Tobacco Hornworm Larvae Research Articles

A hormone from the corpora cardiaca controls fat body glycogen phosphorylase during starvation in tobacco hornworm larvae

Steele has demonstrated the presence of a factor in the corpora cardiaca (Cc) of the adult male cockroach, Periplaneta americana, which increases the trehalose concentration in haemolymph1. This factor, which was shown to be a peptide2, activates fat body glycogen phosphorylase3, and glucose-1-phosphate which is liberated from glycogen is used for trehalose synthesis within the fat body. Peptides having similar properties were found in several other insects, including bees2, locusts4, stick insects5,6 and a lepidopteran, Manduca sexta (tobacco hornworm)7. These peptides have been called hyperglycaemic1 or phosphorylase activating factor7 or trehalogon8. Their physiological significance during an insect's life cycle is unknown9,10, and consequently it is uncertain whether these compounds are hormones or not. We have examined the activity of fat body glycogen phosphorylase during late larval development of M. sexta11. During the moult from the fourth to the fifth instar, when the animals were unable to ingest food for a period of about 25 h, 45% of the enzyme was in an active form. Before and after the moult, only 10% of the enzyme was active. We suggested that reduced feeding activity leads to the activation of fat body glycogen phosphorylase by releasing the phosphorylase activating peptide from the Cc11. We now substantiate this hypothesis in reporting results obtained with fifth instar larvae of M. sexta that were starved during their normally active feeding period.

Isolation of separate apical, lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.

Juvenile hormone titres and metabolism during starvation-induced supernumerary larval moulting of the tobacco hornworm, Manduca sexta L.

When tobacco hornworm ( manduca sexta) larvae are starved for 5 days immediately after ecdysis to the 5th instar, then fed normal diet, they undergo a supernumerary moult instead of metamorphosis. During starvation the titre of juvenile hormone in the haemolymph increased to a maximum of 3 ng juvenile hormone I equivalents/ml (determined by the black Manduca larval bioassay) on the fourth day of starvation, then began a decline which continued through the subsequent feeding period. The changes in juvenile hormone titre were not attributable to changes in haemolymph volume during starvation (only a 5% decrease) and subsequent feeding. During starvation the esterase activity of the haemolymph declined 4-fold with a 2-fold larger decrease in the DFP-insensitive, presumably juvenile hormone specific, esterase activity. Both the total and the juvenile hormone-specific esterase activity then increased as a function of larval weight during the subsequent feeding period. As growth was slow in the prolongedly starved larvae, sufficient juvenile hormone was present at the time of prothoracicotropic hormone (PTTH) and ecdysteroid release at the beginning of the fourth day of feeding to prevent metamorphosis.

Effects of L-canavanine on concentrations of cyclic amp and cyclic GMP in the midgut of Manduca sexta (sphingidae: lepidoptera)

1. Cyclic AMP concentrations of midgut epithelial tissue taken from tobacco hornworm larvae fed a 2.5 mM L-canavanine plus 25 mM L-arginine-supplemented diet (CAAM) were higher than in larvae fed either a 2.5 mM canavanine-supplemented diet (CAV) or control diet. 2. Cyclic AMP accumulation in CAAM-treated larvae over a 6-day period (10.43 pmol/mg protein) was followed by an abrupt decline within 1 day as the larvae entered the wandering stage (4.92 pmol/mg protein). 3. Cyclic GMP concentrations were low (1.10 or less pmol/mg protein) for all treatments during the active feeding period but doubled (2.19 pmol/mg protein) at the beginning of the wandering larval stage. 4. Cyclic GMP concenrations were unaffected by any of the experimental diets.

