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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.

The accumulation of lead, cadmium and mercury in the soft body mass of Biomphalaria glabrata (Say) exposed to dissolved chloride salts was determined. The effects of the heavy-metal exposure on the growth, mortality and reproduction were examined. The ranges of metal concentration tested were established following preliminary studies to determine their relative toxicity. Exposure to PbCl2 (1‐250 � M), CdCl2 (0.05‐0.5 � M,) or HgCl2 (0.1‐2.5 � M) at 28°C for periods up to eight weeks resulted in small, but statistically significant mortality and decreased growth. No significant effects on reproduction were noted. At 17°C, mortality and growth were not affected by heavy-metal exposure over the eight-week observation period. Snails were not reproductively active at the lower temperature. Spectrophotometric analyses demonstrated that snails exposed to Pb, Cd or Hg had significantly elevated levels of these metals in their bodies within four weeks at 17° or 28°C, indicating that this species and/or its close relatives may prove valuable as biomonitors for heavy-metal pollution.

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.

The blood sugar level of the insect Manduca sexta L. is highly variable and appears to be regulated in a non-homeostatic manner. This study examined the roles of gluconeogenesis and dietary carbohydrate as sources of blood sugar in M. sexta L. De novo glucose synthesis from [3-13C]alanine was demonstrated from the fractional 13C enrichment in trehalose, a disaccharide of glucose and the principal blood sugar of insects. 13C was detected by NMR spectroscopy. Gluconeogenesis contributed significantly to blood sugar in insects maintained on a low carbohydrate diet, but on a high carbohydrate diet blood sugar was derived principally from dietary carbohydrate. Starvation failed to induce gluconeogenesis. The relative contributions of the competing pyruvate carboxylation and pyruvate decarboxylation reactions to [3-13C]alanine metabolism under different dietary regimes were indicated from the 13C labeling distribution in glutamine and were consistent with the presence or absence of gluconeogenesis. Despite active gluconeogenesis, the blood sugar level of insects on the low carbohydrate diet was significantly less than that of animals on the high carbohydrate diet. Glucose synthesis was not evident until 6–12 h of feeding on the low carbohydrate diet and maximal gluconeogenesis was not observed until after 48 h. Administration of the transcription inhibitor cordycepin significantly inhibited the initiation of glucose synthesis and it is concluded that long-term regulation over gluconeogenesis through gene expression and enzyme synthesis is the principal mechanism of nutritional regulation.

The effects of dissolved Pb, Cd and Hg on growth and survival of adult Biomphalaria glabrata Say, uninfected and infected with Schistosoma mansoni Sambon, were examined. Pb at concentrations from 0.25-100 M.M, Cd from 0.075-0.25 u.M and Hg from 0.25-1 JJ.M significantly reduced growth and survival of normal snails. With each metal the effects increased with increasing concentration. The LC 25 at 2 wks exposure was 82 ± 19, 0.22 ± 0.04 and 0.94 ± 0.13 M-M (x ± S.E.) for Pb, Cd and Hg, respectively. Snails exposed to heavy metals continued to be reproductively active, but the egg production was highly variable and no significant effect of heavy metal exposure was demonstrated. Infection also decreased survival and growth of snail hosts and infected individuals exposed to heavy metals displayed the greatest mortality. The LC 25 for infected snails at 2 wks exposure was 8 ± 3, 0.9 ± 0.02 and 0.29 ± 0.08 for Pb, Cd and Hg respectively. A significant interaction between heavy metal exposure and infection was apparent. Infected snails were not reproductively active. Cercarial shedding by infected snails was significantly reduced in the presence of heavy metals and by 6 wks shedding had ceased at the highest metal concentrations.

