, 6, 8 h) of static exposure to prochloraz (imidazole fungicide) and nicosulfuron (sulfonylurea herbicide) were recorded in goldfish (Carassius auratus). Observations were also made in an olfactometer to assess the effects of 8-h exposures to these two pesticides and to carbofuran (carbamate insecticide) on the behavioral responses to the flow of a solution of four L-amino acids (glycine, alanine, valine, taurine), mixed in the same relative proportions as in the urine of conspecifics. Each pesticide was tested at three sublethal concentrations (25, 50, 100 microg/L), and the behaviors recorded were related to swimming pattern, social interactions, and comfort movements. Static exposures to prochloraz affected horizontal displacements, burst swimming, grouping, and buccal movements. Static exposures to nicosulfuron affected burst swimming and grouping. In pesticide-unexposed fish (control), the flow of the amino acid solution induced attraction, decreased sheltering, and increased horizontal displacements, burst swimming, buccal movements, and antagonistic interactions. Compared to the controls, some of the behavioral responses to the solution of amino acids were significantly different after 8 h of subacute exposure to prochloraz and carbofuran. Both pesticides decreased attraction and increased sheltering. In addition, carbofuran decreased buccal movements and antagonistic interactions. Contrastingly, exposure to nicosulfuron showed no significant effect. This study further confirms the great vulnerability of fish behavior and chemocommunication processes to exposure to waterborne pesticides.

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The immediate behavioral responses of goldfish (Carassius auratus) to pesticide-contaminated flows were recorded in a countercurrent olfactometer. In addition, electro-olfactograms were recorded from the epithelial surface of the olfactory rosette as a preliminary check for the olfactory sensitivity of the fish to the pesticides tested. All tests were run on prochloraz (imidazole fungicide), bentazone (diazine herbicide), and nicosulfuron (sulfonylurea herbicide). Behavioral effects were assessed, at four concentrations (10 microg/L, 100 microg/L, 1 mg/L, 10 mg/L), on endpoints related to swimming pattern (preference-avoidance responses, burst swimming reactions), comfort activities (buccal movements, feeding attempts), and social relations (antagonistic acts, grouping). The behavior of the fish appeared particularly sensitive to prochloraz exposure. As a whole, prochloraz-contaminated flows showed significant effects on the six behaviors studied; bentazone and nicosulfuron affected three and five, respectively. At the lowest concentration, prochloraz also showed more effects than the two other pesticides. Some of the behavioral endpoints were found particularly sensitive to a given chemical. Pesticide-contaminated flows also induced significant changes in swimming orientation of the fish. Attraction was observed in response to flowing solutions of prochloraz (1 mg/L, 10 mg/L), bentazone (10 microg/L, 10 mg/L), and nicosulfuron (1 mg/L, 10 mg/L). At a concentration of 1 mg/L, none of the pesticides induced a noticeable depolarization of the olfactory epithelium, suggesting that these chemicals are not detected by the olfactory sense of the fish. These results are discussed in the light of the data concerning effects of pesticides on behavior and chemical communication in fish.

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Juvenile goldfish (Carassius auratus) were exposed to three widely used pesticides; carbofuran, diuron, and nicosulfuron. Acetylcholinesterase (AChE) activity and molecular forms of AChE were first characterized in brain and skeletal muscle of unexposed fish. Skeletal muscle had higher AChE activity than brain (306 and 215 nmol/min/mg protein, respectively). In brain, four molecular forms of AChE were found: A12, G4, G2, and G1. In the muscle, three molecular forms were found A12, A8, and G2. AChE activity was then evaluated in both tissues of fish exposed to different concentration of pesticides (5, 50, and 500 microg/L) for 6, 12, 24, and 48 h. In brain, AChE activity was significantly inhibited during all the periods of exposure in response to 50 microg/L (19-28%) and 500 microg/L (85-87%) carbofuran. Such effect was observed in the muscle only at 500 microg/L (86-92%). Carbofuran had no effect on the distribution of molecular forms. Significant inhibitions (9-12%) of brain AChE activity were also observed in response to diuron and nicosulfuron at 500 microg/L during all periods of exposure and for 50 microg/L nicosulfuron after 24 and 48 h. This study pointed out short-term effects of exposure to sublethal concentrations of the three pesticides, ranging among different chemical families, on brain and muscle AChE in goldfish.

