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.

Parasitism of an insect Manduca sexta L. alters feeding behaviour and nutrient utilization to influence developmental success of a parasitoid.

The effects of macronutrient balance on nutrient intake and utilization were examined in Manduca sexta larvae parasitized by Cotesia congregata. Insects fed an artificial diet having constant total macronutrient, but with varied ratios of protein and carbohydrate, with altered diet consumption in response to excesses and deficiencies of the individual macronutrients. Bivariate plots of protein and carbohydrate consumption for non-parasitized larvae demonstrated a curvilinear relationship between points of nutrient intake for the various diets, and the larvae grew best on carbohydrate-biased diets. The relationship was linear for parasitized larvae with the growth uniform across diets. On protein-biased diets, the larvae regulated the nitrogen content, containing similar amounts of nitrogen regardless of consumption. Efficiency of nitrogen conversion in non-parasitized larvae was greatest on carbohydrate-biased diets, while nitrogen conversion by parasitized larvae was greatest with intermediate nutrient ratios. Accounting for carbohydrate consumption, the lipid content decreased as dietary carbohydrate increased, but parasitized larvae contained significantly less lipid. The total biomass of parasites developing in individual host larvae was positively correlated with host protein consumption, but the individual parasites were similar in size. Parasitism influences host nutrient consumption in a manner that achieves uniform host growth under diverse nutritional regimes, thereby constraining blood nutrient concentrations within limits suitable for parasite growth and development.

Nicotine moderates the effects of macronutrient balance on nutrient intake by parasitized Manduca sexta L.

Effects of dietary nicotine and macronutrient ratio on M. sexta larvae were examined. Larvae were fed a carbohydrate-biased, protein-biased or diet having equal amounts of casein and sucrose, with and without nicotine. Without nicotine, larvae displayed compensatory feeding on the low protein diet, but despite consuming more, grew least on this diet. Nicotine at 0.5% had no effect on nutrient consumption. Nicotine at 1.0 and 2.0% reduced overall consumption and thereby also reduced nicotine consumption. Larvae parasitized by C. congregata displayed reduced nutrient intake and growth on all diets. Parasitized larvae responded to 1% nicotine similarly to unparasitized larvae. At 0.5% nicotine, they displayed reduced consumption on all diets, possibly due to altered chemoreceptor sensitivity to nicotine. When offered a choice of two diets having different macronutrient ratios, one with and the other without 0.1% nicotine, all larvae preferred the diet lacking nicotine and failed to regulate nutrient intake such that the nutrient intake target, a ratio of nutrients supporting optimal growth, was achieved. Parasitized larvae consumed less nicotine on a fresh weight basis than unparasitized insects, suggesting that the feeding response of parasitized larvae to nicotine minimizes the exposure of nicotine to developing parasites.

Feeding behaviour and nutrient selection in an insect Manduca sexta L. and alterations induced by parasitism.

Manduca sexta L. larvae exhibit broad food acceptance with regard to nutrient content during the first 3 days of the last stadium. Larvae fed diets with a constant combined level of casein and sucrose, but variable ratios, display a linear relationship between protein and carbohydrate intake. Larvae grow best on a diet with equal nutrients, but will consume an excess of one nutrient in order to obtain an adequate amount of the other, as nutrient ratio shifts. Parasitized larvae feed similarly, but the nutrient ratio does not affect growth. Unparasitized larvae regulate intake of protein and carbohydrate when offered choices of protein-biased and carbohydrate-biased diets having combined nutrient levels of 120 g/l, but with variable ratios. Larvae normally consume equal amounts of nutrients, regardless of ratio, and grow similarly. As combined nutrient level is reduced in one diet, larvae abandon regulation and feed randomly. Parasitized larvae offered choice diets with 120 g/l combined nutrients do not regulate nutrient intake. Consumption of nutrients varies widely, but growth is unaffected. Larvae offered choices of diets having equal amounts of casein and sucrose but variable fat (corn oil), fail to regulate fat intake, although both unparasitized and parasitized larvae prefer a diet containing higher fat.

