Per- and polyfluoroalkyl substances (PFAS) contamination in a microtidal urban estuary: Sources and sinks.

This study evaluates PFAS contamination and determines the major drainage sources to a temperate microtidal estuary, the Swan Canning Estuary, in Perth Western Australia. We describe how variability in these sources influences PFAS concentrations within this urban estuary. Surface water samples were collected from 20 estuary sites and 32 catchment sites in June and December from 2016 to 2018. Modelled catchment discharge was used to estimate PFAS load over the study period. Three major catchment sources of elevated PFAS were identified with contamination likely resulting from historical AFFF use on a commercial airport and defence base. Estuary PFAS concentration and composition varied significantly with season and spatially with the two different estuary arms responding differently to winter and summer conditions. This study has found that the influence of multiple PFAS sources on an estuary depend on the historical usage timeframe, groundwater interactions and surface water discharge.

Gluconeogenesis and effect of nutritional status on TCA cycle activity in the insect Manduca sexta.

Gluconeogenesis from isotopically substituted (3-13C)alanine (Ala) was demonstrated in the last larval instar of an insect, Manduca sexta, when maintained on low carbohydrate diets. 13C was incorporated into all carbons of the blood sugar trehalose (Tre), but enrichments of C1 and C6, and C2 and C5 were greatest. Relative to the amount of [3-13C]Ala metabolized, larvae maintained on a low carbohydrate diet supplemented with casein displayed the greatest enrichment of Tre. Very little de novo synthesis of Tre was observed in larvae maintained on a complete-balanced diet containing calorically equivalent amounts of sucrose and casein. Starvation failed to induce gluconeogenesis and 13C was not incorporated into Tre in starved insects. Activity of the TCA cycle contributed approximately 10% of the 13C incorporated into Tre in larvae on low carbohydrate diets, while the TCA cycle contribution in larvae on the complete diet approached 70%. The pattern of 13C enrichment of glucose in larvae on the low carbohydrate diets indicated that cytoplasmic carboxylation, possibly due to 'malic enzyme'-like activity, contributed significantly to the synthesis of Tre. The pentose phosphate pathway was evidenced in insects on all diets. Glucose labelling ratios indicated a pentose cycling flux of 10 to 20% in insects on the low carbohydrate diets and 50% in larvae on the complete diet. Glutamine together with lesser amounts of glutamate and glutathione were also products of the labelled Ala. The distribution of label in these products under different dietary conditions demonstrated shifts in the relative contribution of pyruvate carboxylase and pyruvate dehydrogenase activities for providing substrate to the TCA cycle. In the expected fashion starved insects and insects on the low carbohydrate diets incorporated a greater proportion of 13C into the TCA cycle via carboxylation while incorporation by the two pathways was similar in insects on the complete diet. The significance of these findings with regard to the regulation of gluconeogenesis in M. sexta and comparison of the present results with those obtained from studies of hepatic gluconeogenesis are discussed.

Glucogenesis in an insect, Manduca sexta L., estimated from the 13C isotopomer distribution in trehalose synthesized from [1,3-13C2]glycerol.

