Formation of insoluble and colloidally dispersed tannic acid complexes in the midgut fluid of Manduca sexta (Lepidoptera: Sphingidae): An explanation for the failure of tannic acid to cross the peritrophic envelopes of lepidopteran larvae

Magnesium and calcium ions, in concentrations comparable to those reported in the midgut fluids of lepidopteran larvae, bring about the precipitation of most of the tannic acid present in simple solutions buffered at pH 8.0 and 10.0, but not at pH 6.5. In contrast, when tannic acid is added to Manduca sexta midgut fluid, less than 31% of the tannic acid added to the gut fluid is converted to a form that can be centrifuged into a pellet. The rest remains in the supernatant solution in the form of a colloidal suspension. Very little of the tannic acid, if any, remains in true solution. We suggest that the tannic acid-containing phase that is produced when tannic acid is added to midgut fluid is a complex multi-molecular aggregate of indefinite chemical composition, incorporating varying amounts of tannic acid, surface-active phospholipids, proteins, and polyvalent metal ions. On the basis of this study, we further suggest that the failure of tannins to diffuse across the peritrophic envelopes of lepidopteran larvae is a result of the capacity of the peritrophic envelope to act as a physical barrier to insoluble and colloidally dispersed particles, not the presence of substances in the matrix that strongly adsorb polyphenols or the presence of an extensive network of fixed anionic sites in the matrix that acts as an electrostatic barrier to the passage of polyphenolate anions. Arch. Insect Biochem. Physiol. 39:109–117, 1998.© 1998 Wiley-Liss, Inc.     

Formation of phenol-protein complexes and their use by two stream invertebrates

Ethanol and water extracts of maple leaves and pine needles were analyzed for proteins, amino acids, sugars and phenolics. Leachates were mixed with a dissolved fungal cellulase. Within 24 h, insoluble particles formed, consisting of phenolics, proteins and amino acids. When exposed to an alkaline pH, or to a 0.04% solution of the surfactant lysolecithin, these particles released amino acids and proteins. Surface tensions of the gut fluids of Gammarus tigrinus and Tipula caloptera were considerably lower than that of distilled water, suggesting the presence of surfactants. Gut fluid of T. caloptera contained enzymes capable of digesting the proteins of particles formed with maple water extracts. The other particles did not appear susceptible to these enzymes. There was no evidence that G. tigrinus was able to digest the proteins of any of the particles examined.     

The immune properties of Manduca sexta transferrin

Transferrins are secreted proteins that bind iron. The well-studied transferrins are mammalian serum transferrin, which is involved in iron transport, and mammalian lactoferrin, which functions as an immune protein. Lactoferrin and lactoferrin-derived peptides have bactericidal activity, and the iron-free form of lactoferrin has bacteriostatic activity due to its ability to sequester iron. Insect transferrin is similar in sequence to both serum transferrin and lactoferrin, and its functions are not well-characterized; however, many studies of insect transferrin indicate that it has some type of immune function. The goal of this study was to determine the specific immune functions of transferrin from Manduca sexta (tobacco hornworm). We verified that transferrin expression is upregulated in response to infection in M. sexta larvae and determined that the concentration of transferrin in hemolymph increases from 2 μM to 10 μM following an immune challenge. It is also present in molting fluid and prepupal midgut fluid, two extracellular fluids with immune capabilities. No immune-induced proteolytic cleavage of transferrin in hemolymph was observed; therefore, M. sexta transferrin does not appear to be a source of antimicrobial peptides. Unlike iron-saturated lactoferrin, iron-saturated transferrin had no detectable antibacterial activity. In contrast, 1 μM iron-free transferrin inhibited bacterial growth, and this inhibition was blocked by supplementing the culture medium with 1 μM iron. Our results suggest that M. sexta transferrin does not have bactericidal activity, but that it does have a bacteriostatic function that depends on its iron sequestering ability. This study supports the hypothesis that insect transferrin participates in an iron withholding strategy to protect insects from infectious bacteria.     

Differential Effects of pH on the Pore-Forming Properties of Bacillus thuringiensis Insecticidal Crystal Toxins

The effect of pH on the pore-forming ability of two Bacillus thuringiensis toxins, Cry1Ac and Cry1C, was examined with midgut brush border membrane vesicles isolated from the tobacco hornworm, Manduca sexta, and a light-scattering assay. In the presence of Cry1Ac, membrane permeability remained high over the entire pH range tested (6.5 to 10.5) for KCl and tetramethylammonium chloride, but was much lower at pH 6.5 than at higher pHs for potassium gluconate, sucrose, and raffinose. On the other hand, the Cry1C-induced permeability to all substrates tested was much higher at pH 6.5, 7.5, and 8.5 than at pH 9.5 and 10.5. These results indicate that the pores formed by Cry1Ac are significantly smaller at pH 6.5 than under alkaline conditions, whereas the pore-forming ability of Cry1C decreases sharply above pH 8.5. The reduced activity of Cry1C at high pH correlates well with the fact that its toxicity for M. sexta is considerably weaker than that of Cry1Aa, Cry1Ab, and Cry1Ac. However, Cry1E, despite having a toxicity comparable to that of Cry1C, formed channels as efficiently as the Cry1A toxins at pH 10.5. These results strongly suggest that although pH can influence toxin activity, additional factors also modulate toxin potency in the insect midgut.     