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Among several biological functions, the epidermal mucus of fish may play an important role in host defense, particularly in the prevention of colonization by parasites, bacteria and fungi. In previous work, two hydrophobic proteins of 27 and 31 kDa were isolated from carp mucus. This study identified a strong antibacterial activity (0.16-0.18 microM) well correlated with pore-forming properties. Here this work was extended to other fish species, four fresh water fish and one sea water fish. After a first step of purification, water-soluble and hydrophobic material were separated, and both fractions were analyzed by SDS-PAGE and capillary electrophoresis. Only the hydrophobic component induced pore-forming activity, when reconstituted in planar lipid bilayers. This pore-forming activity was well correlated to a strong antibacterial activity against several bacteria strains. These results suggest that fish secrete antibacterial proteins able to permeabilize the membrane of the target cell and thus act as a defense barrier.

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Experiments were performed in goldfish to determine the effects of a short-term exposure (24 h) to atrazine or diuron (0.5, 5, 50 microgram/L) on some behavior endpoints related to swimming and social activities. Observations were also made to assess the influence of such exposure on the behavioral responses of fish to the flow of a crude skin extract solution from conspecifics, active in social chemocommunication and producing alarm behaviors. Additive tests were run to check the behavioral responses of previously unexposed goldfish to the flow of a solution of atrazine- or diuron-contaminated water, at three concentrations (0.1, 1, 10 mg/L). Significant burst swimming reactions appeared in response to a 24-h exposure to atrazine, at the lowest concentration tested (0.5 microgram/L). A 24-h exposure to 5 microgram/L atrazine or diuron was found to induce various significant behavioral alterations in fish. At this concentration, both herbicides decreased grouping behavior and atrazine also increased surfacing activity. Herbicide-exposed fish showed a decreased grouping behavior during the flow of the skin extract solution. Sheltering was also decreased during the flow of the biological solution in fish exposed to atrazine. Moreover, fish exposed to diuron clearly displayed attraction responses to the flow of the skin solution. Previously unexposed fish showed a significant increase in burst swimming reactions in response to the flow of a solution of atrazine- or diuron-contaminated water, at all concentrations tested (0.1, 1, 10 mg/L). Furthermore, the diuron-contaminated flow was found to be significantly attractive at the highest concentration. These results indicate that a short-term exposure to a relatively low concentration (5 microgram/L) of atrazine or diuron can affect various behaviors of fish not only directly but also indirectly by altering the chemical perception of natural substances of eco-ethological importance. In consideration of the basic role of olfaction in fish behavior, these results also emphasize the need for further developments on the possible effects of aquatic toxicants on olfactory-mediated behaviors.

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The induction of the sucrose synthase (SuSy) gene (SuSy) by low O2, low temperature, and limiting carbohydrate supply suggested a role in carbohydrate metabolism under stress conditions. The isolation of a maize (Zea mays L.) line mutant for the two known SuSy genes but functionally normal showed that SuSy activity might not be required for aerobic growth and allowed the possibility of investigating its importance during anaerobic stress. As assessed by root elongation after return to air, hypoxic pretreatment improved anoxic tolerance, in correlation with the number of SuSy genes and the level of SuSy expression. Furthermore, root death in double-mutant seedlings during anoxic incubation could be attributed to the impaired utilization of sucrose (Suc). Collectively, these data provide unequivocal evidence that Suc is the principal C source and that SuSy is the main enzyme active in Suc breakdown in roots of maize seedlings deprived of O2. In this situation, SuSy plays a critical role in anoxic tolerance.