Glucogenesis from [3-13C]alanine and [1,3-13C2]glycerol was demonstrated in the insect Manduca sexta by examining the 13C enrichment of trehalose, a non-reducing disaccharide of glucose synthesized in the insect fat body and released into the blood or hemolymph. In insects maintained on a low carbohydrate diet, trehalose synthesized from [3-13C]alanine was selectively enriched at C1 and C6, and C2 and C5. The 13C-labelling pattern indicated the carboxylation of [3-13C]pyruvate, formed by transamination of the [3-13C]alanine followed by randomization of the label at the fumarate step of the tricarboxylic acid cycle and glucose synthesis via the gluconeogenic pathway. 13C enrichment of trehalose was absent in similarly maintained insect larvae administered 3-mercaptopicolinic acid, an inhibitor of hepatic phosphoenolpyruvate carboxykinase. Insects on the low carbohydrate diet also synthesized trehalose from [1,3-13C2]glycerol. 13C multiplets were observed in trehalose C3 and C4 demonstrating the synthesis of three 13C enriched glucose isotopomers from the 13C-labelled glycerol. The relative contributions of 13C-labelled glycerol and unlabelled 3 carbon substrates to the synthesis of the 13C enriched trehalose isotopomers were determined from the multiplet structure at C3, and calculation of minimal rates of glucogenesis were based on the 13C enrichment of C4. The C4/C3 13C enrichment ratio in trehalose synthesized from [1,3-13C2]glycerol was close to unity, and total glucogenesis was calculated after estimation of the expected contribution of unlabelled trehalose synthesis from 3 carbon substrates by comparison of the ratio of unlabelled and labelled contributions to the 13C enriched trehalose isotopomers with the 13C enrichment of [1,3-13C2]glycerol-3-phosphate. The estimated total rates of glucogenesis varied from 0.33 to 2.80 micromol glucose/g fresh weight/h. The blood sugar level of M. sexta was also highly variable. Although the potential importance of glucogenesis from 3 carbon substrates to the maintenance of blood sugar was not established by the present investigation, insects maintained on the low carbohydrate diet had similar blood trehalose levels to those previously reported by others for insects maintained on a natural food.

Dietary fat mediates hyperglycemia and the glucogenic response to increased protein consumption in an insect, Manduca sexta L.

Many insects display non-homeostatic regulation over blood sugar level. The concentration of trehalose varies dramatically depending on physiological and nutritional state. In the absence of dietary carbohydrate, blood trehalose in larvae of the lepidopteran insect Manduca sexta is maintained by gluconeogenesis and is dependent on dietary protein consumption. In the present study, the effect of dietary fat on the glucogenic response of insects to increased dietary protein was examined by NMR analysis of (2-13C)pyruvate metabolism. Last instar larvae were maintained on a carbohydrate-free chemically defined artificial diet having variable levels of casein with and without corn oil. Gluconeogenic flux, the ratio of the rate of gluconeogenesis to the rate of glycolysis, was estimated from the 13C distribution in trehalose arising by gluconeogenesis and the 13C enrichment of alanine due to pyruvate cycling. Insects grew well on carbohydrate-free diets and growth increased with increasing dietary protein level. At all dietary protein levels, larvae grew better on diets with fat. Without dietary fat, larvae were glucogenic but displayed low blood trehalose concentrations, <30 mM, regardless of protein consumption. When fat was included in the diet, however, gluconeogenic flux and blood trehalose level increased sharply in response to increased dietary protein level, with trehalose concentrations >50 mM at higher levels of protein consumption. When offered a choice of a high carbohydrate and a high protein diet, larvae maintained on diets with fat displayed a food preference related to blood sugar level. Those with low blood sugar fed on carbohydrate, while those with high blood sugar preferred protein. Trehalose synthesized from (2-13C)pyruvate exhibited asymmetry in the 13C distribution in individual glucose molecules, indicating a disequilibrium at the triose phosphate isomerase-catalyzed step of the gluconeogenic pathway. In trehalose from larvae on diets with fat, the asymmetric 13C distribution was higher than in trehalose from insects on diets lacking fat. This may partially result from isotopic disequilibrium when unenriched glycerol is metabolized to dihydroxyacetone phosphate following fat hydrolysis. The asymmetry in 13C distribution, however, also occurred in insects on diets without fat and decreased with increased gluconeogenic flux suggesting that true disequilibrium between the triose phosphates is the principal reason for the asymmetry.

Blood sugar formation from dietary carbohydrate is facilitated by the pentose phosphate pathway in an insect Manduca sexta Linnaeus.