Host nutrition determines blood nutrient composition and mediates parasite developmental success: Manduca sexta L. parasitized by Cotesia congregata (Say).

This investigation examined the influence of dietary protein and carbohydrate balance in a chemically defined artificial diet for Manduca sexta larvae on development of the gregarious parasite Cotesia congregata. Normal unparasitized larvae and larvae superparasitized in the fourth stadium were reared to the end of the fifth stadium on six diets, each having the same total amount of casein and sucrose but with different ratios ranging from high protein/no carbohydrate through to low protein/high carbohydrate. Levels of blood protein nitrogen and trehalose, nutrients supporting growth and development of C. congregata, varied with diet and were influenced by parasitism. Different levels of blood metabolites reflected differences in diet consumption, and the relationships between protein nitrogen and trehalose were very similar to those for protein and carbohydrate intake by parasitized and normal larvae on various diets. Dietary nutrient ratio had a significant effect on parasite burden, the numbers of parasites developing in individual host larvae and on parasite biomass. Parasites included individuals that developed and eventually emerged as second instar larvae, moulted to third instars and pupated. Many apparently mature second instar parasites, however, failed to emerge. The proportion of non-emerging individuals varied with diet, and in some cases, parasites failing to emerge were greater in number and total biomass than those that did emerge to complete development. On most diets, the mass of individual parasites was similar regardless of dietary nutrient ratio. Three dimensional models developed to demonstrate the relationships between blood protein nitrogen and trehalose levels and parasite burden and biomass established that the levels of both metabolites are important for supporting growth and development of emerged and non-emerged parasites. In the case of emerged parasites, however, the relationships are linear, and a quadratic function best describes the relationships with non-emerged parasites. Blood metabolite levels supporting the greatest parasite burden and biomass of emerged and non-emerged parasites occupy a region of two dimensional space corresponding to approximately 60-200 mg per insect of protein nitrogen and 60-100 mg per insect of trehalose. Despite the differences in the response of emerged and non-emerged parasites to host nutrition, the present results indicate that host nutrition is not the critical factor determining parasite emergence. The significance of these findings to the biology of C. congregata is discussed.

Nutrition interacts with parasitism to influence growth and physiology of the insect Manduca sexta L.

The influence and interaction of dietary protein:carbohydrate balance and parasitism by Cotesia congregata on nutrient intake and growth were examined over the last two larval stadia of Manduca sexta. Effects of nutritional status on host blood metabolite concentrations were also determined. Six fat-free chemically defined diets were tested, each having the same total level of casein and sucrose, but with casein to sucrose ratios varying from low protein/high carbohydrate to equal levels of both nutrients through to high protein/no carbohydrate. Nutrient ratio and parasitism each affected nutrient consumption and growth. Feeding responses differed between normal and parasitized larvae, as illustrated by nutrient arrays, two-dimensional plots of protein and carbohydrate consumption on diets having different nutrient ratios. Normal larvae consumed more nutrients and took longer to develop as dietary nutrient ratio was displaced from equal levels of both nutrients. Except on the diet having the same amount of protein and carbohydrate, parasitized larvae consumed less nutrients than normal larvae, although on all diets parasitized larvae took longer to develop. When the contribution of parasite biomass was excluded, parasitized larvae showed lower mass gain than normal larvae on all diets. Total mass gain by normal and parasitized larvae with parasite biomass included, however, was similar on diets having intermediate nutrient ratios. Differences in mass gain between diets relative to nutrient consumption were evident from multi-dimensional representations of mass gain with protein and carbohydrate consumption. Three-dimensional plots and contour maps of normal and parasitized larvae were different. When differences in nutrient consumption between diets were taken into account, protein consumption had a greater effect on growth than carbohydrate consumption and normal larvae generally displayed greater mass gain than parasitized larvae on the same diets. Utilization efficiency, the efficiency of conversion of ingested food to body mass, was, therefore, generally reduced in parasitized insects. Concentrations of blood protein, total free amino acids and trehalose were each influenced by dietary nutrient ratio and parasitism. Concentrations of protein and free amino acids generally increased and trehalose concentration decreased as dietary protein increased and carbohydrate decreased. The opposite was the case as dietary carbohydrate increased and protein decreased. Dietary nutrient ratio, however, affected normal and parasitized larvae differently. Parasitized larvae had higher overall trehalose concentrations while normal larvae had higher protein and total free amino acid concentrations. When differences in nutrient consumption between diets were accounted for, protein consumption had a greater effect on blood protein and free amino acid concentrations than did dietary nutrient ratio or parasitism. Protein consumption, however, did not affect trehalose concentration. Carbohydrate consumption had no effect on the concentration of any of the metabolites after differences in nutrient consumption were taken into account. Effects of nutrient consumption on trehalose concentration, therefore, were due to dietary nutrient ratio and parasitism. The potential relevance of the above findings to the biology of parasitized M. sexta larvae is discussed.