Pyruvate cycling from (2- 13 C)pyruvate was detected in vivo in intact 5th instar Manduca sexta larvae by application of NMR spectroscopy. Cycling was evident from the enrichment of C3 in alanine following transamination of recycled pyruvate in larvae maintained on casein-based diets with or without sucrose. This metabolism is assumed to principally occur in the fat body. Analysis of 13 C enriched metabolites released into the hemolymph indicated that isotopic dilution of recycled pyruvate was sufficiently great that further metabolism of the recycled metabolite did not occur to any significant extent under these dietary conditions. The C3/C2 13 C-enrichment ratio of alanine, therefore, accurately reflected the relative degree of pyruvate cycling and indicated that the rate of cycling was approximately three-fold lower in larvae maintained on diets lacking sucrose. Moreover, based on the distribution of 13 C in trehalose, these larvae displayed significantly greater rates of gluconeogenesis. Enrichment of C1, C2, C5 and C6 were principally due to carboxylation of the isotopically substituted substrate catalyzed by pyruvate carboxylase and, therefore, reflected net carbohydrate synthesis. Trehalose C3 and C4 enrichments were principally due to pyruvate dehydrogenase-catalyzed decarboxylation and reflected incorporation of label following metabolism through the TCA cycle. Pentose cycling following glucogenesis significantly affected the 13 C distribution in trehalose in insects on both diets, and the relative intensity of trehalose C6 was, therefore, used for comparing the rates of gluconeogenesis and pyruvate cycling. Based on the 13 C enrichment of trehalose C6 relative to C3 of alanine the mean rate of pyruvate cycling relative to the rate of gluconeogenesis was approximately 60% in larvae on the diet lacking sucrose, while the rate of pyruvate cycling in larvae maintained on the diet supplemented with sucrose was greater than the gluconeogenic flux. The results were consistent with the conclusion that pyruvate kinase likely plays an important role in regulating gluconeogenesis in M. sexta larvae.

Publisher Summary This chapter explores the nutritional habits in insects. The nutritional requirements of insects were at first inferred from knowledge of the chemical compositions of natural foodstuffs. Insects were categorized as carnivorous, omnivorous, or phytophagous, with the appropriate inferences regarding the nutritional content of the animals, plants, and other foods eaten. Studies of food utilization, and the analysis and comparison of foodstuff eaten and excreted or unabsorbed, allowed further assessment of the relative importance of various nutrients for insects with different feeding habits. A thorough knowledge of insect nutrition is essential for understanding the biology of insects. The study of insect nutrition has recently undergone a metamorphosis, in that information gleaned from earlier investigations that focused principally on basic nutritional requirements and rearing technology is now being applied for understanding the feeding strategies, nutritional ecology, and evolution of insects. Nutritional physiology and biochemistry are also advancing, with the molecular arsenal available for Drosophila offering many new opportunities. The neurological bases for food selection and the role of biogenic amines in regulating food choice are beginning to be understood. The chemical composition of the hemolymph is now recognized as a dynamic indicator of nutritional status, affecting food selection and nutrient intake. The metabolic responses of insects to altered nutritional status and the effects of fat body metabolism on hemolymph composition are also being investigated. Future studies employing multidisciplinary approaches will continue to unravel the mysteries of insect nutrition and its consequences and significance to insect biology.

A classical macroscopic description of the NMR phenomenon is presented, and properties of the major NMR active nuclei of importance to metabolic study outlined. Advantages and disadvantages of several biological applications of NMR spectroscopy and the inherent limitation of NMR analysis are discussed. In vitro and in vivo13C, 1H and 31P NMR applications on insects and insect metabolites are reviewed. These include investigations of cuticular hydrocarbon composition and synthesis, cuticular structure, structure and synthesis of silk, analysis of hemolymph metabolites, glucose metabolism, gluconeogenesis and pyruvate metabolism, metabolism of cryoprotectant polyols and analysis and metabolism of high energy phosphorus compounds.

▪ Abstract 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.

Metabolic alterations that accompany parasitism of invertebrate animals can play an important role in parasite development. Employing 13 C 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 13 C 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 13 C distribution in trehalose and alanine. The 13 C-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 13 C 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.

High efficiencies of food utilization have often been predicted for parasitoids. The larval stages that consume food of high nutritional content are relatively inactive within the host and have a limited food supply, both of which may contribute to the selection for efficient food utilization. Non-nutritional factors are intimately and intrinsically involved in food acceptance and intake. These include physical properties such as form and texture, but also non-nutritive chemicals that elicit specific behavioral and/or physiological responses essential for finding and accepting foodstuffs, and in some cases for initiating behaviors associated with the feeding process itself. In addition to feeding on plants and plant products, many parasitoids, particularily idiobiontic species, feed on potential hosts. Host feeding by koinobiontic parasitoids is less common. Adult female hymenopterans puncture or damage host larvae or pupae and feed on the hemolymph and/or internal tissues. Parasitic hymenopterans are often categorized as proovigenic or synovigenic. Females of proovigenic species complete oogenesis prior to, or shortly after, emergence and lay eggs over a relatively short period of time primarily on larval stages of their host. Host feeding is only important for ensuring that the female lives long enough to deposit all her eggs.

