Identification of an allatostatin from the tobacco hornworm Manduca sexta.

A peptide (Manduca sexta allatostatin) that strongly inhibits juvenile hormone biosynthesis in vitro by the corpora allata from fifth-stadium larvae and adult females has been purified from extracts of heads of pharate adult M. sexta by a nine-step purification procedure. The primary structure of this 15-residue peptide has been determined: pGlu-Val-Arg-Phe-Arg-Gln-Cys- Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-OH, where pGlu is pyroglutamate). To our knowledge, this neuro-hormone has no sequence similarity with any known neuropeptide from other organisms. The synthetic free acid and amide forms showed in vitro activity indistinguishable from that of native M. sexta allatostatin. The ED50 of synthetic M. sexta allatostatin on early fifth stadium larval corpora allata in vitro was approximately 2 nM. This inhibition was reversible. In a cross-species study, M. sexta allatostatin also inhibited the corpora allata of adult female Heliothis virescens but had no effect on the activity of corpora allata of adult females of the beetle Tenebrio molitor, the grasshopper Melanoplus sanguinipes, or the cockroach Periplaneta americana.

Isolation and identification of paralytic peptides from hemolymph of the lepidopteran insects Manduca sexta, Spodoptera exigua, and Heliothis virescens.

Seven paralytic peptides were isolated and identified from lepidopteran hemolymph. All of these peptides cause rapid, rigid paralysis when injected into Manduca sexta and some other lepidopteran larvae. Each peptide contains 23 amino acid residues including 2 cysteines and the carboxyl termini are acidic. Synthetic peptides in the disulfide or reduced forms, and as carboxyl-terminal acids or amides were equally paralytic. The most potent paralytic peptide, Mas PP I, has the following sequence: H-Glu-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Tyr-Leu- Arg-Thr-Ala-Asp-Gly-Arg-Cys-Lys-Pro-Thr-Phe-OH. The two peptides from M. sexta hemolymph are remarkable in that they are autoparalytic (i.e. factors in collected hemolymph that are paralytic when injected into the same larvae).

Chemical characterization of acylpolyamine toxins from venom of a trap-door spider and two tarantulas.

Two acylpolyamines are identified from venom of the trap-door spider, Hebestatis theveniti. These toxins (paralytic to lepidopteran insect larvae) are amides containing 3-(3-indolyl)lactic acid joined to spermine or 1,13-diamino-4,10-diazatridecane (Het389 and Het403, respectively). Het389 is also abundant in venom from a tarantual from Mozambique (Harpactirella sp.). Two additional acylpolyamines (Apc600 and Apc728) are partially characterized from venom of another tarantula, Aphonopelma chalcodes.

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

A diuretic hormone (DH) has been isolated from pharate adult heads of Manduca sexta by a nine-step purification procedure. The primary structure of the amino-terminal 40 residues was determined by sequence analysis of intact DH. The structure of an amidated carboxyl-terminal tryptic hexapeptide was characterized by sequence analysis of the peptide, and this hexapeptide was later compared by reversed-phase liquid chromatography with two synthetic hexapeptides with the free acid or amide at the carboxyl terminus. The complete structure of M. sexta DH was established as a 41-residue peptide without disulfide bonds: H-Arg-Met-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Pro-Met-Ser-Val-Leu-Arg-Gln-Lys-Leu-Ser -Leu-Glu-Lys-Glu-Arg-Lys-Val-His-Ala-Leu-Arg-Ala-Ala-Ala-Asn-Arg-Asn-Phe-Leu- Asn-Asp-Ile-NH(2). M. sexta DH was synthesized and shown to have chromatographic and biological properties identical with those of the native material. Synthetic DH stimulated fluid excretion in vivo upon injection into larval M. sexta and newly emerged adult Pieris rapae. M. sexta DH has considerable sequence homology with corticotropin-releasing factor, urotensin I, and sauvagine.

Identification of an allatotropin from adult manduca sexta.

A peptide that strongly stimulates the secretion of juvenile hormone from corpora allata in vitro (allatotropin) has been purified from extracts of heads of pharate adult Manduca sexta. The primary structure of this 13-residue peptide has been determined: H-Gly-Phe-Lys-Asn-Val-Glu-Met-Met-Thr-Ala-Arg-Gly-Phe-NH(2). This neurohormone has no sequence similarity with any known neuropeptide from other organisms. Synthetic allatotropin, as well as truncation fragments, including one with the five amino terminal residues deleted, showed in vitro activity indistinguishable from that of native allatotropin.

