Molecular cloning of the prothoracicotropic hormone from the tobacco hornworm,Manduca sexta

A cDNA encoding a putative precursor of prothoracicotropic hormone (PTTH) from the tobacco hornworm, Manduca sexta, was isolated and sequenced. This clone contains an open reading frame encoding a 226-amino acid prepropeptide hormone. The deduced amino acid sequence is composed of a signal sequence, a precursor domain and a mature hormone and shows similarities to the other PTTHs that have been cloned from closely related lepidopteran species, Bombyx mori, Samia cynthia ricini, Antheraea peryni, and Hyalophora cecropia. Although these cDNAs showed slightly less similarities in predicted amino acid sequences, seven cysteine residues and the hydrophobic regions within those mature peptides were conserved. In situ hybridization using a cDNA probe encoding the Manduca PTTH showed that PTTH mRNA was in two pairs of neurosecretory cells in the Manduca brain. The recombinant putative Manduca PTTH produced in E. coli was biologically active, both causing a larval molt in neck-ligated Manduca 4th instar larvae (ED(50)=50 pM) and the adult molt of diapausing Manduca pupae (ED(50)=79 pM), but was unable to stimulate molting of debrained Bombyx pupae.     

Isolation of ommochromes and 3-hydroxykynurenine from the tobacco hornworm,Manduca sexta

A complex of the two red pigments was prepared from epidermis of the fifth instar larvae of the black mutant of the tobacco hornworm according to the method generally available for ommochromes. The primary component, a brick-red pigment (II), was shown to be identical with synthetic l-dihydroxanthommatin by various physico-chemical tests. The other red pigment (I) was similar to pigment (II), except in the i.r. spectra in 1000–1150 cm⁻¹ region and is thought to be also an ommatin. A red pigment identical with pigment (I) was obtained during the chemical oxidation of 3-hydroxy-l-kynurenine. These same two pigments were found in the red epidermis underlying the black cuticle of larvae either allatectomized or neck-ligatured before the critical period for juvenile hormone action during the last larval molt. Also, they were found in the dorsal epidermis of wandering stage larvae. 3-Hydroxykynurenine, the immediate precursor of the ommochrome pigments, was found to accumulate in the epidermis before the synthesis of the ommochromes began.     

Isolation and identification of a new diuretic peptide from the tobacco hornworm, Manduca sexta.

A 30-amino acid diuretic peptide was isolated from the corpora cardiaca-corpora allata complexes and, separately, from medial neurosecretory cells of the Sphingid moth, Manduca sexta. The peptide was found to have the following sequence, determined by automated Edman degradation and mass spectrometry: SFSVNPAVDILQHRYMEKV AQNNRNFLNRV-NH2. We have named the peptide Mas-DP II. The peptide was synthesized and shown to possess diuretic activity in decapitated moths. Mas-DP II is related by sequence homology to a 41-amino acid diuretic peptide identified previously from M. sexta, and it belongs to the family of corticotropin releasing factor-like peptides.     

Studies on vitellogenin from the tobacco hornworm,Manduca sexta

In the tobacco hornworm moth, Manduca sexta, vitellogenin (Vg) is a very high-density (1.29 g/ml) phosphoglycolipoprotein containing 13% lipids, 3% carbohydrates, and 0.6% protein-bound phosphorus. Vitellogenin (M_r～500,000) has two apoproteins designated apoVg-l (M_r 177,000 ± 3,600) and apoVg-ll (M_r45,000 ± 5,000). ApoVg-l and apoVg-II can be dissociated with 6 M guanidine HCI and separated from each other by gel permeation chromatography. Immunoblotting experiments using antibodies against the apoproteins showed that apoVg-l and apoVg-II antigens were immunologically distinct polypeptides. Antibodies against Vg reacted only with apoVg-l. Antibodies against Vg and apoVg-l reacted with Vg in double immunodiffusion experiments, whereas antibodies against apoVg-II did not. These results suggest that in the native Vg molecule, apoVg-II is positioned inside the molecule away from the aqueous environment. Only apoVg-I contained covalently bound carbohydrate as shown by fluorescein isothiocyanateconjugated concanavalin A, periodate-Schiff reagent, and in vivo labeling with ³H-Man. In vivo labeling with ³²P-inorganic phosphate and chemical determination showed that apoproteins of both Vg and vitellin contain covalently bound phosphate groups.     