Comparison of Potassium Transport in Three Structurally Distinct Regions of the Insect Midgut

ABSTRACT Active potassium transport across the isolated midgut of the Tobacco Hornworm larva, Manduca sexta, was studied by measuring the short circuit current (Isc) and unidirectional 42-potassium fluxes. The midgut is composed of structurally distinct anterior, middle and posterior regions, all of which are shown to transport potassium, so that by comparing and contrasting their structural and functional properties new information on the mechanism of midgut potassium transport was obtained. It has previously been shown that the potassium pump is located on the apical membrane of the goblet cell. In the anterior and middle regions of the midgut the goblet cell has a large cavity and mitochondria are closely associated with the apical membrane while in the posterior midgut the goblet cavity is much smaller, and mitochondria are not associated with the apical membrane. However, the apical membrane particles which have been implicated in active potassium transport in a number of other insect epithelia are present in all three regions. This observation suggests that the particles are a structural requirement for active transport, and that close association between mitochondria and the transporting membrane is not essential. Comparison of the kinetic influx pool size and the differences in the Isc decay profiles between the three midgut regions suggest that part of the influx pool is a transported pool located in the goblet cavity. A new model to explain the driving force for potassium transport in the midgut is proposed, in which the rate of potassium transport controls the entrance of potassium into the cell, rather than the opposite, currently accepted view.

Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains.

Parasporal crystals of Bacillus thuringiensis subspp. kurstaki, tolworthi, alesti, berliner, and israelensis were compared by electron microscopy, polyacrylamide gel electrophoresis, amino acid analysis, tryptic peptide mapping, immunological analysis, and insecticidal activity. Spore coats also were compared by polyacrylamide gel electrophoresis. B. thuringiensis subsp. israelensis crystals were lethally toxic to mosquito larvae and nontoxic to tobacco hornworm larvae. Conversely, crystals from the other subspecies killed tobacco hornworm larvae but were ineffective against mosquitoes. Crystalline inclusion bodies of all subspecies contained a protoxic subunit that had an apparent molecular weight of approximately 1.34 X 10(5). However, polyacrylamide gel electrophoretic patterns of solubilized crystals revealed a small-molecular-weight component (apparent molecular weight, 26,000) in B. thuringiensis subsp. israelensis that was absent in the other subspecies. Also, differences were noted in amino acid composition and tryptic peptide fingerprints. Crystal proteins were found in spore coats of all subspecies. The results suggest that insecticidal specificity is due to unique polypeptide toxins.

Characterization of spore coat proteins of Bacillus thuringiensis and Bacillus cereus

Abstract 1. 1. Spore coat extracts from Bacillus thuringiensis subspecies kurstaki and israelensis and Bacillus cereus T and B. cereus NRRL 569 were characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by amino acid analysis. 2. 2. Both B. cereus spore coats had similar electrophoretic profiles. 3. 3. The B. thuringiensis spore coats contained crystal proteins as major components as well as lower mol. wt proteins. 4. 4. B. thuringiensis subsp. israelensis had a unique coat protein profile which was different from B. cereus and B. thuringiensis subsp. kurstaki coats. 5. 5. Insecticidal activity of spores against the tobacco hornworm, Manduca sexta, and the mosquito, Aedes aegypti, also was determined. 6. 6. B. thuringiensis subsp. kurstaki spores were lethally toxic to the tobacco hornworm (Lepidoptera) larvae, whereas spores of the other subspecies were not. 7. 7. Except for subspecies israelensis, none of the spores was effective against the mosquito (Diptera) larvae.

Effects of l-canavanine on arginine catabolism in manduca sexta (sphingidae: lepidoptera)

1. 1. Hemolymph ornithine concentrations in tobacco horn worm larvae fed a 2.5 mM l -canavanine plus 25 mM l -arginine-supplemented artificial diet (CAAM) were higher than those in larvae fed diets supplemented with 2.5 mM canavanine (CAV), 25 mM arginine (ARG), or controls (CON). 2. 2. Ornithine concentrations in CAV-treated larvae were significantly greater than the control or ARG treatment, but less than the CAAM treatment during the latter part of the wandering larval stage and during the pharate pupal stage. 3. 3. Urea concentrations were greater during the active feeding stage with the CAAM- and ARG-treated larvae having significantly higher levels than control or CAV-treated larvae. 4. 4. Urea concentrations in all treatments never exceeded 36.5% of the ornithine concentration. 5. 5. Canavanine concentrations were higher in CAV-treated larvae than in CAAM-treated larvae. 6. 6. During active feeding, arginine concentrations for all treatments were similar, but were lower in CAV- and CAAM-treated larvae during the pharate pupal stage.

