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PubMed1975

Cyclic nucleotide-stimulable protein kinases in the central nervous sytem of Manduca sexta.

Cyclic nucleotide-stimulable protein kinase (EC 1.7.1.37) has been studied in crude extracts from the central nervous system of the tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae). The insect kinase was fulfhydryl-sensitive and required Mg-2+ for optimal activity. Polyacrylamide gel electrophoresis of supernatants demonstrated the presence of multiple kinases in the larval nerve cord. At low concentrations, cyclic AMP was a much more potent activator of soluble and particulate activities than was cyclic GMP. The specific activity of coluble kinase and the magnitude of its activations by cyclic AMP were greater in the adult than in the larval central nervous system. The exogenous protein substrate specificity of the insect enzyme was similar to that of rat brain kinase with the sole exception that protamine was more readily phosphorylated than histone by nerve cord kinase. It was observed that cyclic AMP lowered the Km of Manduca sexta kinase for ATP, a phenomenon which is apparently nervous tissue=specific in mammals. An effective inhibitor of cyclic AMP-dependent protein kinase was prepared from the larval central nervous system.

Animals↗

PubMed1992

Hormonal regulation and segmental specificity of motoneuron phenotype during metamorphosis of the tobacco hornworm, Manduca sexta.

The abdominal prolegs of Manduca sexta larvae are eliminated at the onset of metamorphosis. Previous work showed that the prepupal peak of ecdysteroids in the hemolymph causes the dendritic arbors of proleg motoneurons to regress and a stereotyped subset of the motoneurons to die. In the present study we investigated the parameters of ecdysteroid exposure that are important for eliciting these responses by directly infusing 20-hydroxyecdysone (20-HE) into the hemolymph of insects deprived of their own endocrine glands. Doses of 20-HE that were near threshold for evoking regression or death were consistently more effective when infused over a longer duration. Theoretical calculations of hemolymph hormone profiles produced by the infusions support a model of ecdysteroid action in which the hormone concentration must remain above a threshold level for a critical duration of time to be physiologically effective. We further found that segmental location can influence both the metamorphic fate and the hormonal sensitivity of Manduca motoneurons.

Animals↗

PubMed1986

Protease inhibitor controls prophenoloxidase activation in Manduca sexta.

Prophenoloxidase from the hemolymph of tobacco hornworm Manduca sexta can be activated by a specific activating enzyme found in the cuticle. Inhibition studies with benzamidine, diisopropyl phosphofluoridate and p-nitrophenyl-p'-guanidinobenzoate indicate that the activating enzyme is a trypsin-like serine protease. An endogenous protease inhibitor, isolated from the hemolymph of Manduca larvae, inhibits the prophenoloxidase activation mediated by this enzyme. These results indicate that the probable physiological role of endogenous protease inhibitor is to control the undesired activation of prophenoloxidase in the hemolymph.

Animals↗

PubMed1992

Primary structure of a cardioactive neuropeptide from the tobacco hawkmoth, Manduca sexta.

The amino acid sequence of the first of a family of insect cardioregulatory peptides from the tobacco hawkmoth, Manduca sexta, has been determined using a combination of Edman degradation microsequencing and mass spectroscopy. This peptide contains 9 amino acid residues and an observed mass for the monoisotopic protonated molecule of 956.4 Da. There are two cysteines at positions 3 and 9 forming a disulfide bridge and the carboxyl-terminus is amidated. The structure of this peptide, Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, is identical to a peptide recently isolated from crabs called crustacean cardioactive peptide (CCAP) and we propose that this peptide be named Manduca CCAP.

Amino Acid Sequence↗

PubMed1987

Regulation of juvenile hormone biosynthesis by 20-hydroxyecdysone during the fourth larval instar of the tobacco hornworm, Manduca sexta.

A previous study of the role of 20-hydroxyecdysone in the regulation of juvenile hormone (JH) biosynthesis during pupal commitment in the last (fifth, V) larval instar of Manduca sexta revealed that the increase in the hemolymph edysteroid titer that elicits commitment also stimulates the corpora allata (CA) to synthesize JHs I and III and/or their acids. This stimulation is exerted indirectly via the brain-corpora cardiaca and results in the postcommitment increase in the JH titer that is important for the molt to a pupa. The possibility that a similar form of interendocrine regulation of JH biosynthesis by 20-hydroxyecdysone is involved in the control of larval molting in Manduca was investigated for the molt from the fourth (IV) to the V larval instar. During the IV instar, the increase in the hemolymph ecdysteroid titer and the capacity of the CA to synthesize JHs I and III in vitro correlated temporally in a manner suggestive of CA regulation by 20-hydroxyecdysone. With an in vitro approach, physiological concentrations of 20-hydroxyecdysone (greater than or equal to 1 microgram ml-1 hemolymph) were shown to have a dose-dependent trophic effect on CA activity, specifically stimulating JH I synthesis. Since neither basally active nor stimulated CA contained significant amounts of JH I, it appears that 20-hydroxyecdysone acts at the level of biosynthesis rather than at the level of release. The specificity of 20-hydroxyecdysone stimulation of JH I synthesis was demonstrated by the inability of the biologically inactive ecdysteroids 22-isoecdysone and 5 alpha-ecdysone to activate the glands.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Properties of the visual pigments of the moth Manduca sexta and the effects of two detergents, digitonin and chaps.