Toxicity of Bacillus thuringiensis spores to the tobacco hornworm, Manduca sexta.

Toxicity of Bacillus thuringiensis spores to the tobacco hornworm, Manduca sexta, is described. The numbers of larvae killed were in relation to spore dry weight. At a surface application of 6.8 ng/cm2, there was an 85 percent survival, but less than 50 percent survived at 68.2 ng/cm2. Striking similarity of spores to parasporal crystals is revealed by slope of mortality curves, inhibition of stadial growth, and 50 percent lethal dose values based on protein content.     

Isolation and primary structure of the eclosion hormone of the tobacco hornworm, Manduca sexta

Eclosion hormone was isolated from trimmed pharate adult heads of Manduca sexta by an eight step purification procedure using a Heliothis virescens in vivo bioassay. The neuropeptide was active in second stadium M. sexta. The primary structure was determined by sequence analyses of the intact peptide and fragment peptides generated by lysyl endopeptidase, endoproteinase Glu-C, and proline-specific endopeptidase. The nature of the carboxyl terminus as a free acid was elucidated by analysis of amino acids from digestion of the intact peptide with lysyl endopeptidase, which liberated leucine, but no leucine amide. The complete primary structure of M. sexta closion hormone is H-Asn-Pro-Ala-Ile-Ala-Thr-Gly-Tyr-Asp-Pro-Met-Glu-Ile-Cys-Ile-Glu-Asn-Cy s-Ala- Gln-Cys-Lys-Lys-Met-Leu-Gly-Ala-Trp-Phe-Glu-Gly-Pro-Leu-Cys-Ala-Glu-Ser- Cys-Ile Lys-Phe-Lys-Gly-Lys-Leu-Ile-Pro-Glu-Cys-Glu-Asp-Phe-Ala-Ser-Ile-Ala-Pro- Phe-Leu-Asn-Lys-Leu-OH.     

Microstructure of feeding by tobacco hornworm caterpillars, Manduca sexta

The microstructure of the feeding activity of tobacco hornworm caterpillars (Manduca sexta Johansson) on tomato leaf was examined by means of an automated cafeteria. In this device each activity of the caterpillar generates a characteristic slow electrical change which can be recorded. The apparatus is therefore both accurate and sensitive. Examination of the activity records indicated that larger animals ate more than smaller ones by increasing both bite frequency and the lengths of meals. Meal frequency did not increase. Correlations amongst a variety of measures indicated that there was regulation of feeding both between and within meals.     

Isolation and characterization of RNA polymerase B from the larval fat body of the tobacco hornworm, Manduca sexta.

DNA-dependent RNA polymerase B has been extensively purified from the larval fat body of the tobacco hornworm (Manduca sexta) by employing chromatography on ion-exchange columns of DEAE-Sephadex, DEAE-cellulose and phosphocellulose and centrifugation on glycerol gradients. The isolated enzyme after electrophoresis on acrylamide gels shows one main band and one minor band, both having enzyme activity sensitive to alpha-amanitin. The catalytic and physicochemical properties of the enzyme are similar to those of other eucaryotic B-type RNA polymerases. The enzyme has an apparent molecular weight of 530000, is inhibited 50% by alpha-amanitin at 0.04 microgram/ml and shows maximum activity on denatured DNA at 5 mM Mn2+ and 100 mM ammonium sulfate. An antibody was obtained that cross-reacts with the pure enzyme and forms a precipitin line. This antibody does not cross react with either Escherichia coli RNA polymerase or with wheat germ RNA polymerase but does react with one of the B polymerases isolated from wing tissue of the silkmoth, Antheraea pernyi.     