Dietary carbohydrate, the principal energy source for insects, also determines the level of the blood sugar trehalose. This disaccharide, a byproduct of glycolysis, occurs at highly variable concentrations that play a key role in regulating feeding behavior and growth. Little is known of how developing insects partition the metabolism of dietary carbohydrate to meet the needs for blood trehalose, ribose sugars and NADPH, as well as energy production. This study examined the effects of varying dietary sucrose levels between 3.4 and 34 g/l in an artificial diet on growth rate, depot fat content and blood sugar formation from (13)C-enriched glucose in Manduca sexta. (2-(13)C)Glucose or (1,2-(13)C(2))glucose were administered to larvae by injection and after 6 h blood was analyzed by nuclear magnetic resonance spectroscopy. [2-(13)C]Trehalose was the principal product of [2-(13)C]glucose, but trehalose was also (13)C-enriched at C1 and C3, demonstrating activity of the pentose phosphate pathway. The trehalose C1/C2 (13)C-enrichment ratio, a measure of the substrate cycled through the pentose pathway, significantly increased with increasing dietary sugar, and reached a mean of 0.22 at the highest level. Blood trehalose concentration increased from approximately 38 mM at the lowest dietary carbohydrate level to 75 mM at the highest. Moreover, blood trehalose, growth rate and depot fat all increased in precisely the same way in relation to the level of pentose cycling. Based on the multiplet (13)C-NMR signal structure of trehalose synthesized from [1,2-(13)C(2)]glucose by insects maintained on a high carbohydrate diet, it was established that the formation of trehalose from glucose phosphate derived directly from the administered substrate, with no involvement of the pentose pathway, was greater than that from glucose phosphate metabolized through the pentose pathway prior to trehalose synthesis. On the other hand, glucose phosphate first metabolized through the pentose pathway contributed more to pyruvate formation than did glucose phosphate formed from the labeled substrate metabolized directly to pyruvate via glycolysis; this finding based on the multiplet (13)C-NMR signal structure in alanine derived from pyruvate. The results suggest that as dietary carbohydrate increases blood sugar synthesis from glucose phosphate derived directly from dietary sugar is facilitated by the pentose pathway which provides an increasing amount of substrate to pyruvate formation.

Interactions of dietary protein and carbohydrate determine blood sugar level and regulate nutrient selection in the insect Manduca sexta L.

The non-homeostatic regulation of blood sugar concentration in the insect Manduca sexta L. was affected by nutritional status. Larvae maintained on diets lacking sucrose displayed low concentrations of trehalose, the blood sugar of insects, which varied from 5 to 15 mM with increasing dietary casein level between 12.5 and 75 g/l. These insects were glucogenic, as demonstrated by the selective 13C enrichment of trehalose synthesized from [3-13C]alanine, and de novo synthesis was the sole source of blood sugar. The distribution of 13C in glutamine established that following transamination of the 13C substituted substrate, [3-13C]pyruvate carboxylation rather than decarboxylation was the principal pathway of Pyr metabolism. The mean blood trehalose level was higher in insects maintained on diets with sucrose. At the lowest dietary casein level blood trehalose was approximately 50 mM, and declined to 20 mM at the highest casein level. Gluconeogenesis was detected in insects maintained on sucrose-free diets at the higher protein levels examined, but [3-13C]pyruvate decarboxylation and TCA cycle metabolism was the principal fate of [3-13C]alanine following transamination, and dietary carbohydrate was the principal source of glucose for trehalose synthesis. Feeding studies established a relationship between nutritional status, blood sugar level and dietary self-selection. Insects preconditioned by feeding on diets without sucrose had low blood sugar levels regardless of dietary casein level, and when subsequently given a choice between a sucrose diet or a casein diet, selected the former. Larvae preconditioned on a diet containing sucrose and the lowest level of casein had high blood sugar levels and subsequently selected the casein diet. Larvae maintained on the sucrose diet with the highest casein level had low blood sugar and self-selected the sucrose diet. When preconditioned on diets with sucrose and intermediate levels of casein, insects selected more equally between the sucrose and the casein diets. It is concluded that blood sugar level may be intimately involved in dietary self-selection by M. sexta larvae, and that in the absence of dietary carbohydrate, gluconeogenesis provides sufficient blood sugar to ensure that larvae choose a diet or diets that produce an optimal intake of dietary protein and carbohydrate.