Major hemolymph proteins from larvae of the black swallowtail butterfly,Papilio polyxenes

Three major hemolymph proteins of Papilio polyxenes larvae were isolated and characterized. Density gradient ultracentrifugation of hemolymph resulted in flotation of the major lipoprotein, lipophorin. P. polyxenes larval lipophorin is composed of two apoproteins, apolipophorin-I and apolipophorin-II, plus a mixture of lipids, to give a density of 1.13 g/ml. Immunoblotting experiments using antisera directed against Manduca sexta apolipophorin-I and apolipophorin-II, respectively, revealed cross-reactivity of apoLp-I with Manduca sexta apoLp-I, and apoLp-II with M. sexta apoLp-II. Gel permeation chromatography of the subnatant obtained following density gradient ultracentrifugation revealed the presence of a major protein peak which was shown to contain three major serum proteins, two of which were isolated and characterized. One of these proteins was purified by lectin affinity chromatography. Both proteins have native molecular weights in the range of 450,000 and appear to be hexamers of a single subunit type. Major serum protein-1 is nonglycosylated and has a subunit molecular weight of 75,000. Major serum protein-2 is glycosylated and has a subunit molecular weight of 74,000. Amino acid analysis of this protein revealed a tyrosine plus phenylalanine content of 20 mole percent, characteristic of the arylphorin class of insect storage proteins. Using antibodies against M. sexta larval hemolymph proteins, both the P. polyxenes major serum proteins were shown to be immunologically related to serum proteins of other lepidopteran species.     

Brush border membrane and amino acid transport

Fundamental differences in midgut structure, physiology, brush border proteins, and transporters among Leptinotarsa decemlineata, lepidopteran caterpillars, other insect taxa, and vertebrates are reviewed. The effects of dietary protein concentration on Manduca sexta midgut amino acid transport and brush border membrane proteins are reported. M. sexta fed diet with reduced protein had elevated levels of leucine aminopeptidase in the brush border membrane. No changes in amino acid transport or alkaline phosphatase activity due to dietary differences were detected. Changes in brush border proteins could affect the toxicity and pathogenicity of microbial agents. © 1996 Wiley-Liss, Inc.     

Properties of Manduca sexta chitinase and its C-terminal deletions

Manduca sexta (tobacco hornworm) chitinase is a molting enzyme that contains several domains including a catalytic domain, a serine/threonine-rich region, and a C-terminal cysteine-rich domain. Previously we showed that this chitinase acts as a biopesticide in transgenic plants where it disrupts gut physiology. To delineate the role of these domains further and to identify and characterize some of the multiple forms produced in molting fluid and in transgenic plants, three different forms with variable lengths of C-terminal deletions were generated. Appropriately truncated forms of the M. sexta chitinase cDNA were generated, introduced into a baculovirus vector, and expressed in insect cells. Two of the truncated chitinases (Chi 1-407 and Chi 1-477) were secreted into the medium, whereas the one with the longest deletion (Chi 1-376) was retained inside the insect cells. The two larger truncated chitinases and the full-length enzyme (Chi 1-535) were purified and their properties were compared. Differences in carbohydrate compositions, pH–activity profiles, and kinetic constants were observed among the different forms of chitinases. All three of these chitinases had some affinity for chitin, and they also exhibited differences in their ability to hydrolyze colloidal chitin. The results support the hypothesis that multiple forms of this enzyme occur in vivo due to proteolytic processing at the C-terminal end and differential glycosylation.     

Degradation of aberrant proteins by larval tobacco hornworm,Manduca sexta L. (Sphingidae)

The tobacco hornworm Manduca sexta (Sphingidae) readily incorporates L-canavanine, the L-2-amino-4-(guanidinooxy)butyric acid structural analog of L-arginine, into newly synthesized proteins. As a result, the developing fifth-instar larva produces structurally aberrant canavanyl proteins that can exhibit severely impaired function. This situation is exacerbated by canavanine's ability to stimulate de novo protein synthesis. M. sexta larvae can respond to anomalous protein production by degrading canavanyl proteins nearly five times faster than normal proteins. The proteases of this insect can distinguish between normal and anomalous proteins and thereby avoid destruction of essential macromolecules. Aberrant protein degradative activity is not dependent upon de novo protein synthesis induced by canavanyl proteins. The fat body appears to be the source of proteases that degrade aberrant proteins; degradation is curtailed in the presence of sulfhydryl protease inhibitors as well as inhibitors of trypsin-like activity.     