The three known visual pigments (P520, P450, P357) of the moth, Manduca sexta (Lepidoptera, Sphingidae), were extracted in two different detergents (2% digitonin, 6 or 12 mM CHAPS). As is the case in unextracted membranes, the metarhodopsins are quite stable in CHAPS extracts, while in digitonin the metarhodopsins of P520 and P450 decay rapidly at 15 degrees C to opsin and free retinal. The relative absorbance ratios are: 1.0:1.6 (P520:M485), 1.0:1.1 (P450:M485), and 1.0:0.8 (P357:M470). The relative amounts of the visual pigments found in digitonin extracts is 100:25:8 (P520:P450:P357); about 60 picomoles of P520 can be extracted from one Manduca retina.

Animals↗

PubMedDate not supplied

Adipokinetic hormone controls lipid metabolism in adults and carbohydrate metabolism in larvae of Manduca sexta.

The peptide hormone which controls activation of fat body glycogen phosphorylase in starving larvae of Manduca sexta was isolated from larval corpora cardiaca and sequenced by FAB tandem mass spectrometry. It was found to be identical with Manduca AKH. This, together with earlier observations, demonstrates that in M. sexta AKH controls glycogen phosphorylase activation in starving larvae while in adults it controls lipid mobilization during flight. Larval corpora cardiaca contain about 10 times less AKH than the corpora cardiaca of adults. The corpora cardiaca of M. sexta appear to contain only one AKH.

Animals↗

PubMed1984

Activation of Manduca sexta corpora allata in vitro by a cerebral neuropeptide.

When an in vitro assay system and radioimmunoassays specific for juvenile hormones (JH) I and III were used to probe the effect of co-incubating pupal brains with last instar larval corpora allata (CA) from the tobacco hornworm, Manduca sexta, a selective activation of JH III synthesis by the CA was observed. This homolog-specific activation suggested the presence of an allatotropic factor for the synthesis of JH III (JH III ATF), and its presence was demonstrated by the ability of a postmicrosomal supernatant of a day 0 pupal brain homogenate to activate the CA in vitro in a dose-dependent manner. This moiety appears to be a protein, based on its heat lability and protease sensitivity, and has an apparent molecular size of 40 kD and an isoelectric point of 5.5 JH III ATF activity is localized in specific neural tissues of the day 0 pupa, the brain and first three abdominal ganglia, with the brain containing 4 times the activity in the ganglia. The existence of this factor suggests that JH III synthesis by the CA of Manduca is regulated by a neuropeptide.

Animals↗

PubMed1987

Inhibition of Manduca sexta corpora allata in vitro by a cerebral allatostatic neuropeptide.

When an in vitro assay system and radioimmunoassays specific for juvenile hormones (JH) I and III were used to probe the effect of day 4 last instar larval brains on JH synthesis by day 0 last instar larval corpora allata (CA) of the tobacco hornworm, Manduca sexta, a selective inhibition of JH I synthesis by the CA was observed. The nature of this inhibition suggested the presence of an allatostatin specific for the synthesis of JH I. Its occurrence in the day 4 brain was demonstrated by the ability of a crude brain extract to inhibit the CA in a dose-dependent manner. The allatostatic factor (ASF) appears to be a protein, based on its heat lability and pronase sensitivity, and it has apparent molecular weights of 6.8 and 13 kDa. Inhibition of JH I synthesis occurs within 1 min of exposure of the CA to the factor and is reversible by 6 h after this exposure. Thus it appears that a cerebral neuropeptide specifically inhibiting JH I synthesis by the CA is present in Manduca on day 4 of the last larval instar, a time when the hemolymph titer of JH must drop to ensure the occurrence of pupal commitment.

Animals↗

PubMed1988

Juvenile hormone regulates the steroidogenic competence of Manduca sexta prothoracic glands.