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Hypoxic pretreatment of tomato (Lycopersicon esculentum M.) roots induced an acclimation to anoxia. Survival in the absence of oxygen was improved from 10 h to more than 36 h if external sucrose was present. The energy charge value of anoxic tissues increased during the course of hypoxic acclimation, indicating an improvement of energy metabolism. In acclimated roots ethanol was produced immediately after transfer to anoxia and little lactic acid accumulated in the tissues. In nonacclimated roots significant ethanol synthesis occurred after a 1-h lag period, during which time large amounts of lactic acid accumulated in the tissues. Several enzyme activities, including that of alcohol dehydrogenase, lactate dehydrogenase, pyruvate decarboxylase, and sucrose synthase, increased during the hypoxic pretreatment. In contrast to maize, hexokinase activities did not increase and phosphorylation of hexoses was strongly inhibited during anoxia in both kinds of tomato roots. Sucrose, but not glucose or fructose, was able to sustain glycolytic flux via the sucrose synthase pathway and allowed anoxic tolerance of acclimated roots. These results are discussed in relation to cytosolic acidosis and the ability of tomato roots to survive anoxia.

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A detergent-solubilized fraction of skin mucus of carp (Cyprinus carpio) induced ion channels after reconstitution into planar lipid bilayers. A differential extraction using a non-ionic detergent followed by electrophoretic separation led to the isolation of two hydrophobic 31-kDa and 27-kDa proteins. In contrast to the 27-kDa protein, which was glycosylated, the 31-kDa did not bind to concanavalin A. The reconstitution of these proteins into a planar lipid bilayer restored the ionophore behavior already observed with the crude mucus. The main unit conductance levels were about 900 pS for the 27-kDa protein and 500 pS for the 31-kDa protein, and selectivity measurements gave Pcl/Pk ratios of 0.6 and 1.0, respectively. These proteins had large potent microbicidal activities (0.018-0.18 microM) against different strains of gram-negative and gram-positive bacteria. This behavior can be compared with insect defensins that are known to form large ion channels in the bacterial membrane. To exclude the eventuality of bacterial origin, the bacterial flora of the crude mucus were analysed and the following were identified: Pseudomonas cepacia; Micrococcus luteus; Micrococcus roseus; Flavobacterium sp.; Aeromonas hydrophila. Antibacterial assays with both proteins were performed against these specific strains and revealed good growth inhibition activities. Furthermore, microsequencing analysis showed that the 31-kDa protein was protected on its N-terminal extremity in contrast to the 27-kDa protein, which had a 19-amino-acid sequence. This last sequence, when compared with sequences in protein data banks, did not reveal any significant similarities to other proteins. These results suggest that these novel proteins could be involved in antibacterial defense processes in fish.

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The effects of concentration (1, 10, 100 microg/L) and duration (4, 8, 12 h) of exposure to carbofuran were assessed on the swimming activity, social interactions, and behavioral responses of goldfish to a flow (0.1 L/min) of water, with or without chironomids. Observations were also made on the behavioral responses of unexposed goldfish to a flow (0.1 L/min) of carbofuran-contaminated water. A 4-h exposure of goldfish 1 microg/L carbofuran produced a significant increase in sheltering, burst swimming, and nipping. Responses were enhanced at 100 microg/L. After a 12-h exposure, the behavioral effects of 1 microg/L carbofuran were less apparent. However, burst swimming at 10 microg/L, and sheltering, nipping and burst swimming at 100 microg/L, were still significantly increased after a 12-h exposure to carbofuran. Grouping was not consistently affected by exposure conditions. Chemical attraction to a filtrate of chironomids was significantly reduced after the 4-h exposure to 1 microg/L carbofuran. Decreased attraction to the food extract was less apparent after the 12-h exposure, except at 100 microg/L carbofuran. A significant decrease in attraction to a flow of uncontaminated water was also observed after a 4-h exposure to 10 and 100 microg/L carbofuran. Unexposed goldfish did not show avoidance reaction to a flow of carbofuran-contaminated water, even at a concentration (10 mg/L) exceeding the mean 96-h LC-50 in cyprinids (0.5-1 mg/L). However, at all concentrations tested (0.1, 1, 10 mg/L), goldfish quickly reacted to the introduction of the solution of carbofuran by increased burst swimming and nipping. These results are discussed in the light of the data concerning behavioral and neurotoxic effects of carbamate and organophosphorous insecticides in fish.