The biochemical basis of antimicrobial responses in Manduca sexta

Innate immunity is essential for the wellbeing of vertebrates and invertebrates. Key components of this defense system include pattern recognition receptors that bind to infectious agents, extra-and intra-cellular proteins that relay signals, as well as molecules and cells that eliminate pathogens. We have been studying the defense mechanisms in a biochemical model insect, Manduca sexta. In this insect, hemolin, peptidoglycan recognition proteins, β-1,3-glucan recognition proteins and C-type lectins detect microbial surface molecules and induce immune responses such as phagocytosis, nodulation, encapsulation, melanization and production of antimicrobial peptides. Some of these responses are mediated by extracellular serine proteinase pathways. The proteolytic activation of prophenoloxidase （proPO） yields active phenoloxidase （PO） which catalyzes the formation of quinones and melanin for wound healing and microbe killing. M. sexta hemolymph proteinase 14 （HP 14） precursor interacts with peptidoglycan or β-1,3-glucan, autoactivates, and leads to the activation of other HPs including HP21 and proPO-activating proteinases （PAPs）. PAP-1, -2 and -3 cut proPO to generate active PO in the presence of two serine proteinase homologs. Inhibition of the proteinases by serpins and association of the proteinase homologs with bacteria ensure a localized defense reaction. M. sexta HP1, HP6, HP8, HP17 and other proteinases may also participate in proPO activation or processing of spatzle and plasmatocyte spreading peptide.     

Tannins in plant-herbivore interactions

Tannins are the most abundant secondary metabolites made by plants, commonly ranging from 5% to 10% dry weight of tree leaves. Tannins can defend leaves against insect herbivores by deterrence and/or toxicity. Contrary to early theories, tannins have no effect on protein digestion in insect herbivores. By contrast, in vertebrate herbivores tannins can decrease protein digestion. Tannins are especially prone to oxidize in insects with high pH guts, forming semiquinone radicals and quinones, as well as other reactive oxygen species. Tannin toxicity in insects is thought to result from the production of high levels of reactive oxygen species. Tannin structure has an important effect on biochemical activity. Ellagitannins oxidize much more readily than do gallotannins, which are more oxidatively active than most condensed tannins. The ability of insects to tolerate ingested tannins comes from a variety of biochemical and physical defenses in their guts, including surfactants, high pH, antioxidants, and a protective peritrophic envelope that lines the midgut. Most work on the ecological roles of tannins has been correlative, e.g., searching for negative associations between tannins and insect performance. A greater emphasis on manipulative experiments that control tannin levels is required to make further progress on the defensive functions of tannins. Recent advances in the use of molecular methods has permitted the production of tannin-overproducing transgenic plants and a better understanding of tannin biosynthetic pathways. Many research areas remain in need of further work, including the effects of different tannin types on different types of insects (e.g., caterpillars, grasshoppers, sap-sucking insects).     

Surfactants in the gut fluids of Porcellio scaber (Isopoda: Oniscidea), and their interactions with phenolics

Fluids from the gut lumen of Porcellio scaber showed significantly reduced surface tension compared to a buffer solution. Tests with several dilutions indicated that the concentration of the surface active substances (surfactants) was about 80-fold higher than the 'critical micelle concentration'. Phenolics, e.g. gallotannins, when ingested in the diet increased the surface tension of the gut fluid, indicating reduced concentrations of free surfactants. The significance of gut surfactants in P. scaber, their role in digestive processes, and their interaction with tannins in this saprophagous soil arthropod are discussed.     

Chemical and immunological properties of lipophorins from seven insect orders

The major insect hemolymph lipoprotein, lipophorin, was isolated from adults of eight insect species representing seven insect orders. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to compare their respective apoprotein components. In all species examined lipophorin was composed of at least two apoproteins, apolipophorin I (M_r ～ 250,000) and apolipophorin II (M_r ～ 78,000), and two species had a third apoprotein, apolipophorin III (M_r ～ 17,000). The density of each isolated lipophorin was determined from the refractive index of KBr following density gradient centrifugation. Immunoblotting with anti-larval Manduca sexta apolipophorin I and II of the apoproteins separated by SDS-PACE indicated cross reactivity between anti-M sexta apoLp-ll and apoLp-ll in all species tested. Anti-M sexta apoLp-l exhibited no cross reactivity for any species tested. Fluorescent lectin staining of the apoproteins separated on SDS-PAGE gels revealed the presence of covalently bound carbohydrate residues.     