In vivo flow 31P NMR spectroscopy of a microscopic nematode, Steinernema carpocapsae, is described. Long-term viability was maintained during analysis by continuous circulation of an oxygenated suspension of the parasite through an NMR spectrometer. Saturation transfer and inversion recovery were employed under flowing conditions to investigate the kinetics of phosphoarginine ⇌ adenosine triphosphate exchange. The kinetic constants for the forward and reverse reactions were 0.37/s and 1.45/s, respectively. This report is the first to demonstrate a functional phosphagen kinase in the metabolism of a parasitic helminth.© 1992 Academic Press,Inc.

31P NMR spectroscopy of a parasitic nematode, Steinernema carpocapsae, is described. In vivo spectra were generated from nematode suspensions continuously circulating through the spectrometer. By flowing the organism, saturation effects were avoided and short in-terpulse delays significantly reduced spectral generation time. To maximize sensitivity, 90° pulses were employed. Changes in energetic status in response to oxygen availability were easily and rapidly monitored in circulating nematodes. © 1990 Academic Press, Inc.

Moderate activity of arginine kinase was found in Steinernema carpocapsae, an entomopathogenic nematode. In the forward reaction, 4.60 and 3.12 pLmol 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 pxmol phosphoarginine/min/mg protein was produced by J3s and adults, respectively. The Kms for phosphoarginine and ADP were 0.73 and 0.42 mM, respectively, in the forward reaction, whereas in the reverse reaction, the Kms 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. Thompson et al. (1990) observed a phosphagen, phosphoar- ginine, in infectious third-stage juveniles (J3s) of Steinerema carpocapsae (an entomopathogenic nematode) by employing in vivo flow 31P-NMR spectroscopy. In this procedure, suspen- sions of juvenile nematodes were circulated continuously through the spectrometer. Phosphoarginine was the major peak in the phosphorous spectrum of S. carpocapsae maintained un- der aerobic conditions, but its level declined rapidly during hyp- oxic conditions. As oxygen tension decreased, changes in the relative levels of phosphoarginine and ATP were consistent

Phosphorus resonances consistent with phosphoarginine and ATP were observed in the in vivo 31 P-nuclear magnetic resonance (NMR) spectra of infective larvae of Haemonchus contortus. The level of phosphoarginine quickly declined when nematode suspensions were purged with nitrogen and was restored upon return to aerobic conditions. Saturation transfer NMR demonstrated forward and reverse exchange of phosphorus between phosphoarginine and ATP.

The basis of the nuclear magnetic resonance (NMR) phenomenon is described in a classical frame- work with emphasis on magnetic nuclei of 1/2 spin, including IH, 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. Nuclear magnetic resonance (NMR) spectros- copy has long been an important method in an- alytical and organic chemistry (Martin et al., 1980). Recent development of high field instru- ments has made the analysis of complex biolog- ical samples practical. NMR offers new and ex- citing ways for examining biological phenomena in vivo (Williams and Gadian, 1986; Shulman, 1988), and 'H, 13C, and 31p NMR spectroscopy have been employed in metabolic studies on in- tact cells, tissues, and organs, as well as on whole

NMR spectroscopy was applied to the study of gluconeogenesis and pyruvate metabolism in Manduca sexta. [13C]2-Pyruvate injected into early 5th instar larvae was incorporated into a variety of metabolic intermediates including trehalose. The labelled pyruvate was completely metabolized within 4 hr and no significant label was detected in any metabolite after 24 hr. The predominant flux of 13C into the Krebs cycle appreared to result from the formation of [13C]1-acetyl CoA and 13C quickly accumulated in C5 of glutamate and glutamine. [13C]2-Pyruvate was directly incorporated into alanine C2, citrate C3, α ketoglutarate C3 and C3 of glutamate and glutamine. As a result of reverse malate-fumarate exchange and isomerization, 13C was also incorporated, but to a far lesser degree, into citrate C4 and C2 of glutamate and glutamine. 13C was incorporated into C1-6 of glucose in trehalose. The distribution of label indicated gluconeogenesis directly from [13C]2-pyruvate and following reverse isomerization with approximately equal incorporation into C2,5 and C1,6, respectively. Synthesis from [13C]1-acetyl CoA following passage through the Krebs cycle was also noted. Starvation for 24 hr had little apparent effect on the metabolism of [13C]2-pyruvate or on the distribution of label in glucose, indicating that insects may not regulate de novo carbohydrate synthesis in response to diet in a manner similar to higher animals.