Identification of an arginine vasopressin-like diuretic hormone from Locusta migratoria.

We have identified two neuropeptides (F1 and F2) from suboesophageal and thoracic ganglia of Locusta migratoria, which we isolated earlier based on their immunological similarity to arginine vasopressin. The more abundant and hydrophilic factor, F1, has sequence Cys-Leu-Ile-Thr-Asn-Cys-Pro-Arg-Gly-NH2, but its biological role is unknown. The less abundant factor, F2, is an antiparallel dimer of F1, and functions as a diuretic hormone of this species. It appears to act through the intermediacy of cyclic AMP. The properties of the native neuropeptides were identical with those of samples synthesized from appropriately protected L-amino acids.

Structures and biological activities of three synaptic antagonists from orb weaver spider venom.

The venom of Argiope aurantia, an orb weaver spider, contains a mixture of low molecular weight "argiotoxins", which block neuromuscular transmission in insects. Complete structure elucidation of three argiotoxins reveals common features; a hydrophilic, basic domain of arginine, a polyamine and asparagine is connected to an aromatic moiety contributed either by 4-hydroxyindole-3-acetic acid or 2,4-dihydroxyphenylacetic acid. Structural assignments of two argiotoxins are verified by chemical synthesis. The argiotoxins cause reversible paralysis when injected into insects and this is correlated with a stimulus-dependent inhibition of skeletal neuromuscular transmission at submicromolar concentrations.

Response of northern corn rootworm,Diabrotica barberi Smith and Lawrence, to stereoisomers of 8-methyl-2-decyl propanoate.

The four stereoisomers of 8-methyl-2-decyl propanoate were tested in South Dakota for attractiveness to the northern corn rootworm,Diabrotica barberi Smith and Lawrence (NCR). Only the 2R,8R configuration was attractive to the NCR. Inhibition of the NCR response to 2R,8R occurred when either the 2S,8R or 2S,8S isomers were components of the pheromone source. The 2R,8S configuration elicited no behavioral activity in the NCR.

Synthesis of stereoisomers of 8-methyl-2-decanol and esters attractive to severalDiabrotica sp.

Details of the syntheses of the four stereoisomers of 8-methyl-2-decanol and its propanoate ester are given. The racemic ester, two of its stereoisomers, and one stereoisomer as an acetate are attractive to several species ofDiabrotica. The key steps in the syntheses involve high-performance liquid chromatograpic resolutions of diastereomers to achieve high configurational enrichment of each site and generation of (R)-2-methylbutyric acid by chemical degradation ofD-isoleucine.

Metabolism of proctolin, a pentapeptide neurotransmitter in insects.

The in vitro metabolism of [tyrosyl-3, 5-3H]proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) was studied in the following tissues from the American cockroach, Periplaneta americana: proctodeum, midgut, hemolymph, brain, terminal ganglion, and coxal depressor muscles. In all tissues assayed, the Tyr-Leu bond is the primary cleavage site, but scission of the Arg-Tyr bond is also significant. Greater than 90% of the degradative activity is found in the 100,000 X g supernatant from homogenates. In vivo studies with the tobacco hornworm, Manduca sexta, show that topically applied proctolin does not penetrate larval cuticle; proctolin is readily degraded to constituent amino acids (at least to Tyr) upon ingestion.

Response ofDiabrotica virgifera virgifera, D. v. Zeae, andD. porracea to stereoisomers of 8-methyl-2-decyl propanoate.

The four stereoisomers of 8-methyl-2-decyl propanoate were tested in the United States and Mexico for attractiveness toDiabrotica virgifera virgifera LeConte, the western corn rootworm,D. v. zeae Krysan and Smith, the Mexican corn rootworm, andD. porracea Harold. Males ofD. v. virgifera andD. v. zeae responded strongly to the (2R,8R)-isomer and secondarily to (2S,8R), whileD. ponacea responded exclusively to the (2S,8R)-isomer. The (2S,8S)- and (2R,8S)-isomers were inactive in all tests. Synergism or inhibition was not detected when various mixtures of the isomers were tested withD. v. virgifera. These phenomena were not tested withD. v. zeae andD. ponacea.