Characterization of a diuretic hormone receptor from the tobacco hornworm,Manduca sexta

We have characterized a diuretic hormone receptor from the tobacco hornworm, Manduca sexta. A single high affinity binding site for the 41 amino acid M. sexta diuretic hormone was found in membranes prepared from Malpighian tubules of fifth stadium larvae. The site has a Kd = 79 pM and Bmax = 3.1 pmol/mg protein. The dissociation rate constant was determined to be 0.11 min^?1 with a corresponding half-life of 6.4 min. Receptor binding of the hormone is inhibited by Ca²⁺ and Mg2⁺, while Na⁺ and K⁺ inhibit binding to a lesser extent. Truncated diuretic hormone analogs in which up to 20 amino acids were removed from the N-terminus maintain high affinity for the receptor. A diuretic hormone from Locusta migratoria which has 43% sequence identity with the M. sexta diuretic hormone also possesses a high affinity for the receptor. Conformational analysis of the M. sexta diuretic hormone indicates the core region of the peptide assumes a helical conformation, which may have implications in the binding of the peptide to the receptor. © 1993 Wiley-Liss. Inc.     

Proteinases in molting fluid of the tobacco hornworm,Manduca sexta

Manduca sexta pharate pupal molting fluid contains more than 10 proteolytic enzymes that differ in relative mobility during electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and gelatin. The major gelatin digesting enzyme was an endoprotease with an apparent molecular weight of 100 kDa. Gel filtration on a Sephacryl S-300 column resolved another endoprotease of similar size that digests azocoll and [³H]casein. In addition we found an aminopeptidase-like enzyme (MW_app 500 kDa) and at least three carboxypeptidase-like enzymes (MW_app 10–60 kDa). Use of pseudosubstrates and inhibitors suggested the presence of both trypsin-like and chymotrypsin-like enzymes with the former activity approx. 10-fold greater than the latter. However, none of the proteolytic enzymes were substantially inhibited by diisopropylphosphorofluoridate or phenylmethylsulfonyl fluoride which are poteint inhibitors of trypsin and chymotrypsin. No carboxyl or sulfhydryl proteases were detected. The enzymes were most active in the neutral to alkaline pH range, but they were relatively unstable during storage which precluded their purification to homogeneity. Proteolysis of Manduca cuticular protein appears to involve a rather complex and unique mixture of endo- and exo-cleaving proteolytic enzymes.     

Prostaglandin biosynthesis by midgut tissue isolated from the tobacco hornworm, Manduca sexta

We describe prostaglandin (PG) biosynthesis by isolated midgut preparations from tobacco hornworms, Manduca sexta. Microsomal-enriched midgut preparations yielded four PGs, PGA/B(2), PGD(2), PGE(2) and PGF(2alpha), all of which were confirmed by analysis on gas chromatography--mass spectrometry (GC--MS). PGA and PGB are double bond isomers which do not resolve on TLC but do resolve by GC; for convenience, we use the single term PGA(2) for this product. PGA(2) was the major product under most conditions. The midgut preparations were sensitive to reaction conditions, including radioactive substrate, protein concentration (optimal at 1mg/reaction), reaction time (optimal at 0.5 min), temperature (optimal at 22 degrees C), buffer pH (highest at pH 6), and the presence of a co-factor cocktail composed of reduced glutathione, hydroquinine and hemoglobin. In vitro PG biosynthesis was inhibited by two cyclooxygenase inhibitors, indomethacin and naproxen. Subcellular localization of PG biosynthetic activity in midgut preparations, determined by ultracentrifugation, revealed the presence of PG biosynthetic activity in the cytosolic and microsomal fractions, although most activity was found in the cytosolic fractions. This is similar to other invertebrates, and different from mammalian preparations, in which the activity is exclusively associated with the microsomal fractions. Midgut preparations from M. sexta pupae, adult cockroach, Periplaneta americana, and corn ear worms, Helicoverpa zea, also produced the same four major PG products. We infer that insect midguts are competent to biosynthesize PGs, and speculate they exert important, albeit unrevealed, actions in midgut physiology.     

The molecular structure of insecticyanin from the tobacco hornworm Manduca sexta L. at 2.6 A resolution.