The aquatic parasitic fungus, Coelomomyces dodgei (Chytridiomycetes; Blastocladiales), has a life cycle of the Euallomyces type with the gametophyte and sporophyte generations alternating obligately between a copepod host and a mosquito host, respectively. In infected copepods, the fungus produces gametophytes which are either bright orange or colorless. Subsequently, each gametophyte forms gametangia and gametes of a corresponding color. The colors are indicative of opposite mating types, and zygotes are only formed by fusion of orange and colorless gametes. In the present investigation the orange pigment was extracted from copepods containing orange gametangia and subjected to uv spectral analysis and thinlayer chromatography. The uv absorption profile in hexane, including maxima at 454 and 480 nm, and the chromatographic properties of the pigment indicate it is β-carotene.

The parasite Exeristes roborator grew rapidly and completed larval development on fatty acid free chemically defined diets. Dietary supplements of palmitoleate, oleate, linoleate, and linolenate were detrimental to parasite development with most larvae dying in the first instar. Palmitate supplements were also toxic, but a small percentage of larvae consistently completed development and survival and development time on diets supplemented with free stearate did not differ significantly from results obtained with fatty acid free diets. Supplements of a mixture of all six free fatty acids were as toxic as the unsaturated free fatty acids. Dietary supplements of the triglycerides, tripalmitin, tripalmitoleate, tristearin, and trioleate had no positive nutritional value for larval growth and development but were not detrimental. Development time was increased when the parasite was reared on fatty acid free diets lacking carbohydrate, but survival was not affected. The parasite, therefore, appears to have the ability to utilize dietary free amino acids as the sole energy source at this stage. Under these nutritional conditions, supplements of triglycerides did not replace the nutritional value of carbohydrate and some of the supplementary triglycerides were detrimental to larval survival.

Parasitization of Trichoplusia ni by Hyposoter exiguae resulted in elevations in the haemolymph trehalose concentration and fat body glycogen level. These increases in tissue carbohydrate reserves were accompanied by an elevation in the maximal velocities of fat body phosphofructokinase and fructose 1,6 bisphosphatase. The activity of the latter, however, was several-fold greater than that of phosphofructokinase and the potential gluconeogenic flux through the fructose phosphate step was, therefore, markedly increased following parasitization. The changes in adenylate levels in parasitized fat body, specifically an increase in AMP and decrease in ATP, were consistent with an elevation in phosphofructokinase activity in vivo but not in fructose 1,6 bisphosphatase activity. The latter enzyme, however, was shown to be less sensitive to both AMP and fructose 2,6 bisphosphate inhibition in fat body of parasitized individuals. Decreased haemolymph glucogenic amino acid concentrations and an increase in faecal uric acid level accompanied the above effects. It was concluded that parasitization of T. ni by H. exiguae caused a stress-induced hyperglycemia that may result from an alteration in the hormonal regulation over carbohydrate synthesis.

1. 1. Parasitization of late 3rd instar Trichoplusia ni by Hyposoter exiguae resulted in a marked reduction in weight gain as well as delayed development. Similar effects were observed in partially starved larvae reared on nutrient deficient media. 2. 2. The total body lipid concentration and level in T. ni larvae were reduced following parasitization or starvation. 3. 3. The total body glycogen concentration and level in T. ni larvae were also reduced as a result of starvation. Parasitized individuals, however, had glycogen concentrations similar to control unparasitized larvae, but their small size reduced the total level considerably. 4. 4. The reduction of total body glycogen and lipid reserves in parasitized T. ni appeared to occur in a similar fashion to that which results from partial starvation, suggesting that these effects of parasitization may result from the competition between the developing parasite and host tissues for available nutrients.

Exponential weight gain equations and growth rates were determined during larval development of Brachymeria lasus reared aseptically on chemically defined media with varying amino acid and glucose levels. The rate of weight gain increased from 0 to 2% dietary glucose and mean growth rate increased from 160 to 260 μg/mg · day. Similar results were observed for larvae reared on media containing 1 to 4% free amino acids. At higher nutrient levels, growth rate and rate of weight gain declined and maximal growth occurred at dietary osmolalities of 500–700 mOsm/kg. A further experiment utilizing nutritionally inert l -glucose suggested that this osmolality range does not reflect the optimal for growth but rather the point at which the detrimental effects of dietary osmotic pressure exceed the nutritive benefit of the dietary ingredients.