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Several enzyme activities were measured in extracts from acclimated and nonacclimated maize (Zea mays) root tips at pH 6.5 and 7.5, corresponding to cytoplasmic pH in anaerobiosis or aerobiosis, respectively, to determine what causes the decline of the glycolytic flux observed in anoxia in nonacclimated tips. We found that phosphorylation of hexoses by kinases was a major limiting step of glycolysis in anoxia. When fructose was substituted for glucose, glycolysis was slightly enhanced and survival improved, but neither matched that of acclimated tips. Decrease of kinase activities was not the result of proteolytic degradation but was more likely the result of inhibition by internal factors (low pH and low ATP). There was no evidence of induction during the hypoxic pretreatment of isoenzymes better adapted to the anoxic cellular environment. Maintenance of the glycolytic flux in acclimated tissues is explained by a combination of a rise in kinase activities and decreased inhibition resulting from a higher cytoplasmic pH and ATP content. The behavior of intact root tips is discussed in comparison with the behavior of excised root tips.

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We tested the hypothesis that ATP levels and energy charge determine the resistance of maize (Zea mays) root tips to anoxia. We focused on root tips of whole maize seedlings that had been acclimated to low O2 by exposure to an atmosphere of 3% (v/v) O2 in N2. Acclimated anoxic root tips characteristically have higher ATP levels and energy charge and survive longer under anoxia than nonacclimated tips. We poisoned intact, acclimated root tips with either fluoride or mannose, causing decreases in ATP and energy charge to values similar to or, in most cases, below those found in nonacclimated anoxic tips. With the exception of the highest fluoride concentration used, the poisoned, acclimated tips remained much more tolerant of anoxia than nonacclimated root tips. We conclude that high ATP and energy charge are not components critical for the survival of acclimated root tips during anoxia. The reduced nucleotide status in poisoned, acclimated root tips had little effect on cytoplasmic pH regulation during anoxia. This result indicates that in anoxic, acclimated root tips either cytoplasmic pH regulation is not dominated by ATP-dependent processes or these processes can continue in vivo largely independently of any changes in ATP levels in the physiological range. The role of glycolytic flux in survival under anoxia is discussed.

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Hypoxic pretreatment (3 kPa oxygen) of maize (Zea mays L.) root tips improved their survival time in a subsequent anoxic incubation from 10 h to more than 3 d, provided that glucose was added to the medium to sustain metabolism. The glycolytic flux (lactate + ethanol) was the same in both pretreated and untreated root tips during the 1st h after transfer to anoxia. It was only after 2 h that it declined sharply in untreated tips, but was sustained in pretreated ones. Right after the transition from normoxia to anoxia of untreated root tips, the only fermentative product detected was lactic acid, which accumulated in a 7:1 proportion after 30 min in tissue and medium, respectively. It took 10 min before ethanol could be detected and 20 min for it to be produced at its maximum rate at the expense of lactate production, which slowed down. In contrast, in hypoxically pretreated root tips, ethanol was produced at a maximum rate right after the transfer to anoxia. Concurrently, low amounts of lactic acid were produced that accumulated in a 1:1 proportion after 30 min in tissue and medium, respectively. This large efflux of lactic acid could account for the higher cytoplasmic pH values always found in pretreated tissues. The presence of cycloheximide during pretreatment abolished this difference, suggesting that the greater efficiency of lactate efflux was linked to protein synthesis. The role of lactate in cytosolic pH regulation and in sensitivity to anoxia is discussed.

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The relationship between changes in H(+) flux and sugar transport in maize Zea mays L. DEA root tips have been investigated using two methods for controlling the cellular nucleotide level: (a) incubation in the presence of a glucose analog, the 2-deoxyglucose, which decreased the ATP level to less than 15% of its initial value within 60 minutes without changing the ADP and AMP levels; (b) an hypoxic treatment which also decreased the ATP level but with a concomitant rise in ADP and AMP. In both cases the rate of hexose transport was not modified until ATP had dropped to 70% of its initial value; then it decreased with the cellular ATP level. The residual uptake rate at very low ATP concentrations still represented 50% of the maximum rate with the dGlc treatment but only the diffusion rate in anoxia. H(+) efflux was abolished in anoxia but not by the 2-deoxyglucose treatment, in spite of a lower cellular ATP concentration. Our results are consistent with an inhibition of H(+)-ATPase activity in anoxia by the high levels of cellular ADP and AMP, and provide in vivo evidence that sugar uptake is dependent upon the proton motive force rather than cellular ATP concentration. The absence of stimulation of H(+) extrusion by ferricyanide in either normoxic or hypoxic conditions suggests that a redox system does not appear to contribute to H(+) secretion under the conditions of this investigation.