The acquisition of steroidogenic competence by prothoracic glands of last instar Manduca sexta larvae is regulated by juvenile hormone (JH). Topical treatment of pre-commitment larvae with JH I or (7S)-hydroprene (a JH analog) delays development by increasing the time to pupal commitment and wandering. Prothoracic gland competence is suppressed in JH-treated larvae: Unstimulated and prothoracicotropic hormone (PTTH)-stimulated rates of in vitro ecdysone secretion are decreased relative to rates of secretion by competent glands. (7S)-Hydroprene also suppresses the competence of glands in head-ligated pre-commitment larvae, suggesting the hormone acts directly on the glands. Two results indicate PTTH plays a role in controlling competence, and that JH regulates competence indirectly by inhibiting PTTH release: (1) head-ligation prevents the acquisition of full competence, and (2) cAMP levels are elevated in glands from JH-treated larvae. Thus, the decrease in the JH titer that precedes pupal commitment in Manduca is permissive for the acquisition by prothoracic glands of steroidogenic competence.

Animals↗

PubMed1993

A cuticle-degrading proteinase from the moulting fluid of the tobacco hornworm, Manduca sexta.

Moulting fluid of pharate adult tobacco hornworm moths, Manduca sexta, contains a novel cuticle-degrading proteinase, designated as MFP-1. The enzyme has been purified using heparin affinity chromatography and partially characterized. Before purification MFP-1 is associated with a large complex having an apparent native molecular mass > 669 kDa. After purification MFP-1 has a molecular mass of 41 kDa. The pI of the enzyme is 5.54. MFP-1 can be classified as generally trypsin-like on the basis of its substrate specificity and inhibition by soybean trypsin inhibitor. The enzyme's preferred substrate, Tos-Gly-Pro-Arg-pNA, its inhibition by hirudin, and its affinity for heparin, all indicate that MFP-1 has some characteristics in common with the vertebrate blood-clotting enzyme thrombin. MFP-1 is probably a serine protease, since it is inhibited by both DFP and PMSF (specific inhibitors of serine proteinases). However, the enzyme was also inhibited by a number of agents that affect cysteine proteinases. Purified MFP-1 degrades Manduca cuticle in vitro. We suggest that the enzyme may act as the first step in the degradation of the cuticle during the moulting process.

Amino Acid Sequence↗

PubMed1993

Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta.

Insects use chitinolytic enzymes to digest chitin in the exoskeleton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the tobacco hornworm, Manduca sexta, compared its sequence with genes encoding chitinolytic enzymes from other sources, and studied chitinase gene expression and hormonal regulation during the larval-pupal transformation. The insert DNA in this clone is 2452 nucleotides long with an open reading frame of 1662 nucleotides that encodes a protein of 554 amino acids with a molecular weight of 62 kDa. Several regions of the amino acid sequence in this protein are similar to sequences in yeast, cucumber and bacterial endo-beta-N-acetylglucosaminidases. Hybrid-selection of mRNA and in vitro translation yielded an immunoreactive protein with an apparent molecular mass of 75 kDa, which is similar to the size of a chitinase present in pharate pupal molting fluid. Southern blot analysis indicated that one or two genes related to the cDNA clone are encoding chitinases in the Manduca genome. The major tissues expressing chitinase genes were the epidermis and gut with mRNA levels highest on c. days 5-7 during the fifth larval instar. Injection of 20-hydroxyecdysone into ligated fifth instar abdomens caused about a 10-fold increase in mRNA levels in both epidermis and gut, and topical application of the juvenile hormone mimic, fenoxycarb, suppressed the ecdysteroid-induced accumulation of chitinase RNA.

Amino Acid Sequence↗

PubMed1993

The E75 gene of Manduca sexta and comparison with its Drosophila homolog.

The ecdysone-inducible E75 gene responsible for the 75B puff of Drosophila melanogaster encodes a family of proteins which are members of the steroid receptor superfamily. These proteins are believed to be involved in the regulation of ecdysone response. In order to investigate the evolutionary conservation of E75, we have identified the E75 gene of Manduca sexta. We show here the structure of a cDNA believed to encode the Manduca homolog of the E75B protein, and demonstrate that the putative DNA binding, hormone binding and amino and carboxy terminal flanking domains are conserved. However, due to a relative reduction in intron size and number and the absence of homopolymeric amino acid repeats, the E75 B transcription unit and protein are considerably smaller in M. sexta than in D. melanogaster. These findings have implications for the identification of critical structural features of E75 and also suggest that E75 has a conserved function and a shared ligand in Lepidoptera.

Amino Acid Sequence↗

PubMed1997

Life cycle expression of a bombyxin-like neuropeptide in the tobacco hornworm, Manduca sexta.