Glucose metabolism in an insect Manduca sexta and effects of parasitism.

The metabolism of [1-13C]glucose was examined during the last larval stadium of an insect Manduca sexta (Lepidoptera: Sphingidae) parasitized by Cotesia congregata (Hymenoptera: Braconidae). Following injection, the isotopically substituted glucose was metabolized at a significantly lower rate by parasitized larvae than by normal, control insects. 13C enrichment was principally observed in [1-13C]trehalose in both groups. Randomization of the label at the triose phosphate step was evidenced by incorporation of 13C into C6 of trehalose. Parasitized and control larvae both synthesized [1,6-13C]glycogen but the relative amount of label observed in parasitized larvae was greater. The ratio of C6/C1 enrichment in trehalose and glycogen was significantly less in parasitized larvae. The rate of labelled trehalose and glycogen synthesis was relatively high when compared with the estimated rate of glycolytic glucose oxidation, and the difference in C6/C1 enrichment ratio between normal and parasitized insects was, therefore, not reflective of a difference in the rate of substrate cycling, but rather, was due to the increased synthesis of [1-13C]glycogen in parasitized larvae when compared with controls. Inhibition of glycolysis by administration of iodoacetate to normal larvae resulted in an increase in the incorporation of 13C into glycogen relative to glucose metabolized, suggesting that inhibition of glycolysis may be responsible for the higher level of glycogen synthesis observed in parasitized insects. In control larvae, significant 13C enrichment from [1-13C]glucose was observed in fat, but no evidence of lipogenesis was observed in parasitized insects. Iodoacetate had no observable effects on the relative amount of 13C incorporated into fat. Malonic acid and cyanide resulted in accumulation of 13C from [1-13C]glucose in several TCA cycle intermediates of normal larvae, but had little effect on the relative enrichments of trehalose, glycogen and fat.

Metabolic fate of alanine in an insect Manduca sexta: effects of starvation and parasitism.

The fate of [3-13C]alanine administered to last instar larvae of an insect Manduca sexta was investigated in vivo by 13C-NMR spectroscopy. Following injection of the isotopically substituted substrate and conversion to [3-13C]pyruvate 13C was principally incorporated into C2, C3 and C4 of glutamate and glutamine in unparasitized ad libitum-fed larvae, insects starved 48 hr prior to injection and larvae parasitized by the insect parasite Cotesia congregata. Selective labeling at C2 and C3 of glutamate/glutamine resulted from carboxylation of [3-13C]pyruvate to [2,3-13C]oxaloacetate catalyzed by pyruvate carboxylase, randomization of the label in fumarate, and synthesis of glutamate and glutamine after condensation with acetyl CoA to [2 proR,3-13C]citrate. In contrast, enrichment at C4 of glutamate and glutamine resulted from oxidation [3-13C]pyruvate to [2-13C]acetyl CoA catalyzed by pyruvate dehydrogenase followed by condensation with oxaloacetate. The ratio of enrichment (C2 + C3): C4 provided a measure of the relative contributions of the pyruvate dehydrogenase and pyruvate carboxylase catalyzed pathways of substrate utilization by the tricarboxylic acid cycle. The mean ratio was 0.6 and 0.7 in control and parasitized larvae, respectively, and 2.4 in starved insects. The latter result demonstrated that substrate utilization by the TCA cycle was markedly altered by starvation. In addition, the rate of labeled alanine metabolism was significantly reduced by starvation. The concentrations of glutamate and glutamine in the blood (hemolymph) were similar in all three groups of insects. No evidence for gluconeogenesis was observed in any group. Starved larvae incorporated label into C6 of glucose and trehalose but no complementary enrichment at C1 was observed. This result was consistent with the activity of the non-oxidative phase of the pentose phosphate pathway during which labeled glyceraldehyde-3-phosphate arising from [3-13C]alanine reacts with sedoheptulose-7-phosphate yielding erythrose-4-phosphate and [6-13C]fructose-6-phosphate catalyzed by transaldolase. Specifically labeled fructose-6-phosphate then gives rise to glucose and trehalose labeled at C6. Preliminary analysis of the hemolymph of starved insects indicated the presence of several hexose phosphates labeled at C6. The hemolymph level of trehalose was significantly reduced in both starved and parasitized insects. Lipogenesis from [3-13C]alanine was evident in unparasitized control larvae but was absent in parasitized and starved insects. The pattern of labeling in fatty acid was consistent with de novo pathway utilizing [2-13C]acetyl CoA derived by oxidation of [3-13C]alanine.