EFFECTS OF JUVENILE HORMONE ON ECDYSONE-DEPENDENT DEVELOPMENT IN THE TOBACCO HORNWORM,MANDUCA SEXTA

1. Juvenile hormone delayed or prevented the onset of metamorphosis by neck-ligated fourth-instar tobacco hornworm larvae and by fifth instars neck-ligated prior to the cessation of feeding.2. After the onset of the wandering period J H had precisely the opposite effects in that it accelerated the onset of metamorphosis. This was the case both for wandering larvae and for pupae, irrespective of whether they were intact or subjected to brain removal or neck ligation.3. These findings point to a previously unsuspected shifting role of JH in the control of metamorphosis.4. Fourth-instar larvae underwent no further development after brain removal unless they were also effectively allatectomized by neck ligation. Evidently the secretion and inhibitory action of JH persists in brainless fourth-instar larvae.5. Brainless fifths, by contrast, were often able to initiate metamorphosis despite the continued presence of their CA. Development could be prevented by daily application of JH. The disappearance of effective levels of JH, which normally occurs in the late fifth instar as a necessary prelude to metamorphosis, can evidently take place in the absence of the brain. This implies that the brain is not prerequisite for the decline in JH.6. Since the ability of JH first to delay and subsequently to accelerate the development of intact individuals also persists after brain removal, the brain and its PTTH cannot be the sole targets for these effects of JH, if in fact they are targets at all.7. The shifting effects of JH on development appear to be ecdysone-dependent. Four possible sites of JH action are identified.

Possible origin and function of the parasporal crystals in [formula omitted

Acrystalliferous strains of Bacillus thuringiensis subsp. kurstaki were isolated at a high frequency following heat treatment of spores. Spores of these strains lacked a 130,000 dalton glycoprotein, the major component common to both parasporal crystals and coats and were nontoxic to tobacco hornworm larvae. Moreover, the deficiency of this glycoprotein resulted in lysozyme sensitivity of the spores of some mutants and the presence of new spore coat proteins in others. All nontoxic acrystalliferous mutants lacked the complete array of at least six plasmids present in the wild type, implying a relationship between presence of plasmid(s) and toxicity. The unique capacity of this species to alter the surface coating of spores which appears to be related to crystal formation may provide flexibility for germination and growth in diverse soil environments.

Reduction of Manduca sexta Fecundity and Fertility by L-Canavanine122

Agar-based artificial diets containing various concentrations of L-canavanine werc fed to 5th-instar tobacco hornworm larvae. The number of chorionated oocytes in the 4-day-old moths, resulting from these larvae, was concentration-dependent between 0.75 and 2.5 mM canavanine. The mean number of chorionated oocytes from the 2.5 mM treatment was only 7.5% of that obtained from the untreated control. Fecundity and fertility of moths from 1 mM canavanine were 14 and 21% of untreated control values, respectively. Matings between treated and untreated moths show that only female reproductive capacity was significantly reduced by canavanine under these conditions.

Hormonal control of epidermal detachment during the final feeding stage of the tobacco hornworm larva

During the final feeding stage of the tobacco hornworm, Manduca sexta, the abdominal epidermis was found to undergo a transient apolysis coincident with the first surge of ecdysone that initiates metamorphosis. During this detachment, the epidermal cells become committed to form pupal cuticle, but morphological and histological studies indicated that larval endocuticle was still being deposited. The cells reattached to the larval cuticle with the decline in ecdysone titre. Infusion of as little as 0.01 μg β-ecdysone ( ca. 2.6 ng/ml haemolymph) over an 8-hr period into larval abdomens isolated before the ecdysone surge was sufficient to initiate this detachment provided that juvenile hormone was absent.

Laboratory Studies on the Antifeeding Effect of a Fungicide, Guazatine, on Eleven Species of Phytophagous Insects

Effects of a fungicide, Guazatine, designated SN 513 (9-aza, 1, 17 diguanidinoheptadecane triacetate, Keno Card AB, Stockholm, Sweden), on feeding of various insect species were investigated in the laboratory. The reduction in feeding of all species tested, except the southern green stink bug, Nezara viridula (L.), and the velvetbean caterpillar, Anticarsia gemmatalis Hübner, was significant, predictable, and directly related to the log 10% concentration. Insects listed in approximate order of most to least sensitive to SN 513 were the Mexican bean beetle adult, Epilachna varivestis Multan, Colorado potato beetle adult and Lana, Leptinotarsa decemlineata (Say), tobacco hornworm larva, Manduca sexta (L.), cabbage looper larva, Trichoplusia ni (Hübner), corn earworm larva, Heliothis zea (Baddie), diamondback moth larva, Plutella xylostella (L.), beet armyworm larva, Spodoptera exigua (Hübner), brown stink bug adult, Euschistus servus (Say), and soybean looper larva, Pseudoplusia incluclens (Walker).

