Insect Manduca Sexta Research Articles

Modulation by eicosanoid biosynthesis inhibitors of immune responses by the insect Manduca sexta to the pathogenic fungus Metarhizium anisopliae.

Metarhizium anisopliae conidia (spores) reduced weight gain and caused death when injected into Manduca sexta larvae. When the fungus was co-injected with the eicosanoid biosynthesis inhibitor dexamethasone, larval weight gain was further reduced and mortality increased. These effects were reversed when dexamethasone was given together with the eicosanoid precursor arachidonic acid (AA). Similarly, treatment with other eicosanoid biosynthesis inhibitors (esculetin, phenidone, ibuprofen, and indomethacin) with differing modes of action enhanced the reduction in weight gain caused by mycosis. Injection of M. anisopliae conidia induced nodule formation in vivo; nodule numbers were reduced by dexamethasone, and restored by AA. Incubation of hemocytes with conidia caused microaggregation of hemocytes (indicative of nodule formation) in vitro and this was inhibited by dexamethasone, suggesting that dexamethasone acts directly on hemocytes, although inhibition was only partially reversed by AA. We suggest that the M. sexta immune response to fungal pathogens is normally modulated by physiological systems that include eicosanoid biosynthesis. This is the first demonstration that the virulence of a fungal entomopathogen can be enhanced by compromising the insect host's immune system.

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.

Nitric Oxide Activates the β2 Subunit of Soluble Guanylyl Cyclase in the Absence of a Second Subunit

Previously characterized mammalian soluble guanylyl cyclases form alpha/beta heterodimers that can be activated by the gaseous messenger, nitric oxide, and the novel guanylyl cyclase modulator YC-1. Four mammalian subunits have been cloned named alpha(1), beta(1), alpha(2), and beta(2). The alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzyme isoforms have been rigorously characterized. The role of the beta(2) subunit has remained elusive. Here we isolate a novel variant of this subunit and show that the beta(2) subunit does not need to form heterodimers for catalytic activity because enzyme activity can be measured when it is expressed alone in Sf9 cells. In analogy to the beta(3) subunit recently isolated from the insect Manduca sexta, activity was dependent on the presence of 4 mm free Mn(2+). The EC(50) values for the NO-donor diethylamine/NO were shifted to the left by 1 order of magnitude as compared with the alpha(1)/beta(1) heterodimeric form. In the presence of the detergent Tween, NO sensitivity of beta(2) was abolished, but the enzyme could be activated by protoporphyrin IX, indicating removal of a prosthetic heme group and exchange for the heme precursor. We suggest that the beta(2) subunit is the first mammalian NO-sensitive guanylyl cyclase lacking a heterodimeric structure.

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- 13C)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- 13C)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- 13C)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.

Proteolytic activation of prophenoloxidase in an insect Manduca sexta.

Phenol oxidase (PO) (EC1.14.18.1) is important in cuticular sclerotization and in defense against pathogens and parasites (Söderhäll et al., 1996; Sugumaran, 1996; Ashida and Brey, 1998). PO catalyzes hydroxylation of monophenols to o-diphenols and oxidation of o-diphenols to quinones. The quinones take part in sclerotization of cuticle and serve as precursors for synthesis of melanin. PO is synthesized in arthropods as an inactive zymogen, prophenoloxidase (proPO), which can be activated by proteolytic cleavage at a specific site in the protein. Wounding of an insect or exposure to certain microbial polysaccharides leads to the activation of a proteinase that specifically activates proPO, perhaps at the last step of a proteinase cascade (Ashida and Brey, 1998). The reactions catalyzed by PO then can result in melanization of foreign organisms trapped in capsules or hemocyte nodules (Gillespie et al., 1997).KeywordsHorseshoe CrabPhenol OxidaseAmidase ActivityPhenol Oxidase ActivityMicrobial PolysaccharideThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Signal Transduction in Eclosion Hormone-induced Secretion of Ecdysis-triggering Hormone