Blood sugar formation due to abnormally elevated gluconeogenesis: aberrant regulation in a parasitized insect, Manduca sexta Linnaeus.

Alterations of carbohydrate metabolism associated with parasitism were examined in an insect, Manduca sexta L. In insect larvae maintained on a low carbohydrate diet gluconeogenesis from [3-13C]alanine was established from the fractional 13C enrichment in trehalose, a disaccharide of glucose and the blood sugar of insects and other invertebrates. After transamination of the isotopically substituted substrate to [3-13C]pyruvate, the latter was carboxylated to oxaloacetate ultimately leading to de novo glucose synthesis and trehalose formation. Trehalose was selectively enriched with 13C at C1 and C6 followed by C2 and C5. 13C enrichment of blood sugar in insects parasitized by Cotesia congregata (Say) was significantly greater than was observed in normal animals. The relative contributions of pyruvate carboxylation and decarboxylation to trehalose labeling were determined from the 13C distribution in glutamine, synthesized as a byproduct of the tricarboxylic acid cycle. The relative contribution of carboxylation was significantly greater in parasitized larvae than in normal insects providing additional evidence of elevated gluconeogenesis due to parasitism. Despite the increased gluconeogenesis in parasitized insects the level of blood sugar was the same in all animals. Because de novo glucose synthesis does not normally maintain blood sugar level in insects maintained under these dietary conditions the findings suggest an aberrant regulation over gluconeogenesis. The 13C labeling in trehalose was nearly symmetric in all insects but the mean C1/C6 13C ratio was higher in parasitized animals suggesting a lower activity of the pentose phosphate pathway that brings about a redistribution of 13C in trehalose following de novo glucose synthesis. Additional studies with insects maintained on a high carbohydrate diet and administered [1,2-13C2]glucose confirmed a decreased level of pentose cycling during parasitism consistent with a lower level of lipogenesis. It is suggested, however, that the pentose pathway may facilitate the synthesis of trehalose from dietary carbohydrate by directing hexose phosphate cycled through the pathway to the production of energy.

The glucogenic response of a parasitized insect Manduca sexta L. is partially mediated by differential nutrient intake.