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.

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.

Dietary nutrient levels regulate protein and carbohydrate intake, gluconeogenic/glycolytic flux and blood trehalose level in the insect Manduca sexta L.

This study examined the effects of dietary casein and sucrose levels on nutrient intake, and distinguished the effects of carbohydrate and protein consumption on growth, fat content, pyruvate metabolism and blood trehalose level of 5th instar Manduca sexta larvae. Growth increased with increasing casein consumption but was unaffected by carbohydrate intake. Fat content also increased with carbohydrate consumption, but on carbohydrate-free diets fat content increased with increased protein consumption. Blood trehalose level and pyruvate metabolism were examined by nuclear magnetic resonance spectroscopy analysis of blood following administration of (3-(13)C)pyruvate. On diets containing sucrose, blood trehalose increased with increasing carbohydrate intake, and on most diets trehalose was synthesized entirely from dietary sucrose. Pyruvate cycling, indicated by the alanine C2/C3 (13)C enrichment ratio, increased with carbohydrate consumption reflecting increased glycolysis, and pyruvate decarboxylation exceeded carboxylation on all sucrose diets. Larvae that consumed <75 mg/day sucrose were gluconeogenic, based on the [2 (trehalose C6)(Glx C3/C2)]/alanine C2] (13)C enrichment ratio. On carbohydrate-free diets, blood trehalose levels were low and maintained entirely by gluconeogenesis. Blood trehalose level increased with increasing protein intake. Pyruvate cycling was very low, although many insects displayed higher levels of pyruvate decarboxylation than carboxylation. All gluconeogenic larvae displayed alanine (13)C enrichment ratios <0.35 and had blood trehalose levels <50 mM.

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.

Altered dietary nutrient intake maintains metabolic homeostasis in parasitized larvae of the insect Manduca sexta L.