A soluble fatty acid synthetase complex was isolated from whole insect homogenates of Lucilia sericata by high speed centrifugation. Like synthetases isolated from other organisms, that from L. sericata catalyzed the conversion of acetyl and malonyl-CoA to long chain fatty acids. The purification of the insect complex by salt fractionation differed from that displayed by most previously isolated synthetases. The insect synthetase consistently precipitated between 40 and 50% ammonium sulphate saturation compared to 24 to 40% saturation for previously isolated complexes. The sole pyridine nucleotide requirement for synthetase activity was NADPH. NADH was ineffective alone or in combination with NADPH. The major products of synthetase activity were palmitic acid, 40% and stearic acid, 60%. In contrast, the major products of other animal synthetases are myristic and palmitic acids. However, like other animal synthetases the products of the insect synthetase appear to be released from the complex as free fatty acids rather than thioester derivatives. Lineweaver-Burk plots of enzyme velocity vs malonyl-CoA and NADPH concentrations were nonlinear suggesting the role of these substrates as homotrophic effectors of synthetase activity. A modified plot of enzyme velocity vs NADPH concentration squared resulted in a linear relationship with an extrapolated K m value of 17 nmoles. The Lineweaver-Burk plot of enzyme velocity vs acetyl-CoA concentration was linear with an extrapolated K m value of 11 nmoles. The K m values for acetyl-CoA and NADPH were much lower than those obtained for other animal synthetases suggesting a far greater enzymesubstrate affinity for the insect synthetase. The ratio of the K m values of acetyl-CoA and NADPH were, however, similar to those of other animal synthetases.

The levels of adenylate nucleotides were examined in 4th-instar Trichoplusia ni larvae 3 days after parasitization by the insect parasite Hyposoter exiguae. In general, parasitization caused a decrease in the level of ATP and increased ADP and AMP levels. These changes resulted in alteration of the adenylate kinase mass-action ratio. The overall energy status of parasitized larvae, however, as indicated by energy ratios, including the “energy charge,” was affected only slightly. The result demonstrates that the host maintained an active and viable metabolic state despite extensive alterations in physiology which occur at this stage of the parasite-host association.

Brachymeria lasus and Pachycrepoideus vindemiae failed to develop in vitro on sterol-free artificial media, and dietary acetate and squalene failed to maintain and/or support growth. The sterols, cholesterol, cholestanol, β-sitosterol, 7-dehydrocholesterol, and cholesterol linoleate were all utilized and maintained larvae of both species. Larval survival and development rate were greatest with cholesterol followed by cholestanol, β-sitosterol and 7-dehydrocholesterol. Although cholesterol linoleate maintained larvae little growth occurred and mortality was high. Cholestanol followed by β-sitosterol and 7-dehydrocholesterol displayed partial cholesterol sparing activity. Cholesterol linoleate had little effect on larval growth when fed with suboptimal levels of cholesterol or cholestanol. Both species contained 5 to 10% of the total body lipids as free sterol with traces of sterol ester. The major free sterol appears to be cholesterol.

1. 1. The concentration of total phospholipid in the parasite, Exeristes roborator, reared on chemically defined media increased in response to increased dietary glucose level, although the proportion of phospholipid to total lipid decreased. The proportion of radioactivity incorporated into phospholipid from [2-14C] acetate also decreased although the absolute amount of activity increased. 2. 2. [14C(U)] glycerolphosphate was incorporated only into phospholipid and triglyceride indicating their de novo synthesis by esterification of glycerolphosphate, and the proportion of radioactive glycerolphosphate incorporated into triglyceride increased with increasing dietary glucose. 3. 3. Dietary glucose also had a marked effect on the degree of saturation in both lipid fractions and on the proportion of radioactive glycerolphosphate incorporated into phosphatidyl choline and phosphatidyl ethanolamine.

Melanization of artificial media accompanied feeding by the parasite Exeristes roborator and was found to be due to phenyloxidase activity secreted by the developing larvae into the diet. The enzyme was localized in the salivary glands and its possible significance to the host-parasite relationship discussed.

Dietary glucose had pronounced effects on lipogenesis in the parasite, Exeristes roborator , reared on ‘fatty acid-free’ artificial diets. Larvae reared on such media, but carbohydrate-free, contained little triglyceride, and radioactivity from [2- 14 C] acetate was incorporated largely into phospholipid. On media containing from 2 to 6% (w/v) glucose increasing amounts of radioactivity were incorporated into the triglyceride fraction and at the 6% glucose level more was incorporated into triglyceride than phospholipid. Radioactivity was found in both the glycerol and fatty acid moieties of the triglyceride and phospholipid fractions, but the proportion in the fatty acids of the triglyceride fraction increased with increasing dietary glucose, while that in the phospholipid fraction remained little changed. Increased dietary glucose resulted in marked alteration in the degree of saturation in the fatty acids of both the triglyceride and phospholipid fractions. On carbohydrate-free media the fatty acid composition of the triglycerides was composed primarily of the saturates, palmitic and stearic acids, while that of the phospholipid fraction contained higher levels of the corresponding unsaturates, palmitoleic and oleic acids. The degree of unsaturation increased sharply with increasing glucose levels in the diet and at the 6% level both the phospholipid and triglyceride fractions were at least 50% unsaturated. Isolated fatty acid synthetase activity was greater in larvae reared on diets with glucose than without. Glucose had no short-term effector action on synthetase activity in vitro .