Immunocytochemistry was used to investigate the developmental expression of the insulin-like neuropeptide bombyxin in the tobacco hornworm, Manduca sexta. A mouse monoclonal antibody raised against a synthetic peptide corresponding to bombyxin's A-chain N-terminus was used to localize a bombyxin-like peptide to a group of cerebral medial neurosecretory cells, the M-NSC IIa(2). Immunostaining was first detected on day 0 of the second larval instar, localized in the M-NSC IIa(2) somata and in the neurohemal organ, the corpora allata (CA). By day 0 of the fourth larval instar, the peptide was present throughout the M-NSC IIa(2) somata, axons, dendritic fields and CA. Between days 7 and 9 of the fifth instar, a dramatic reduction in the dendritic fields and CA staining occurred, suggesting the peptide is released. After day 2 of the pupal period, only M-NSC IIa(2) somata immunostained, a pattern that persisted through day 2 of the adult stage. The specificity of immunostaining was demonstrated by using a synthetic bombyxin peptide to block staining. These developmental data reveal times of potential Manduca bombyxin-like peptide release which should provide insight into the peptide's function.

Journal Article↗

PubMed1984

Photoaffinity labeling of the hemolymph juvenile hormone binding protein of Manduca sexta.

A synthetic analogue of the insect juvenile hormone (JH) III, 10,11-epoxy[10-3H]farnesyl diazoacetate [( 3H]-EFDA), binds to several proteins in a partially purified preparation of hemolymph protein from fourth instar larvae of Manduca sexta when irradiated with UV light. Approximately 80% of this binding could be inhibited by the addition of excess unlabeled JH I. To compare the relative affinity of EFDA for the juvenile hormone binding protein (JHBP) with that of the various JH homologues, the ability of unlabeled EFDA and JH homologues to displace [3H]JH I from binding sites was measured. The relative affinities were EFDA greater than JH I greater than JH II greater than JH III. When Scatchard analysis of the binding of [3H]EFDA or [3H]JH I to the larval JHBP was performed, an estimated apparent KD of 4.5 X 10(-8)M was found for EFDA, whereas for JH I a slightly higher KD of 8.8 X 10(-8) M was calculated. To determine if [3H]EFDA bound at the JH I binding site, displacement of [3H]JH I from the JHBP complex with unlabeled JH I, JH II, and JH III was compared to the displacement of [3H]EFDA with the same homologues. The results demonstrated that the photoaffinity label bound covalently at the JH I binding site on the hemolymph binding protein of Manduca sexta. Fluorescence autoradiography of [3H]EFDA photoaffinity labeled proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that [3H]EFDA bound covalently to two major proteins in the absence of JH I.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

PubMed1994

Drosophila mode of metamerization in the embryogenesis of the lepidopteran insect Manduca sexta.

Insect embryos have been classified as intermediate- and short-germ embryos, in which posterior segments are thought to be generated sequentially from an uncommitted growth zone, or as long-term embryos, such as Drosophila melanogaster, which develop primordia for all segments simultaneously. In Drosophila the coordinated activities among a three-tiered cascade of zygotic segmentation genes subdivide the embryo into progressively smaller units along the anterior-posterior axis. The mode of pattern specification in lepidopteran embryos has not been determined, although on morphological grounds they have been characterized as intermediate-germ insects. We have cloned orthologues of Drosophila segmentation genes from the tobacco hawkmoth Manduca sexta and have found that the blastoderm expression patterns of these genes show a molecular prepatterning typical of Drosophila. Thus, successive segment formation in Manduca embryos may not be due to sequential addition but rather may be the consequence of a lateral compression of the embryo proceeding in an anterior-to-posterior progression. These data challenge the view that the classification of insect development according to morphological criteria can serve as a reliable indicator of the molecular mechanisms underlying segmentation.

Amino Acid Sequence↗

PubMed1990

Characterization of a ubiquitin-fusion gene from the tobacco hawkmoth, Manduca sexta.

A gene encoding a ubiquitin-fusion protein was isolated from a cDNA library made from the intersegmental muscles (ISM) of the moth, Manduca sexta. The predicted amino acid sequence of this fusion protein is highly conserved when compared to the sequence of homologous proteins from diverse species. The Manduca clone encoding this ubiquitin fusion gene hybridized with a single, abundantly expressed transcript in all tissues examined. In the ISM, the transcript was present at high levels, independent of the developmental stage or hormonal treatment of these muscles. Data from other species indicate that ubiquitin-fusion genes participate in ribosome biogenesis.

Amino Acid Sequence↗

PubMed1992

Complete structure of eclosion hormone of Manduca sexta. Assignment of disulfide bond location.

The locations of the three disulfide bonds of eclosion hormone (EH) isolated from Manduca sexta were assigned by sequence analysis of thermolysin fragments and by comparison of a key heterodimeric fragment to regiospecifically synthesized parallel and antiparallel isomers. We elucidated the complete structure of Manduca EH as a 62-residue peptide which has three disulfide bonds between Cys14-Cys38, Cys18-Cys34, and Cys21-Cys49.

Amino Acid Sequence↗