Inka cells of insect epitracheal glands (EGs) secrete preecdysis and ecdysis-triggering hormones (PETH and ETH) at the end of each developmental stage. Both peptides act in the central nervous system to evoke the ecdysis behavioral sequence, a stereotype behavior during which old cuticle is shed. Secretion of ETH is stimulated by a brain neuropeptide, eclosion hormone (EH). EH evokes accumulation of cGMP followed by release of ETH from Inka cells, and exogenous cGMP evokes secretion of ETH. The secretory responses to EH and cGMP are inhibited by the broad-spectrum kinase inhibitor staurosporine, and the response to EH is potentiated by the phosphatase inhibitor calyculin A. Staurosporine did not inhibit EH-evoked accumulation of cGMP. Changes in cytoplasmic Ca2+ in Inka cells during EH signaling were monitored via fluorescence ratioing with fura-2-loaded EGs. Cytoplasmic Ca2+ increases within 30-120 s after addition of EH to EGs, and it remains elevated for at least 10 min, corresponding with the time course of secretion. Secretion is increased in dose-dependent manner by the Ca2+-ATPase inhibitor thapsigargin, a treatment that does not elevate glandular cGMP above basal levels. The secretory response to EH is partially inhibited in glands loaded with EGTA, while cGMP levels are unaffected. These findings suggest that EH activates second messenger cascades leading to cGMP accumulation and Ca2+ mobilization and/or influx and that both pathways are required for a full secretory response. cGMP activates a staurosporine-inhibitable protein kinase. We propose that Ca2+ acts via a parallel cascade with a time course that is similar to that for cGMP activation of a cGMP-dependent protein kinase.

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. (2-13C)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 13C in blood metabolites, including trehalose, the blood sugar of insects, and alanine. Pyruvate cycling was evident from the 13C enrichment of alanine C3, synthesized by transamination of pyruvate following carboxylation to oxaloacetate and cycling through phosphoenolpyruvate. Based on the relative 13C 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 13C enrichment in trehalose. The level of gluconeogenesis relative to glycolysis was indicated by the 13C 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 13C-enriched glucose in Manduca sexta. (2- 13C)Glucose or (1,2- 13C 2)glucose were administered to larvae by injection and after 6 h blood was analyzed by nuclear magnetic resonance spectroscopy. [2- 13C]Trehalose was the principal product of [2- 13C]glucose, but trehalose was also 13C-enriched at C1 and C3, demonstrating activity of the pentose phosphate pathway. The trehalose C1/C2 13C-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 13C-NMR signal structure of trehalose synthesized from [1,2- 13C 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 13C-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.

Soluble guanylyl cyclases in Caenorhabditis elegans: NO is not the answer

Soluble guanylyl cyclases in Caenorhabditis elegans: NO is not the answer

Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N.

Three types of binding assays were used to study the binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to brush border membrane vesicle (BBMV) membranes and a purified putative receptor of the target insect Manduca sexta. Using hybrid proteins consisting of Cry1Ac and the related Cry1C protein, it was shown that domain III of Cry1Ac is involved in specificity of binding as observed by all three techniques. In ligand blotting experiments using SDS-PAGE-separated BBMV proteins as well as the purified putative receptor aminopeptidase N (APN), the presence of domain III of Cry1Ac in a hybrid with Cry1C was necessary and sufficient for specific binding to APN. Using the surface plasmon resonance (SPR) technique with immobilized APN, it was shown that the presence of domain III of Cry1Ac in a hybrid is sufficient for binding to one of the two previously identified Cry1Ac binding sites, whereas the second site requires the full Cry1Ac toxin for binding. In addition, the role of domain III in the very specific inhibition of Cry1Ac binding by the amino sugar N-acetylgalactosamine (GalNac) was determined. Both in ligand blotting and in surface plasmon resonance experiments, as well as in binding assays using intact BBMVs, it was shown that the presence of domain III of Cry1Ac in a toxin molecule is sufficient for the inhibition of binding by GalNAc. These and other results strongly suggest that domain III of delta-endotoxins play a role in insect specificity through their involvement in specific binding to insect gut epithelial receptors.