1. 1. The nature of metabolic interaction during parasitic infection was discussed and the concept of metabolic integration outlined. The subjective nature of the integrative argument was noted. 2. 2. The parasite-host relationships of larval trematodes of the genus Schistosoma with their intermediate molluscan hosts, the nematode Trichnella spiralis and cestode Hymenolepis diminuta, with their definitive hosts, as well as the hymenopterous insect parasite, Hyposoter exiguae, with its insect host, Trichoplusia ni, were examined. 3. 3. The significance of the immune system in the establishment of the parasite-host association and the means by which parasites evade host defense were discussed. 4. 4. The involvement of microorganisms or “hyperparasites” during the host associations of multicellular parasites was described. 5. 5. The importance of evolutionary considerations in assessing the nature of metabolic interaction and its significance to the success of the parasite-host relationship was emphasized. The use of teleological assessment and anthropomorphic description was discussed.

Microsomes from two species of parasitic Hymenoptera, Exeristes roborator and Itoplectis conquisitor, exhibited little or no de novo glyceride synthesis but actively acylated endogenous mono- and diacylglycerides. It is suggested that this lack of de novo synthesis is related to the fact that the fatty acid composition of these parasitoid species closely resembles that of the hosts on which they are reared. Microsomes from three other species of parasitic Hymenoptera, Aphaereta pallipes, Brachymeria lasus, and Hyposoter exigua, whose fatty acid compositions are little influenced by the host species, exhibited active de novo glyceride synthesis as well as acylation of endogenous mono- and diacylglycerides. Radiotracer studies indicated that E. roborator microsomes and cytosol did not contain noncompetitive or uncompetitive inhibitors of glycerophosphate acyltransferase. E. roborator microsomes acylated exogenous phosphatidic acid but not dihydroxyacetone phosphate or glycerol. The maximum rate of glycerophosphate acylation was less than 0.1 nmole/min/mg microsomal protein after 15 min incubation. The incorporation was subject to rapid lipolysis on further incubation. The addition of bovine serum albumin (BSA) reduced the ability of E. roborator microsomes to acylate mono- and diacylglycerides with endogenous acyl groups. In the absence of BSA, palmitoyl-CoA was a more effective substrate than stearoyl-CoA for both mono- and diacylglyceride acyltransferases.

1. 1. The metabolism of the major chemical fractions of Romanomermis culicivorax postparasites was studied under aerobic and anaerobic conditions. 2. 2. Postparasites contained approximately 42% protein, 35% lipid, and 14% carbohydrate on a dry weight basis. 3. Essentially, all of the carbohydrate storage was glycogen, but small quantities of glucose and trehalose were found. 4. 4. Lipid, primarily triacylglycerol, was catabolized and glycogen synthesized under aerobic conditions, but in the absence of oxygen only glycogen was catabolized. 5. 5. The relative proportions of fatty acids from total lipids extracted from postparasites developing under aerobic and anaerobic conditions were the same indicating nonpreferential degradation. 6. 6. The results suggested that lipid was converted to glycogen via a glyoxylate pathway and that glycogen was utilized for energy production only in the absence of oxygen.

High resolution 31 P nuclear magnetic resonance spectroscopy (NMR) was successfully applied to 5th instar larvae of Manduca sexta . Conditions for in vivo analysis under non-saturating conditions are described. The 31 P NMR spectrum of intact larvae was composed of six peaks. Their resonance frequencies are reported relative to orthophosphoric acid. Analysis of tissue extracts demonstrated the in vivo peaks to be composed of the β phosphorus resonance of nucleotide triphosphates (NTP) at −19.36 ppm; α phosphorus of NTP and nucleotide diphosphates (NDP) at −10.51 ppm; β and γ phosphorus of NDP and NTP, respectively, at −5.42 ppm; phosphoarginine (PA) at −3.45 ppm; inorganic phosphate (Pi) at +2.76 ppm and sugar phosphates at +3.34 ppm. The major sugar phosphate present in fat body extracts was trehalose-6-phosphate and this was the major phosphorus component of the spectrum of hemolymph. The spin-lattice relaxation times for each in vivo peak were determined. Titration of aqueous fat body and hemolymph extracts was carried out and the relationship between the chemical shift of Pi and pH determined. On this basis the pH of the hemolymph was estimated at approx. 6.7. The metabolic inhibitors, iodoacetate and dinitrophenol, had significant effects on the 31 P NMR spectrum of intact larvae. Administration of iodoacetate caused a rapid increase in the levels of sugar phosphates together with decreases in NTP and PA. Dinitrophenol also caused declines in the relative levels of NTP and PA but sugar phosphates decreased as well. The experiments demonstrated the potential of in vivo NMR analysis for metabolic studies on high energy phosphate metabolites in M. sexta .