Manduca sexta larvae exhibited altered food selection over a 2- or 3-day feeding period when parasitized by Cotesia congregata, and offered a choice of two chemically defined diets, one containing casein without sucrose and a second with sucrose but no casein. While normal larvae consumed the diets in a ratio of approximately 2:1 protein:carbohydrate (w/w), parasitized insects consumed a ratio of approximately 1:1. The altered nutrient ratio consumed by parasitized insects was principally due to a decrease in consumption of the protein diet, and was only partially explained by their lower growth. Conditioning larvae for 1 day to either one of the choice diets had little effect on subsequent dietary intake over a 2-day feeding period. Conditioned larvae, regardless of parasitism, initially fed on the opposite diet immediately after conditioning. Although this suggests that the altered nutrient intake displayed by parasitized insects was not due to any failure in their capacity for dietary selection, these results do not definitively demonstrate an altered nutrient intake target by parasitized larvae. Rather, parasitism may compromise dietary selection, resulting in random feeding. When parasitized larvae were maintained on several isocaloric diets with a varying ratio of casein and sucrose, those larvae feeding on the diet with a ratio of 1:1 of these nutrients supported the largest parasite population. Previous investigation of larvae maintained on a single artificial diet established that parasitized insects display an aberrant induction of gluconeogenesis, so that haemolymph trehalose is maintained at a level equivalent to that of normal insects. In contrast, the present results demonstrated that parasitized larvae offered a choice of diets, and feeding at the altered nutrient ratio above, maintain haemolymph sugar but have the same level of gluconeogenesis as normal larvae given the same dietary choice. These investigations suggest that altered food selection by parasitized M. sexta larvae maintains metabolic homeostasis and, moreover, may be adaptive for C. congregata, potentially maximizing the number of parasites developing in a single host larva.

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.

Pyruvate cycling and implications for regulation of gluconeogenesis in the insect, Manduca sexta L.

Pyruvate cycling was examined in the insect Manduca sexta L. (2-(13)C)pyruvate was injected into 5th instar larvae maintained on a semisynthetic high sucrose, low sucrose, or sucrose-free diet. Pyruvate cycling and gluconeogenesis were determined from the distribution of (13)C in blood metabolites, including trehalose, the blood sugar of insects, and alanine. Pyruvate cycling was evident from the (13)C enrichment of alanine C3, synthesized by transamination of pyruvate following carboxylation to oxaloacetate and cycling through phosphoenolpyruvate. Based on the relative (13)C enrichments of alanine C2 and C3, insects maintained on the high sucrose diet displayed higher levels of cycling than insects on the other diets. Insects on all the diets, when subsequently starved, displayed low levels of cycling. Gluconeogenesis was evident in insects on sucrose-free or low sucrose diets from the selective (13)C enrichment in trehalose. The level of gluconeogenesis relative to glycolysis was indicated by the (13)C enrichment of trehalose C6 and alanine C3, both enrichments metabolically derived in the same manner. Insects starved after maintenance on the sucrose-free or low sucrose diets remained glucogenic. Insects on the high sucrose diet were not glucogenic, and subsequent starvation did not induce gluconeogenesis. The results indicate that pyruvate kinase plays a critical role in regulating the gluconeogenic/glycolytic balance, and that inhibition of pyruvate kinase is a principal regulatory event during induction of de novo trehalose synthesis. Gluconeogenesis failed to maintain homeostatic levels of blood trehalose, supporting the conclusion that blood sugar level may be important for mediating nutrient intake. Possible factors involved in the regulation of gluconeogenesis in insects are discussed.

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.

Arginine kinase and phosphoarginine, a functional phosphagen, in the rhabditoid nematode steinernema carpocapsae.

Moderate activity of arginine kinase was found in Steinernema carpocapsae, an entomopathogenic nematode. In the forward reaction, 4.60 and 3.12 micromol ATP/min/mg protein was produced in infectious third-stage juveniles (J3s) and adult nematodes, respectively. For the reverse reaction, 3.20 and 2.27 micromol phosphoarginine/min/mg protein was produced by J3s and adults, respectively. The K(m)s for phosphoarginine and ADP were 0.73 and 0.42 mM, respectively, in the forward reaction, whereas in the reverse reaction, the K(m)s were 0.37 and 2.35 mM for arginine and ATP, respectively, for the enzyme from J3s. The pH optimum for the forward reaction was 7.2 and 7.3 in J3s and adults, respectively. The pH optimum was elevated for the reverse reaction, 7.8 and 7.9-8.5 in J3s and adults, respectively. In the J3s, the in vitro optima for arginine kinase activity was correlated with the in vivo tissue pH in hypoxic (6.9) and aerobic (7.5) J3s estimated by in vivo flow 31P-NMR.