Identification and Characterization of a Novel β Subunit of Soluble Guanylyl Cyclase That Is Active in the Absence of a Second Subunit and Is Relatively Insensitive to Nitric Oxide

Previously characterized soluble guanylyl cyclases form alpha-beta heterodimers that can be activated by the gaseous messenger, nitric oxide. In mammals, four subunits have been cloned, named alpha1, alpha2, beta1, and beta2. We have identified a novel soluble guanylyl cyclase isoform from the nervous system of the insect Manduca sexta that we have named M. sexta guanylyl cyclase beta3 (MsGC-beta3). It is most closely related to the mammalian beta subunits but has several features that distinguish it from previously identified soluble cyclases. Most importantly, MsGC-beta3 does not need to form heterodimers to form an active enzyme because guanylyl cyclase activity can be measured when it is expressed alone in COS-7 cells. Moreover, this activity is only weakly enhanced in the presence of the nitric oxide donor, sodium nitroprusside. Several of the amino acids in rat beta1 subunits, previously identified as being important in heme binding or necessary for nitric oxide activation, are substituted with nonsimilar amino acids in MsGC-beta3. There are also an additional 315 amino acids C-terminal to the catalytic domain of MsGC-beta3 that have no sequence similarity to any known protein. Northern blot analysis shows that MsGC-beta3 is primarily expressed in the nervous system of Manduca.

Long-term regulation of glucogenesis by dietary carbohydrate and relevance to blood sugar level in an insect Manduca sexta L.

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.

Glucogenesis in an insect, Manduca sexta L., estimated from the 13C isotopomer distribution in trehalose synthesized from [1,3- 13C 2]glycerol

Glucogenesis from [3- 13C]alanine and [1,3- 13C 2]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- 13C 2]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- 13C 2]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- 13C 2]glycerol-3-phosphate. The estimated total rates of glucogenesis varied from 0.33 to 2.80 μmol 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.

Expression of a 26S proteasome ATPase subunit, MS73, in muscles that undergo developmentally programmed cell death, and its control by ecdysteroid hormones in the insect Manduca sexta

MS73, an ATPase regulatory subunit of the 26S proteasome in the moth Manduca sexta, is shown to be expressed at a high level only in muscles that are undergoing developmentally programmed cell death, or which are destined to do so. The amount of MS73 is increased by more than two-fold just before death in each of three different muscles that die at different times, under different developmental controls. An ecdysteroid (moulting hormone) agonist, RH-5849, that prevents the occurrence of programmed cell death in two of these muscles also prevents the normally occurring rise in level of MS73 in these muscles. This evidence is consistent with a role for MS73 in programmed cell death.

Combined effects of night-time temperature and allelochemicals on performance of a Solanaceae specialist herbivore

:To assess the influence of temperature on the impact of allelochemicals on the growth of insect herbivores, we examined the interactions between allelochemicals and warmer night-time temperatures on an insect herbivore, Manduca sexta (L.) and tomato plants. We compared the effects of four thermal regimes with a standard day-time temperature of 26°C and the night-time temperature varying from 14 to 26°C by increments of 4°C. Increasing concentrations of chlorogenic acid and tomatine reduced developmental rate linearly. At 26:22°C, there was also a quadratic effect, such that intermediate concentrations had a disproportionately large negative effect. Furthermore, the negative effects of the allelochemicals were less at the 26:14°C regime. These results differ from another study examining a different insect species, which suggests that the response to incremental changes in night-time temperature will vary with the allelochemicals and insect species tested. However, some consistent patterns for M. sexta have emerged. The negative effects on caterpillar development of increasing allelochemical concentration changed across the gradient in night-time temperatures. At one thermal regime (26:22°C), M. sexta growth was disproportionately reduced by intermediate concentrations of each of three allelochemicals tested.

Merck Frosst award lecture 1995. La conference Merck Frosst 1995. Structural studies of lipoproteins and their apolipoprotein components.