An unusual effect of N-octylamine on the cytochrome b5 spectrum of insect and mammalian microsomes

The addition of n-octylamine to microsomes prepared from the midgut of tobacco hornworm ( Manduca sexta) larvae causes an unusual spectral interaction. The initial optical difference spectrum appears to be the sum of reduced cytochrome b 5 and a type II difference spectrum of cytochrome P-450. This initial spectrum is unstable and diminishes in size, with a concurrent shift in peak (424 to 428 nm) and trough (409 and 392 to approx. 400 nm) positions, to yield a stable spectrum identical to the type II spectrum of cytochrome P-450. Thus, in addition to its interaction with cytochrome P-450, n-octylamine causes a reduction of cytochrome b 5 which subsequently becomes reoxidized. The casual factor for this unusual spectral interaction occurs in the cytoplasm and appears to be protein-bound. It was also present in similar preparations from the tobacco budworm ( Heliothis virescens) but not in those from rat or mouse liver or abdomens from insecticide-resistant or susceptible houseflies ( Musca domestica). Microsomes from rat and mouse liver, but not those from housefly abdomens, exhibit similar unusual spectral interactions with n-octylamine when supplemented with the soluble factor from the hornworm.

Some Factors Influencing Development, Behavior and Survival of Hornworms on Postharvest Tobacco1,2

Foliage consumption and survival of late-stage larvae of the tobacco hornworm, Manduca sexta (L.), were studied in relation to intraspecific competition and parasitization by Apanteles congregatus (Say) on postharvest tobacco. Over 70% of 5th-stage larvae which had attained only 1/2 the expected prepupal weight were able to pupate. Reductions in feeding were as much as 20-fold for some parasitized larvae. The rate of development and survival of hornworm larvae were affected by the amount and quality of sucker growth, as influenced by the preharvest use of maleic hydrazide (a systemic plant growth inhibitor). Depletion of foliage under heavy feeding pressure induced feeding on flowers and unopened seed heads, food of marginal suitability for development and survival. Compensation by the host plant for foliage loss to hornworm feeding was displayed by an increased succulence of foliage and an abundance of more suckers following defoliation.

Requirements for molting of the crochet epidermis of the tobacco hornworm larva in vivo and in vitro.

In the tobacco hornworm,Manduca sexta, the epidermis which underlies the larval crochets is the first tissue to become independent of the prothoracic glands (PG) in a larval molt. In each successive larval molt, crochet forming cells increase in size, form hooks at their distal ends and, finally, secrete cuticle. This paper examines the endocrine requirements for competence to molt and describes parallel cultures in vivo and in vitro to define the hormonal control of crochet molting. When implanted into a fourth instar host larva prior to initiation of the last larval molt, competent crochet epidermis molted, forming crochets synchronously with its host. In the fourth instar, competence to form crochets is attained slowly during the first two days following ecdysis from the third instar. During the feeding phase of the fifth (last) instar, the crochet epidermis remains competent to molt (to form an extra "sixth instar" set of crochets) until the larva attains a weight of about 4.5 gm. Then, concurrent with the decline in the titer of juvenile hormone (JH) in the hemolymph, competence to form crochets declines. A similar loss of competence did not occur when fourth instar crochet epidermis was exposed to a declining JH titer by culture in either fourth instar isolated abdomens for 72 h or in fifth instar host larvae between 4 and 7 gm. Responses of crochet epidermis cultured in vitro also were examined. Competent fourth instar crochet epidermis formed crochets following 3-6 h exposure to ecdysone in vitro. Six ×10-7M β-ecdysone was required for 50% response, whereas a 10-50-fold higher concentration of α-ecdysone was necessary. Although formation of morphologically complete crochets in vitro proceeded with similar time course to that in situ, no molt-induced growth occurred in vitro. When crochet epidermis was exposed to ecdysone in vitro immediately after explantation, exogenous JH was not required for molting. But when tissue was first cultured for 72 h without hormones, subsequent molting in vitro could not be elicited, although molting still could occur when the tissue subsequently was implanted into a fourth instar host. Exposure to corpora allata or to JH during the 72 h of culture in vivo partially prevented the loss in capacity to respond to ecdysone in vitro, suggesting that JH may be one factor involved directly or indirectly in maintenance of tissue responsiveness.