Induction of gluconeogenesis is accelerated in larvae of the insect Manduca sexta L. parasitized by Cotesia congregata (Say), maintaining the concentration of the blood sugar trehalose, an important nutrient for parasite development. Investigation has demonstrated that when host larvae are offered a choice of diets with varying levels of sucrose and casein, parasitized insects consume a different balance of these nutrients, principally due to a decrease in protein consumption. The result is metabolic homeostasis, with normal unparasitized and parasitized larvae exhibiting similar levels of gluconeogenesis and blood sugar level. In the present study, normal unparasitized and parasitized larvae were maintained on individual chemically defined diets having the balance of protein and carbohydrate consumed by each when offered a dietary choice. Total dietary nutrient, the sum of carbohydrate and protein, was provided at six levels, composed of three pairs of diets. Each diet pair consisting of diets having equivalent overall nutrient ratios of 2:1 and 1:1 casein/sucrose. Host growth and diet consumption were significantly affected by dietary nutrient level and the magnitude of these effects was influenced by parasitism. Due to the effects of dietary nutrient level on diet consumption, none of the unparasitized and parasitized larvae within any of the three diet pairs consumed protein and carbohydrate at the levels predicted by the earlier choice experiments. Among insects on all of the diets, however, two groups of unparasitized and parasitized larvae consumed the expected levels of protein and carbohydrate. In each case, gluconeogenesis, as measured by 13C nuclear magnetic resonance spectroscopy (NMR) analysis of pyruvate cycling and trehalose synthesis from [2-13C]pyruvate, was evident in unparasitized and parasitized insects, confirming the conclusions of the earlier experiments. Generally, all larvae that consumed less than approximately 250 mg of sucrose over the 3-day feeding period, were gluconeogenic, regardless of diet. Differential carbohydrate consumption, therefore, was an important factor in inducing gluconeogenesis in both unparasitized and parasitized insects. The selective 13C enrichment in trehalose displayed by non-gluconeogenic larvae on some diets demonstrated trehalose formation from [2]pyruvate. The absence of net carbohydrate synthesis in these insects was likely due to an elevation of glycolysis. There was no significant effect of diet consumption or parasitism on blood trehalose level. Parasitized larvae displayed higher levels of gluconeogenesis than did unparasitized insects, a finding consistent with the conclusion that blood sugar is rapidly sequestered by developing parasites. The parasite burden, the total number of parasites developing within host larvae, as well as the number of parasites emerging from host larvae to complete development, was significantly less at the lowest dietary nutrient level, but was otherwise similar at all dietary nutrient levels. Moreover, the number of parasites that emerged increased with increasing diet consumption as reflected by host final weight.

Parasitism enhances the induction of glucogenesis by the insect, Manduca sexta L.

Metabolic alterations that accompany parasitism of invertebrate animals can play an important role in parasite development. Employing 13C NMR, this study examined pyruvate cycling from (2-(13)C)pyruvate in the lepidopteran insect Manduca sexta, and the effects of parasitism by the hymenopteran Cotesia congregata on the gluconeogenic formation of trehalose, the haemolymph or blood sugar of insects. Larvae were maintained on a semi-synthetic sucrose-free diet, or on the same diet with sucrose at 8.5 g/l. Pyruvate cycling was evident from the 13C enrichment in C3 of alanine, derived following carboxylation to oxaloacetate, and was similar in parasitized and normal insects regardless of diet. Trehalose was formed following de novo synthesis of glucose, and net synthesis was estimated from the 13C distribution in trehalose and alanine. The 13C-enrichment ratio [2trehalose C6/alanine C3] is an indicator of the level of gluconeogenesis relative to glycolysis, both enrichments were derived from (2-(13)C)pyruvate in the same manner. The ratio was greater than unity in all insects, regardless of diet, but was significantly greater in parasitized larvae, demonstrating an enhanced level of gluconeogenesis. This was confirmed by analysis of the 13C distribution in trehalose and glutamine derived from (3-(13)C)alanine. Despite enhanced de novo trehalose formation in parasitized insects, the haemolymph sugar level was similar to that of normal larvae. Because haemolymph trehalose regulates dietary carbohydrate intake, but not gluconeogenesis, the results suggest that accelerated induction of gluconeogenesis is an adaptive response to parasitism that provides increased carbohydrate for parasite growth and simultaneously maintains nutrient intake.

The adenylate nucleotide pool in the digestive gland-gonad complex of Biomphalaria glabrata infected by Schistosoma mansoni.

The levels of adenylate nucleotides were examined in the digestive gland and ovotestes of Biomphalaria glabrata during cercarial shedding of Schistosoma mansoni, 10 weeks post-infection. In general, parasitization resulted in decreases in the adenylate levels in both tissues, but the results were not statistically significant. Moreover, the energy charge ratio was not significantly altered. The mean energy charge in ovotestes from uninfected and infected individuals was 0.81 and 0.77, respectively, and that in the digestive gland, 0.70 and 0.66, respectively. Extensive variation was observed in energy charge of digestive gland from infected individuals and this was attributed to possible differences in the degree of infection.

In vivo 31P NMR spectrum of Hymenolepis diminuta and its change on short-term exposure to mebendazole.