Insecticyanin, a blue biliprotein isolated from the tobacco hornworm Manduca sexta L., is involved in insect camouflage. Its three-dimensional structure has now been solved to 2.6 A resolution using the techniques of multiple isomorphous replacement, non-crystallographic symmetry averaging about a local 2-fold rotation axis and solvent flattening. All 189 amino acids have been fitted to the electron density map. The map clearly shows that insecticyanin is a tetramer with one of its molecular 2-fold axes coincident to a crystallographic dyad. The individual subunits have overall dimensions of 44 A X 37 A X 40 A and consist primarily of an eight-stranded anti-parallel beta-barrel flanked on one side by a 4.5-turn alpha-helix. Interestingly the overall three-dimensional fold of the insecticyanin subunit shows remarkable similarity to the structural motifs of bovine beta-lactoglobulin and the human serum retinol-binding protein. The electron density attributable to the chromophore is unambiguous and shows that it is indeed the gamma-isomer of biliverdin. The biliverdin lies towards the open end of the beta-barrel with its two propionate side chains pointing towards the solvent and it adopts a rather folded conformation, much like a heme.     

Annotation and expression analysis of cuticular proteins from the tobacco hornworm,Manduca sexta

The insect cuticle is a unique material that covers the exterior of the animal as well as lining the foregut, hindgut, and tracheae. It offers protection from predators and desiccation, defines body shape, and serves as an attachment site for internal organs and muscle. It has demonstrated remarkable variations in hardness, flexibility and elasticity, all the while being light weight, which allows for ease of movement and flight. It is composed primarily of chitin, proteins, catecholamines, and lipids. Proteomic analyses of cuticle from different life stages and species of insects has allowed for a more detailed examination of the protein content and how it relates to cuticle mechanical properties. It is now recognized that several groups of cuticular proteins exist and that they can be classified according to conserved amino acid sequence motifs. We have annotated the genome of the tobacco hornworm, Manduca sexta, for genes that encode putative cuticular proteins that belong to seven different groups: proteins with a Rebers and Riddiford motif (CPR), proteins analogous to peritrophins (CPAP), proteins with a tweedle motif (CPT), proteins with a 44 amino acid motif (CPF), proteins that are CPF-like (CPFL), proteins with an 18 amino acid motif (18 aa), and proteins with two to three copies of a C-X₅-C motif (CPCFC). In total we annotated 248 genes, of which 207 belong to the CPR family, the most for any insect genome annotated to date. Additionally, we discovered new members of the CPAP family and determined that orthologous genes are present in other insects. We established orthology between the M. sexta and Bombyx mori genes and identified duplication events that occurred after separation of the two species. Finally, we utilized 52 RNAseq libraries to ascertain gene expression profiles that revealed commonalities and differences between different tissues and developmental stages.     

Studies on the carbohydrate moiety of vitellogenin from the tobacco hornworm,Manduca sexta

Vitellogenin can be isolated in large quantities from the hemolymph of the tobacco hornworm, Manduca sexta by a combination of KBr density gradient ultracentrifugation, gel permeation and cation exchange chromatography. Glycopeptides generated by exhaustive pronase digestion of vitellogenin were separated by gel permeation chromatography on a Bio-Gel P-6 column. The major glycopeptide was shown to bind to concanavalin A-Sepharose but not to lentil lectin-Sepharose and was sensitive to treatment with endo-β-N-acetylglucosaminidase H. Analysis of the glycopeptide by high field proton NMR spectroscopy revealed that the primary structure is of the high mannose class containing nine mannose and two N-acetylglucosamine units. These results suggested that N-glycosylation of insect proteins, as in mammals, yeasts and plants, involves the en bloc transfer of an oligosaccharide containing the unit (Glu)₃(Man)₉(GlcNAc)₂ from a lipid intermediate to an asparagine residue followed by removal of glucose residues. Processing to more complex structures does not seem to occur in M. sexta vitellogenin. In vitro uptake with isolated M. sexta follicles showed that deglycosylation had no significant effect on uptake of ¹²⁵I-labeled vitellogenin.     