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Soybean (Glycine max cv Hodgson) nitrogenase activity (C(2)H(2) reduction) in the presence or absence of nitrate was studied at various external O(2) tensions. Nitrogenase activity increased with oxygen partial pressure up to 30 kilopascals, which appeared to be the optimum. A parallel increase in ATP/ADP ratios indicated a limitation of respiration rate by low O(2) tensions in the nodule, and the values found for adenine nucleotide ratios suggested that the nitrogenase activity was limited by the rate of ATP regeneration. In the presence of nitrate, the nitrogenase activity was low and less stimulated by increased pO(2), although the nitrite content per gram of nodules decreased from 0.05 to 0.02 micromole when pO(2) increased from 10 to 30 kilopascals. Therefore, the accumulation of nitrite inside the nodule was probably not the major cause of the inhibition. Instead, inhibition by nitrate could be due to competition for reducing power between nitrate reduction and bacteroid or mitochondrial respiration inside the nodule. This is supported by the observation of decrease in ATP/ADP ratios from 1.65, in absence of nitrate, to 0.93 in the presence of this anion at 30 kilopascals O(2). Furthermore, the inhibition was suppressed by the addition, to the plant nutrient solution, of 15 millimolar l-malate, a carbon substrate that is considered to be the major source of reductant for the bacteroids in the symbiosis.

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Sugar-depleted excised maize (Zea mays L.) root tips were used to study the kinetics and the specificity of hexose uptake. It was found that difficulties induced by bulk diffusion and penetration barriers did not exist with root tips. Several lines of evidence indicate the existence of a complex set of uptake systems for hexoses showing an overall biphasic dependence on external sugar concentrations. The results suggest that the high and the low affinity components might be located on the same carrier. One uptake system was specific for fructose, but the high affinity component was repressed by high concentrations of external glucose. A second system was specific for glucose and its analogs (2-deoxy-d-glucose and 3-O-methyl-d-glucose), and a third one, more complex, had a high affinity for glucose and its analogs but could transport fructose when glucose was not present in the external solution. A simple method is proposed to determine the inhibitor constants in competition experiments.

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Young intact plants of maize (Zea mays L. cv INRA 508) were exposed to 2 to 4 kilopascals partial pressure oxygen (hypoxic pretreatment) for 18 hours before excision of the 5 millimeter root apex and treatment with strictly anaerobic conditions (anoxia). Hypoxic acclimation gave rise to larger amounts of ATP, to larger ATP/ADP and adenylate energy charge ratios, and to higher rates of ethanol production when excised root tips were subsequently made anaerobic, compared with root tips transferred directly from aerobic to anaerobic media. Improved energy metabolism following hypoxic pretreatment was associated with increased activity of alcohol dehydrogenase (ADH), and induction of ADH-2 isozymes. Roots of Adh1(-) mutant plants lacked constitutive ADH and only slowly produced ethanol when made anaerobic. Those that were hypoxically pretreated acclimated to anoxia with induction of ADH2 and a higher energy metabolism, and a rate of ethanol production comparable to that of nonmutants. All these responses were insensitive to the presence or absence of NO(3) (-). Additionally, the rate of ethanol production was about 50 times greater than the rate of reduction of NO(3) (-) to NO(2) (-). These results indicate that nitrate reductase does not compete effectively with ADH for NADH, or contribute to energy metabolism during anaerobic respiration in this tissue through nitrate reduction. Unacclimated root tips of wild type and Adhl(-) mutants appeared not to survive more than 8 to 9 hours in strict anoxia; when hypoxically pretreated they tolerated periods under anoxia in excess of 22 hours.