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.

Nutrition and culture of entomophagous insects.

This article reviews the current state of knowledge concerning the nutrition of entomophagous insects and other arthropods. Early nutritional investigations focused principally on the basic dietary and nutritional requirements for beneficial insects. Based on these findings, the first attempts were made to develop mass culture methods and to formulate artificial diets that would allow rearing of parasitoids and predators in the absence of host or prey, as well as the latter's food sources. Although limited success was achieved, these studies established the nutritional foundation for later advances. Recently, attention has focused on the interaction between nutrition, physiology, behavior, and ecology of the different life stages of entomophagous species within a unified concept of nutritional ecology. This approach has resulted in more comprehensive understanding of nutrition, which in turn has facilitated the continuous artificial culture of several parasites and predators. Additional studies have confirmed the importance of supplemental feeding of the adult stages of beneficial insects for maximizing reproduction and longevity. Applications of nutritional ecology to biological control are discussed.

Effects of lead, mercury and cadmium on the extramolluscan larval stages of Schistosoma mansoni (Sambon).

The effects of the heavy metal pollutants, lead, mercury and cadmium on the survival and behaviour of miracidia and cercariae of Schistosoma mansoni were examined. Concentration ranges of 100-900 microM lead chloride, 0.25-20 microM mercuric chloride and 0.25-9.0 microM cadmium chloride were tested. Inversely proportional relationships between metal concentration and survival were evident with all the metals. The LC50 of miracidia was 600 microM for lead, 1 microM for mercury and 2.5 for cadmium. The LC50 of cercariae was 600 microM for lead, 6 microM for mercury and 2.5 microM for cadmium. Movement and behavior of miracidia and cercariae were severely affected by exposure at the higher end of the concentrations of heavy metals tested. Infectivity of cercariae to mice was reduced by nearly half when cercariae were pre-exposed to lead, mercury or cadmium for five hours at the LC50 concentrations, compared with the infectivity of unexposed cercariae.

Aberrant nutritional regulation of carbohydrate synthesis by parasitized Manduca sexta L.

The present studies confirm that storage carbohydrate synthesis from [1-(13)C]glucose is elevated in Manduca sexta parasitized by Cotesia congregata, despite a decrease in the rate of metabolism of the labeled substrate. Further, the results demonstrate that a similar pattern of carbohydrate synthesis and glucose metabolism was induced in normal larvae by administration of the glycolytic inhibitor, iodoacetate. (13)C enrichment of C6 of trehalose and glycogen demonstrated randomization of the C1 label at the triose phosphate step of the glycolytic/gluconeogenic pathway and suggested that gluconeogenesis, that is, de novo carbohydrate formation, contributed to the synthesis of carbohydrate in both normal and parasitized insects. Accounting for differences in the (13)C enrichment in C1 of trehalose and glycogen due to direct labeling from [1-(13)C]glucose, the mean C6/C1 labeling ratios in trehalose and glycogen of parasitized larvae and insects treated with iodoacetate were greater than the mean ratio observed in normal larvae, suggesting a greater contribution of gluconeogenesis to trehalose labeling in parasitized insects. This conclusion was confirmed by additional investigations on the metabolism of [3-(13)C]alanine by normal and parasitized insects. The pattern of (13)C enrichment in hemolymph trehalose observed in normal larvae maintained on a low carbohydrate diet indicated a large contribution of gluconeogenesis, while gluconeogenesis contributed very little to trehalose labeling in normal insects maintained on a high carbohydrate diet. Parasitized insects maintained on a high or a low carbohydrate diet displayed a significantly greater contribution of gluconeogenesis to trehalose labeling than was observed in normal larvae maintained on the same diets. In conclusion, these investigations indicate that regulation over the utilization of dietary glucose for trehalose and glycogen synthesis as well as the dietary regulation of de novo carbohydrate synthesis were altered by parasitism.