Lipoprotein biosynthesis in the larvae of the tobacco hornworm, Manduca sexta

Lipoprotein biosynthesis in larvae of the tobacco hornworm (Manduca sexta) was investigated. By immunoblotting, it was shown that the apoproteins are present in the fat body, but not in the midgut. Fat body incubated in vitro with [35S]methionine secreted labeled apoproteins. However, when the density of the secreted particle was determined, it was found at 1.24-1.28 g/ml instead of 1.15 g/ml, which is the density of the circulating lipoprotein. Lipid analysis of immunoprecipitated lipoprotein secreted by the fat body showed a phospholipid/diacylglycerol ratio of 8.3 rather than 0.9, the ratio found in the circulating lipoprotein. When labeled oleic acid or triolein was fed to larvae, it was found that greater than 98% of the label in the circulating lipoprotein was in diacylglycerol. In studies using animals raised on a fat-free diet, it was shown that the circulating lipoprotein has properties comparable to those of the material secreted in vitro by the fat body and that this diacylglycerol-poor particle can be converted to the normal lipoprotein by feeding a bolus of triolein. These data support the hypothesis that the fat body makes and secretes a "nascent" lipoprotein which contains apoproteins and phospholipid, but is devoid of diacylglycerol. The diacylglycerol is then picked up from the midgut to complete assembly of the mature circulating lipoprotein.     

Eicosanoids influence in vitro elongation of plasmatocytes from the tobacco hornworm, Manduca sexta

Nodule formation is the predominant insect cellular defense reaction to bacterial challenges, responsible for clearing the largest proportion of infecting bacteria from hemolymph circulation. Hemocyte spreading behavior is a critical step in the nodulation process. It has been suggested that eicosanoids mediate several steps in the process. However, the influence of eicosanoids on hemocyte spreading has not been investigated in detail. To test the hypothesis that eicosanoids mediate hemocyte spreading behavior, I treated larvae of the tobacco hornworm, Manduca sexta, with eicosanoid biosynthesis inhibitors and later assessed plasmatocyte elongation on glass slides. Plasmatocytes from larvae treated with dexamethasone did not elongate to the extent of plasmatocytes from untreated control larvae. The dexamethasone effect on plasmatocyte elongation was expressed in a dose-dependent manner and was reversed by injecting dexamethasone-treated larvae with the eicosanoid-precursor fatty acid, arachidonic acid. Palmitic acid, which is not substrate for eicosanoid biosynthesis, did not reverse the influence of dexamethasone on plasmatocyte elongation. Finally, plasmatocytes from larvae treated with a range of eicosanoid biosynthesis inhibitors did not elongate to the extent of plasmatocytes from control larvae. Plasmatocyte width did not appear to be influenced in this study. These findings strongly support the idea that insect plasmatocyte elongation is influenced by eicosanoids.     

Expression and evolution of hexamerins from the tobacco hornworm,Manduca sexta,and other Lepidoptera

Hexamerins are large hemolymph-proteins that accumulate during the late larval stages of insects. Hexamerins have emerged from hemocyanin, but have lost the ability to bind oxygen. Hexamerins are mainly considered as storage proteins for non-feeding stages, but may also have other functions, e.g. in cuticle formation, transport and immune response. The genome of the hornworm Manduca sexta harbors six hexamerin genes. Two of them code for arylphorins (Msex2.01690, Msex2.15504) and two genes correspond to a methionine-rich hexamerin (Msex2.10735) and a moderately methionine-rich hexamerin (Msex2.01694), respectively. Two other genes do not correspond to any known hexamerin and distantly resemble the arylphorins (Msex2.01691, Msex2.01693). Five of the six hexamerin genes are clustered within ∼45 kb on scaffold 00023, which shows conserved synteny in various lepidopteran genomes. The methionine-rich hexamerin gene is located at a distinct site. M. sexta and other Lepidoptera have lost the riboflavin-binding hexamerin. With the exception of Msex2.01691, which displays low mRNA levels throughout the life cycle, all hexamerins are most highly expressed during pre-wandering phase of the 5th larval instar of M. sexta, supporting their role as storage proteins. Notably, Msex2.01691 is most highly expressed in the brain, suggesting a divergent function. Phylogenetic analyses showed that hexamerin evolution basically follows insect systematics. Lepidoptera display an unparalleled diversity of hexamerins, which exceeds that of other hexapod orders. In contrast to previous analyses, the lepidopteran hexamerins were found monophyletic. Five distinct types of hexamerins have been identified in this order, which differ in terms of amino acid composition and evolutionary history: i. the arylphorins, which are rich in aromatic amino acids (∼20% phenylalanine and tyrosine), ii. the distantly related arylphorin-like hexamerins, iii. the methionine-rich hexamerins, iv. the moderately methionine rich hexamerins, and v. the riboflavin-binding hexamerins.