Lipid transport processes via the circulatory system of animals are a vital function that utilizes highly specialized lipoprotein complexes. These complexes of protein and lipid impart solubility to otherwise insoluble lipids. The apoprotein components of lipoprotein complexes serve to stabilize the lipid components and modulate particle metabolism and function as ligands for receptor-mediated endocytosis of lipoproteins. We have used an insect (Manduca sexta) model system for studies of lipid transport. In this system, flight activity elicits a dramatic increase in the demand for glycerolipid fuel molecules by flight muscle tissue. These lipids are mobilized from a storage organ and transported through the hemolymph (blood) to the flight muscle by the lipoprotein, lipophorin. This system possesses the unique property that lipids are loaded onto pre-existing high density lipophorin through the action of a lipid transfer particle (LTP). LTP is a high molecular weight hemolymph component that facilitates net vectorial lipid transfer from fat body tissue to lipophorin. The increase in lipid content of the lipoprotein induces association of a low molecular weight amphipathic exchangeable apolipoprotein, apolipophorin III (apoLp-III). ApoLp-III is a 18 kDa protein that normally exists as a water-soluble monomeric hemolymph protein. The structural properties of apoLp-III have been investigated by X-ray crystallography. ApoLp-III from Locusta migratoria adopts a five helix bundle conformation wherein each of the amphipathic helices orients with its hydrophobic face directed toward the interior of the bundle. It has been hypothesized that lipid association requires a dramatic conformational change wherein the helix bundle opens about putative hinge domains located in the loops between helices. The data accumulated support the concept that apoLp-III is a member of the broad class of exchangeable apolipoproteins and structural information learned from this system is directly applicable to analogous proteins in higher organisms.

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.

Molecular Cloning of an Insect Aminopeptidase N That Serves as a Receptor for Bacillus thuringiensis CryIA(c) Toxin

The Bacillus thuringiensis CryIA(c) insecticidal delta-endotoxin binds to a 120-kDa glycoprotein receptor in the larval midgut epithelia of the susceptible insect Manduca sexta. This glycoprotein has recently been purified and identified as aminopeptidase N. We now report the cloning of aminopeptidase N from a M. sexta midgut cDNA library. Two overlapping clones were isolated, and their combined 3095-nucleotide sequence contains an open reading frame encoding a 990-residue pre-pro-protein. The N-terminal amino acid sequence derived from the glycoprotein is present in the open reading frame, immediately following a predicted cleavable signal peptide and a pro-peptide. There are four potential N-linked glycosylation sites. The C-terminal sequence contains a possible glycosylphosphatidylinositol (GPI) anchor signal peptide, which suggests that, unlike most other characterized aminopeptidases, the lepidopteran enzyme is anchored in the membrane by a GPI anchor. This was confirmed by partial release of aminopeptidase N activity from M. sexta midgut brush border membranes by phosphatidylinositol-specific phospholipase C. The deduced amino acid sequence shows significant similarity to the zinc-dependent aminopeptidase gene family, particularly in the region surrounding the consensus zinc-binding motif characteristic of these enzymes.

Identification and localization of two distinct microenvironments for the diacylglycerol component of lipophorin particles by 13C NMR

13C nuclear magnetic resonance spectroscopy of lipoproteins, isolated from the insect Manduca sexta, has been employed to probe the microenvironment of diacylglycerol (DG), their major neutral lipid component. Natural abundance 13C NMR spectra of high density lipophorin exhibited several well-separated resonances derived from its lipid moiety, including those for the carbonyl carbon atoms of phospholipid and DG fatty acyl chains in the region of 175-180 ppm. To verify the assignment of the DG acyl chain carbonyl carbon resonances, di[1-13C]oleoylglycerol high density lipophorin was isolated after instilling a bolus of tri[1-13C]oleoylglycerol into the midgut of larvae fed a fat-free diet. 13C NMR spectra of the isolated lipoprotein revealed a specific and dramatic enrichment of resonances at 175.5 ppm. Expansion of this region revealed two resonances separated by 0.08 ppm. These were assigned as 1,2- and 1,3- isomers of DG, the latter presumably arising from spontaneous acyl chain migration of 1,2-DG following lipoprotein isolation. On the basis of compositional and structural analysis of this lipoprotein, it is postulated that these DG species are localized predominantly in the hydrophobic core of the particle. By contrast, natural abundance 13C NMR spectra of the DG-rich, low density lipophorin (LDLp) subspecies revealed two additional resonances, separated by 0.2 ppm, that were tentatively assigned as 1,2- and 1,3-DG present at the surface of the particle. The verify this assignment, experiments employing phospholipase C, to convert lipophorin surface associated phospholipid into DG, were performed.(ABSTRACT TRUNCATED AT 250 WORDS)