Larval growth rates of the insect parasite, Exeristes roborator reared in vitro were calculated from egg hatching to the fourth instar. Dry weight gain and mean wet weight during feeding were considered. Dry weights of developing larvae were estimated as the sum of the total protein and lipid levels, eliminating the contribution of stored waste. Dry/wet weight ratios of larvae were found to decrease markedly during development. Growth rates of parasite larvae increased from approximately 1 μg/mg·day on carbohydrate-free media to 50 μg/mg·day on artifical diets containing 8% glucose.

1. 1. The concentration of triglyceride in Trichoplusia ni larvae increased during larval development. 2. 2. In larvae parasitized late in the 3rd stadium by Hyposoter exiguae the concentration remained constant throughout the remainder of larval development. 3. 3. Partially starved larvae maintained from the late 3rd stadium on nutrient deficient media and displaying a pattern of growth similar to parasitized individuals became lipid depleted. 4. 4. The fatty acid composition of the neutral lipids was altered during development and the relative proportions of palmitic and oleic acids increased in both parasitized and unparasitized larvae. 5. 5. In contrast, the composition of nutrient deprived larvae had lower proportions of palmitic and oleic acids and higher levels of linoleic and linolenic acids. 6. 6. The results suggest that although the parasitized host does not accumulate lipid, active synthesis may be occurring. 7. 7. Nutrient-deprived larvae, however, appeared to be utilizing lipid as an energy source and thus became lipid depleted.

1. 1. After short exposure periods to 14 C-1-acetate, Galleria mellonella incorporates activity into octadecanoic (steraic) acid, consistent with the elongation synthetic mechanism. No de novo synthesis, involving the incorporation of 14 C-1-acetate was detected until a few hours after initial exposure. It appears, therefore, that the two synthetic mechanisms of elongation and de novo synthesis can be studied independently, by varying the exposure periods to 14 C-1-acetate. 2. 2. The desaturation reaction yielding 9-octadecenoic acid corresponds with the incorporation of 14 C-1-acetate into octadecanoic acid in a typical precursor-product fashion but the rate of incorporation is much slower. 3. 3. No elongation of unsaturated fatty acids with 14 C-1-acetate was detected in G. mellonella , in the first 30 minutes of exposure. 4. 4. The incorporation rate of acetate into 9-octodecenoic acid is much lower than that of octadecanoic acid, indicating that the unsaturates may be storage products with lower fractional turnover rates. This is consistent with the percentage composition, in which the unsaturates, particularly 9-octadecenoic acid, comprise the largest pool. This is generally true in all animals and plants. 5. 5. Accurate calculations of fractional turnover rates in vivo are impractical at this time.

1. 1. The fatty acid compositions of the seven insect orders, Lepidoptera, Hemiptera, Orthoptera, Diptera, Hymenoptera and Dictyoptera differ quantitatively in a number of respects with regard to the relative percentages of the commonly occurring fatty acids, myristic, myristoleic, palmitic, palmitoleic, stearic, oleic, linoleic and linolenic acids. 2. 2. Major characteristics which appear to be taxonomically related include the relatively high levels of palmitoleic acid in dipterous insects and high levels of myristic acid in some families of hemipterous insects. The exceptions are discussed. 3. 3. The apparent significance of the fatty acid composition, in general, is discussed.

1. 1. In all three species minor qualitative and quantitative differences in fatty acid composition were found between cuticle and fat body tissue. However, little correlation could be found between these differences among the species. 2. 2. In T. molitor, quantities of C 14:1, ∗ C 16:2 and C 18:3 fatty acids were found in the fat body, and not detected in the cuticle. The fat body contained a higher percentage of stearic acid and less palmitic and C 18:1 fatty acids. 3. 3. In P. americana, the cuticle contained C 18:3 fatty acids not detected in the fat body. The fat body contained a lower percentage of stearic and C 18:2 fatty acids, and a higher percentage of C 18:1 fatty acids. 4. 4. There was very little difference in the fatty acid composition of the cuticle and fat body of S. gregaria.