Comparison of the localization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1A δ-endotoxins and their binding proteins in larval midgut of tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry toxins on lepidopterans. The proteins, which bind Bt toxins to midgut epithelial cells, are key factors involved in the insecticidal functions of the toxins. Three Cry1A-binding proteins, viz., aminopeptidase N (APN), the cadherin-like Bt-R₁, and membrane-type alkaline phosphatase (m-ALP), were localized, by immunohistochemistry, in sections from the anterior, middle, and posterior regions of the midgut from second instar M. sexta larvae. Both APN and m-ALP were distributed predominantly along microvilli in the posterior region and to a lesser extent on the apical tip of microvilli in the anterior and middle regions. Bt-R₁ was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle regions. Thus, binding sites for Bt proteins and Cry1A toxins are co-localized on the microvilli of M. sexta midgut epithelial cells.     

A Novel Insecticidal Toxin from Photorhabdus luminescens, Toxin Complex a (Tca), and Its Histopathological Effects on the Midgut of Manduca sexta

Photorhabdus luminescens is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from Bacillus thuringiensis (δ-endotoxins and Vip’s), P. luminescens toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran Manduca sexta. In view of the difference in the normal mode of delivery of P. luminescens toxin and the apparent communality in the histopathological effects of other gut-active toxins from B. thuringiensis, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of M. sexta. Here we report that the histopathology of the M. sexta midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by M. sexta, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.     

Regulation of fat body glycogen phosphorylase activity during refeeding in Manduca sexta larvae

In 12-h-starved larvae of the tobacco hornworm, Manduca sexta, fat body glycogen phosphorylase was quickly inactivated when insects were refed with normal diet and agar which contained 3% sucrose. Only the first 2 min of refeeding were necessary to induce enzyme inactivation. During this short period, larvae did not ingest enough sucrose to increase the hemolymph glucose concentration. This may indicate that the gut released a hormone(s) which directly or indirectly led to the inactivation of fat body glycogen phosphorylase. Inactivation of the enzyme could also be induced by injection of glucose (30 mg) into the hemolymph of starving M. sexta larvae suggesting that there may be separate control from a neuroendocrine site such as the brain or the corpora cardiaca. Trehalose was less effective. Bovine insulin (2 and 4 μg/starved larva) did not induce phosphorylase inactivation over 20 min or decrease hemolymph carbohydrate or lipid concentrations within 60 min. It is, therefore, necessary to screen insect tissues for substances which could bring about inactivation of fat body glycogen phosphorylase. © 1992 Wiley-Liss, Inc.     

Functional interpretation of APN receptor from M.sexta using a molecular model

Insect pests are the major cause of damage to commercially important agricultural crops. The continuous application of synthetic pesticides resulted in severe insect resistance by plants. This causes irreversible damage to the environment. Bacillus thuringiensis (Bt) emerged as a valuable biological alternative in pest control. However, insect resistance against Bt has been reported in many cases. Insects develop resistance to insecticides through mechanisms that reduce the binding of toxins to gut receptors. Nonetheless, the molecular mechanism of insect resistance is not fully understood. Therefore, it is important to study the mechanism of toxin resistance by analyzing amino‐peptidase‐N (APN) receptor of the insect M. sexta. A homology model of APN was constructed using Insight II molecular modeling software and the model was further evaluated using the PROCHECK program. Oligosaccharides participating in post translational modification were constructed and docked onto specific APN functional sites. Post analyses of the APN model provide insights on the functional properties of APN towards the understanding of receptor and toxin interactions. We also discuss the predicted binding sites for ligands, metals and Bt toxins in M. sexta APN receptor. These data help in the development of a roadmap for the design and synthesis of novel insect resistant Cry toxins.     

Mode of action studies on a Manduca sexta diuretic hormone

The mode of action of a diuretic hormone from pharate adult Manduca Sexta heads, which triggers fluid loss in M. sexta larvae and Pieris rapae adults, was studied. In vivo, Mas-DH (M. sexta diuretic hormone) decreased fluid absorption from larval recta, and increased levels of the second messenger cAMP in recta and Malpighian tubules (Mt) from larvae, and in fat body of larvae and adult M. sexta. In vitro, Mas-DH triggered minor changes in fluid loss from adult Mt, but did not affect levels of cAMP in Mt from larvae, pharate adults, or adults, though it elevated cAMP levels in fat body of these stages. © 1992 Wiley-Liss, Inc.