The 31P NMR spectrum of the adult tapeworm, Hymenolepis diminuta, at 37 degrees C during perfusion with physiological saline was composed of 10 peaks. Based on chemical shifts and analysis of worm extracts, the phosphorus components included glucose-6-phosphate, fructose-6-phosphate, phosphorylcholine, glycerophosphoryl choline and -ethanolamine, nucleotide monophosphate-diphosphate and -triphosphate, nicotinamide adenine dinucleotide and uridine diphosphate glucose. The mean level of nucleotide triphosphate was 0.86 nmol (mg fresh weight)-1 and the nucleotide triphosphate/-diphosphate ratio 3.9. Based on the nucleotide triphosphate level, worms were viable for at least 3 h and the intracellular pH was maintained constant at approximately 6.7. Short-term exposure to mebendazole perfused at 11 or 27 microM solubilized in physiological saline containing 0.5% Tween 80 or 0.1% dimethyl sulphoxide had little effect on the nucleotide triphosphate level. Some cytological changes, however, were evident following perfusion of mebendazole. In contrast, exposure to 2,4-dinitrophenol caused a rapid decline in nucleotide triphosphate level. It was concluded that mebendazole does not exert its primary effect on oxidative phosphorylation.

In vivo gluconeogenesis under different nutritional regimens during murine schistosomiasis.

The effect of schistosome infection on in vivo gluconeogenesis was studied in mice. Mice infected for 6 weeks displayed no nutritional pathology, regardless of nutritional treatment. All mice with patent infections (9 weeks) displayed decreased rates of growth and food consumption as well as significantly reduced gross conversion efficiencies. The glucogenic capacity, % pyruvate incorporated/g liver, was markedly reduced in infected fasted mice when compared with fasted controls. Total glucogenesis, % pyruvate incorporated/100 g body water, was the same in infected and control groups and the compensatory response in infected individuals was correlated with increased liver mass. The level of hepatic glycogen was significantly greater in infected fasted mice than in their unparasitized counterparts.

Glucogenic blood sugar formation in an insect Manduca sexta L.: asymmetric synthesis of trehalose from 13C enriched pyruvate.

Gluconeogenesis and blood sugar formation were examined in Manduca sexta, fed carbohydrate- and fat-free diets with varying levels of casein. De novo carbohydrate synthesis was examined by nuclear magnetic resonance spectroscopy of the 13C enrichment in blood trehalose and alanine derived from (2-(13)C)pyruvate and (2,3-(13)C(2))pyruvate administered to 5th instar larvae. Gluconeogenic flux and blood trehalose concentration were positively correlated with protein consumption. On all diets, the 13C distribution in trehalose was asymmetric, with C6 more highly enriched than C1. The C6/C1 13C enrichment ratio, however, decreased with increased protein consumption and gluconeogenic flux. Although the asymmetric 13C enrichment pattern in trehalose is consistent with pentose cycling via the pentose phosphate pathway following de novo synthesis, experiments employing [2,3-(13)C(2)]pyruvate demonstrated that pentose cycling is not detected in insects under these nutritional conditions. Analysis of the multiplet NMR signal structure in trehalose due to spin-spin coupling between adjacent 13C enriched carbons showed the absence of uncoupling expected by pentose phosphate pathway activity. Here we suggest that the asymmetric 13C distribution in trehalose results from a disequilibrium of the triose phosphate isomerase-catalyzed reaction.

Applications of nuclear magnetic resonance in parasitology.

The basis of the nuclear magnetic resonance (NMR) phenomenon is described in a classical framework with emphasis on magnetic nuclei of 1/2 spin, including 1H, 13C, and 31P. Biological applications of NMR spectroscopy and magnetic resonance imaging (MRI) are outlined briefly. NMR spectroscopic studies on parasitic protozoa, cestodes, nematodes, trematodes, and hymenopterous insect parasites are reviewed. NMR and MRI investigations on the pathophysiology of the host are also discussed, and the potential future of NMR applications in parasitology outlined.

Effects of Schistosoma mansoni on the nutrition of its intermediate host, Biomphalaria glabrata.