During flight, male beetles lost weight primarily due to oxidation of fatty acids. RQ values of 0·7 and 0·8 were obtained for non-flying and flying insects respectively, indicating that Dendroctonus pseudotsugae is predominantly a fat utilizer, with some carbohydrate utilization during early flight periods. Selective oxidation of fatty acids was evident. The monounsaturates, C16:1 and C18:1 were oxidized at the greatest rate, followed by the saturates, palmitic (C16:0), stearic (C18:0), and myristic (C14:0) acids, respectively.

1. 1. The total lipid level of the digestive gland-gonad complex of Biomphalaria glabrata was reduced by starvation and elevated following infection with Schistosoma mansoni. 2. 2. The lipid classes of B. glabrata were phospholipid, monoglyceride, free fatty acid, triglyceride, sterol ester and wax and a single unidentified lipid. The major lipid present in control, starved and infected tissue was sterol. Triglyceride was a minor component of control tissue. 3. 3. Starvation reduced the relative level of the unidentified lipid but had little effect on triglyceride. 4. 4. Infection also caused a decrease in the unidentified component but the triglyceride level was sharply elevated. 5. 5. The phospholipid fraction of tissue from all groups was composed of phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl serine. Infection and starvation had little effect on the composition.

The costs for injecting 3 million gallons of waste/grout mix in 200,000-gallon batches over 2 years are estimated to be 9.75 million dollars (including direct, indirect, operational, and decommissioning costs). (DLC)

Exeristes roborator contains a wide variety of free amino acids, and the composition of all developmental stages was quantitatively dominated by proline and glutamic acid. The latter occurred together with lesser amounts of glycine, alanine, valine, leucine, tyrosine, and histidine which varied between developmental stages. Minor and trace amounts of most other commonly occurring amino acids were also found. The percentages or relative amounts of major constituents were not influenced when the parasite was reared on the alternate hosts, Pectinophora gossypiella and Gnorimoschema operculella . Likewise, the major characteristics of the latter hosts' free amino compositions could not be accounted for, in either case, by their diets. Differences in the relative distribution of the major amino acids between the developmental stages of E. roborator indicate large decreases in the percentage of proline occur during development from the larval to the adult stage with corresponding increases in the percentages of glycine in the pupal stage and alanine as well as other amino acids in the adult stage. The results suggest that proline may play an important role in E. roborator , probably as an energy reserve. The amino acid compositions of the total proteins of E. roborator and its hosts were similar and all quantitatively dominated by glutamic acid, aspartic acid, and amides. However, the disk gel electropherograms of the proteins of both hosts and parasite were different. Quantitative changes were evident in the protein pattern of the parasite when reared on the alternate hosts. E. roborator incorporated radioactivity from 14 C(U)-glucose into the amino acids, glutamic acid, aspartic acid, serine, glycine, alanine, and proline. Furthermore, 14 C(U)-glutamic acid was incorporated into a wide variety of proteins. The data suggest that the above amino acids may be non-essential dietary components for E. roborator . However, quantitative determinations indicate that the amount of glutamic acid synthesized does not account for the amount incorporated into protein over the same time period.

1. The amino acid requirements for larval development of E. roborator were determined by amino acid deletion from a holidic diet. The results demonstrated that the parasitoid has an absolute requirement for the 10 amino acids, that is, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. 2. No statistically significant difference in survival was found between insects reared on the control diet and diets lacking alanine, aspartate, cysteine, glutamate, glycine, hydroxyproline, serine or tyrosine and although there were significant differences in development time these were slight. E. roborator , therefore, has no dietary requirement for these amino acids under the present rearing conditions. 3. E. roborator did not survive on diets containing only the 10 essential amino acids.

The composition and formulation of an artificial medium and handling techniques are described for rearing the chalcid insect endoparasite, Brachymeria lasus (Walker) from the egg to adult stage under in vitro conditions. The medium was devoid of host materials, prepared aseptically, and contained 15% ( w v ) albumin, 1% amino acids, 4% glucose, and approximately 2.5% lipids, 0.3% inorganic salts, 0.01% B vitamins, and 0.2% lipogenic growth factors. Eggs were obtained by host dissection and developing larvae were reared in tissue culture plates. Adults were fecund.