Larval Manduca sexta Hemolymph Carboxylesterase Activity During Chronic Exposure to Insecticide-containing Diets123

Tobacco hornworm larvae were fed diets containing 1, 2 or 4 ppm monocrotophos, methomyl or endosulfan. Total hemolymph serum protein was elevated by 1 ppm monocrotophos, but 2 and 4 ppm reduced the level when compared with the control. Similar, but less distinct, responses were observed in respect to methomyl and endosulfan. The specific activity and total serum carboxylesterase increased in tobacco hornworms fed 1 ppm monocrotophos-containing diet, but the specific activity was reduced at 2 and 4 ppm. Methomyl and endosulfan had no effect on carboxylesterase specific activity. Disc electrophoresis of hemolymph serum revealed 6 carboxylesterase-positive bands, the activity of which were differentially affected by the insecticide treatments.

Olfactory capabilities of the ?gustatory? chemoreceptors of the tobacco hornworm larvae

Gustatory chemoreceptors on the maxillae ofManduca sexta responded to natural stimuli from distances of up to 600 μm (average is 100 μm). In the lateral sensilla styloconica, at least three of the four known chemoreceptive cells responded, indicating that different compounds could be involved in the stimulus. The medial sensilla did not show a comparable olfactory capability. Thus, chemoreceptors classed as contact receptors on a morphological basis (thick walls, single apical pore) were responsive to vapors of normal food substances. Adaptation of the receptor was observed prior to contact with the stimulus. This has important implications for experiments on gustatory receptors.

Non-protein amino acid-insect interactions—I. Growth effects and symptomology of l-canavanine consumption by tobacco hornworm, Manduca sexta (L.)

1. 1. Incorporation of l-canavanine, a naturally occurring structural analogue of arginine, into the artificial diet of the tobacco hornworm larvae, Manduca sexta (L.), produced severely toxic effects. 2. 2. The toxic effects included enhanced larval mortality, decreased larval growth rates and malformed pupae and adults. 3. 3. Larval response to canavanine was concentration dependent at least over a range of 3–45 mM. 4. 4. Diets supplemented with arginine, ornithine, glycine and lysine were not toxic to fifth instar hornworms.

Studies on hypo- and hypercholesterolemia induced in insects by filipin

The polyene antibiotic, filipin, can produce both hypocholesterolemic and hypercholesterolemic effects in insects. The type of cholesterolemia induced is dependent on the dietary molar ratio of filipin to cholesterol. When present in the diet at a filipin to cholesterol ratio of 38 : 1, cholesterol uptake by Manduca sexta larvae is reduced 99% and the concentration of haemolymph cholesterol is lowered 90%. However, at a dietary filipin to cholesterol ratio of 1 : 2, cholesterol uptake by tobacco hornworm larvae ( M. sexta) is increased by 50% but the haemolymph cholesterol concentration is lower than control values. In addition, the concentration of sterol-carrying lipoproteins in the haemolymph is increased two-fold. At filipin to cholesterol ratios of 38 : 1, filipin increases excretion of unesterified cholesterol 8-fold. No effect on cholesterol esterification is noted with filipin, but cholesterol-sulfate excretion is reduced by 60 per cent. Excess dietary cholesterol (filipin to cholesterol ratio of 1 : 2) prevents these alterations in cholesterol excretion, but cholesterol-sulfate excretion remains lower than control values. Filipin is not absorbed in the digestive tract of Galleria mellonella or M. sexta larvae. More than 99% of ingested dietary [ 14C]filipin appears in the faeces. Most of the remaining radioactivity is found bound to the intestine. Injected filipin is rapidly excreted; 95 per cent of the injected [ 14C]filipin appears in faeces in less than 2 hr. Filipin is not metabolized to CO 2.