Biomphalaria glabrata infected with Schistosoma mansoni and maintained on an artificial diet of Spirulina alga displayed reduced growth during the 5 wk following patency. Food consumption per unit snail weight was unaffected. Infected snails also failed to lay eggs. No difference in percentage of assimilation was observed between control and infected individuals, but infected snails had significantly decreased gross conversion efficiencies. The effects of infection on nutrition of B. glabrata were similar to those observed in nutrient-deprived snails fed diets containing low Spirulina levels. Nutrient deprivation, however, did not alter conversion efficiency.

Characterization of the 31P NMR spectrum of the schistosome vector Biomphalaria glabrata and of the changes following infection by Schistosoma mansoni.

The 31P nuclear magnetic resonance (NMR) spectrum of the digestive gland-gonad complex (DGG) of the schistosome vector Biomphalaria glabrata was characterized and the effects of infection by Schistosoma mansoni noted. The in vivo spectrum was comprised of 11 peaks, 5 downfield and 6 upfield of an external 85% phosphoric acid standard. Based on a variety of analytical procedures, the upfield peaks from the standard were demonstrated to be composed of carbamoyl phosphate + a mixture of 3 phosphatides and sphingomyelin, the gamma + beta phosphorus resonances of nucleotide triphosphate (NTP) and nucleotide diphosphate (NDP), respectively, the alpha phosphorus resonances of NTP + NDP, NAD(H) + the phosphorus resonance of uridine phosphate from uridine diphosphoglucose (UDPG), the phosphorus resonance of glucose phosphate from UDPG and, last, the beta phosphorus resonance of NTP. The downfield peaks were assigned as glycerophosphoryl choline, intracellular inorganic phosphate (Pi), sugar phosphates + phosphoryl choline, aminoethyl phosphonate (AEP), and ceramide AEP. T1 values for the in vivo NMR components were determined by inversion recovery. Infection produced distinct alterations in the levels of nonnucleotide components of the in vivo 31P NMR spectrum and the spectra of tissue extracts. Specifically, the levels of phosphonate, phospholipids, and carbamoyl phosphate were markedly reduced, and the relative level of Pi was increased. The potential significance of these changes to the parasite-host relationship was discussed. In contrast, starvation resulted in a decreased level of phosphonate only. The pH of the intact DGG was estimated by titrating the inorganic phosphate component of tissue extracts. The mean pH was 6.9 for both control and infected material.

31P NMR studies on adenylates and other phosphorus metabolites in the schistosome vector Biomphalaria glabrata.

31P nuclear magnetic resonance spectroscopy was applied successfully to whole intact and de-shelled snails and to the isolated digestive gland-gonad tissue complex as well as other tissues of Biomphalaria glabrata. Several phosphorus metabolites, including ATP and ADP, were observed. The mean ATP/ADP ratio calculated for the tissue complex was 3.1 and the ATP concentration was 0.73 nmoles/mg tissue fresh weight. Assignments for AMP, sugar phosphates, and inorganic phosphate peaks were tentatively made. A major phosphorus component was identified as a phosphonate and this metabolite was also present in egg masses and the albumin gland. Phosphoarginine was not observed in the tissue complex but was present in whole animals. Infection by Schistosoma mansoni resulted in marked alteration in the relative levels of phosphorus metabolites in the digestive gland-gonad complex during the course of infection. The decrease in phosphonate was particularly notable. The relative level of a metabolite occurring at -1.1 ppm was also decreased but its identity remained unknown. The ATP/ADP ratio was not affected by infection, but an increase in the relative level of inorganic phosphate suggested a possible decrease in phosphorylation potential.

Phosphoglycerides and derivatives in Taenia crassiceps examined by 31P NMR spectroscopy.

Examination of the larval stage of the tapeworm, Taenia crassiceps, by 31P NMR spectroscopy revealed the presence of a major phosphoglyceride component. However, using saturation transfer, no exchange between glycerophosphorylcholine and phosphoglyceride or any other NMR-detectable phosphorus metabolites was detected.