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The growth of scions and rootstocks of compatible (Prunus persica L. Batsch cv. Springtime/Prunus cerasifera L. Ehrh. cv. myrobolan P2032) and incompatible (Prunus persica L. Batsch cv. Springtime/Prunus cerasifera L. Ehrh. cv. myrobolan P18) peach/plum grafts were compared. The composition of soluble carbohydrates in phloem and cortical tissues of both peach/plum grafts and ungrafted plums and the translocation of these compounds across the union of grafted plants were examined. Sorbitol and sucrose were the dominant sugars in the phloem and cortical tissues of plum. A cyanogenic glycoside, prunasin, was present in peach tissues in amounts equivalent to those of sorbitol or sucrose, whereas only small amounts of prunasin were detected in plum tissues. The concentration of prunasin was significantly higher in the phloem of the P18 rootstock of the incompatible graft. Sorbitol was the only sugar significantly depleted in rootstock tissues of the incompatible graft when the first foliar symptoms of graft incompatibility became evident. Translocation studies with 1-(14)C-deoxyglucose showed that the relative distribution of radioactivity across the graft union was similar in both compatible and incompatible grafts. However, the total amount of radioactivity translocated across the incompatible graft was less than one-third of that translocated across the compatible graft. The results are consistent with the hypothesis of a progressive poisoning of the root system in the incompatible graft by a compound synthesized in peach foliage. The role of prunasin as a possible candidate is discussed.

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After feeding the scutellum of young maize seedlings with a labeled analog of glucose, 2-deoxy-d-glucose, the progress of radioactivity along the root was followed when either 70% of the path or the whole root were in strict anoxic conditions, and was compared with the translocation pattern of aerobic seedlings. Special care was taken to suppress the internal O(2) transport and to control its occurrence.In air, the radioactive compounds accumulated from 30 minutes in the root tip mainly as an analog of sucrose. When the whole root was anoxic, the progress of the radioactivity was very slow and never reached the tip which did not survive more than 8 hours. When 70% of the path was in strict anoxia and the sink (root tip) in air, the translocation was not impaired and the radioactivity accumulated in the tips as fast as in aerobic controls. The addition of 3 millimolar NaF, which inhibits the fermentative energy production, did not modify these results. It is concluded that long distance transport in maize sieve tubes has no special energy requirements and is controlled by source-sink relationships. The inhibition of sugar supply in anoxic root tips is attributed to an effect on unloading processes rather than on sink metabolism.

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Free amino acid contents in skin extracts and influence of food and starvation on free amino acid content in skin mucus were analysed in sexually immature goldfish. Free amino acid concentration in skin mucus (91.1 mumol/g dry wt) was higher than in deep skin (54 mumol/g) or in whole skin (56.6 mumol/g) extracts. Free amino acid compositions were very similar in the latter extracts. They both differed from skin mucus extract in taurine, glutamic acid, glycine and histidine relative contents. Free amino acid composition in zooplankton used to feed goldfish was close to the composition found in corresponding skin mucus extracts, except in taurine content. Goldfish weighing 3 g (6 months old) and 17 g (1 year old) reared on zooplankton showed similar patterns of free amino acid composition in skin mucus. Comparison with free amino acid composition in skin mucus from goldfish fed on commercial food had big differences in glutamic acid, valine, methionine and lysine relative contents. During fasting, we observed an increase in the amount of mucus secreted and a concomitant decrease of the free amino acid concentration in the secretion. The origin of free amino acids found in skin mucus and their possible role in pheromonal and allelochemical communications of goldfish are discussed.

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Internal transport of O2 from the aerial tissues along the adventitious roots of intact maize plants was estimated by measuring the concentrations of adenine nucleotides in various zones along the root under an oxygen-free atmosphere. Young maize plants were grown in nutrient solution under conditions that either stimulated or prevented the formation of a lysigenous aerenchyma, and the roots (up to 210 mm long) were then exposed to an anaerobic (oxygen-free) nutrient solution. Aerenchymatous roots showed higher values than non-aerenchymatous ones for ATP content, adenylate energy charge and ATP/ADP ratios. We conclude that the lysigenous cortical gas spaces help maintain a high respiration rate in the tissues along the root, and in the apical zone, by improving internal transport of oxygen over distances of at least 210 mm. This contrasted sharply with the low energy status (poor O2 transport) in non-aerenchymatous roots.