A unique charged tyrosine-containing member of the adipokinetic hormone/red-pigment-concentrating hormone peptide family isolated and sequenced from two beetle species.

An identical neuropeptide was isolated from the corpora cardiaca of two beetle species, Melolontha melolontha and Geotrupes stercorosus. Its primary structure was determined by pulsed-liquid-phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal pyroglutamate residue. The C-terminus was also blocked, as indicated by the lack of digestion when the peptide was incubated with carboxypeptidase A. The sequence of this peptide, which is designated Mem-CC, is pGlu-Leu-Asn-Tyr-Ser-Pro-Asp-Trp-NH2. It is a new member of the adipokinetic hormone/red-pigment-concentrating hormone (AKH/RPCH) family of peptides with two unusual structural features: it is charged and contains a tyrosine residue at position 4, where all other family members have a phenylalanine residue. Structure-activity studies in the migratory locust (Locusta migratoria) and the American cockroach (Periplaneta americana) revealed that the peptide was poorly active, owing to its structural uniqueness.

Isolation, identification and synthesis of novel oviductal motility stimulating head peptide in the Colorado potato beetle, Leptinotarsa decemlineata.

A novel myotropic peptide was isolated from an extract of 10,000 heads of adult Leptinotarsa decemlineata by means of high performance liquid chromatography (HPLC). The peptide stimulates the contractions of the oviduct of Leptinotarsa as well as that of Locusta migratoria. Gas phase sequencing and comparison of candidate synthetic peptides in the amide and acid form revealed the following primary structure: Ile-Ala-Tyr-Lys-Pro-Glu-NH2. This new peptide has a molecular weight of 720 Da and has been named Led OVM. Led OVM does not exhibit significant sequence homology with any known vertebrate or invertebrate peptide. Sixteen additional myotropic factors were also separated by means of HPLC, but were as yet not recovered in amounts large enough for them to be sequenced.

The primary structure of the hypertrehalosemic neuropeptide from tenebrionid beetles: a novel member of the AKH/RPCH family.

A hypertrehalosemic neuropeptide from the corpora cardiac of the two tenebrionid beetle species, Tenebrio molitor and Zophobas rugipes, was purified by high performance liquid chromatography, and its sequence determined by pulsed-liquid phase sequencing employing Edman degradation after deblocking enzymatically the N-terminal pyroglutamate residue. Additionally, the C-terminus of the peptide was blocked as shown by the lack of breakdown using carboxypeptidase. In both species an identical octapeptide, designated Tem-HrTH, with the following amino acid sequence, was found: pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-NH2. This primary sequence has an 88% homology with the hypertrehalosemic hormone I (Pea-CAH-I) from the American cockroach as well as with the red pigment-concentrating hormone (RPCH) of prawns. Injection of the synthetic peptide into larvae or young adults of T. molitor or adult Z. rugipes increases the hemolymph carbohydrate levels in a dose-dependent manner. Thin layer chromatography identified the elevated sugar component of the hemolymph as the disaccharide trehalose. Carbohydrate release from larval fat body in vitro was also shown upon administration of a low concentration of synthetic Tem-HrTH.

[Ultraviolet spectrophotometric determination of cantharidin in Mylabris].

In this study the content of cantharidin in Mylabris was analyzed quantitatively by UV. Analytical results thus obtained agree well with those by the neutralization method set forth in ChP. The average recovery of cantharidin is 99.98% and the coefficient of variation is 0.719%.

Etiologic agents, incidence, and improved diagnostic methods of cantharidin toxicosis in horses.

In addition to the 3-striped blister beetles (Epicauta temexa and E occidentalis), other sources of equine cantharidin toxicosis were identified at the Texas Veterinary Medical Diagnostic Laboratory and included E albida and E attrivittata and the previously incriminated E pardalis and E pennsylvanica. Improved methods for diagnosing cantharidin or blister beetle toxicosis involve partial purification of urine and gastric content extracts, using silica cartridges, followed by analysis, using capillary gas chromatography/mass spectrometry. During a 26-month period, 53 episodes of cantharidin toxicosis in horses were confirmed at our diagnostic laboratory. Concentrations of cantharidin in urine and gastric contents ranged from 0.0003 to 3.50 micrograms/g. Peak incidences were observed in late summer and early fall.

A sensitive IL-1 thymocyte co-stimulator assay using a novel beta-D-galactoside specific lectin from the beetle, Allomyrina dichotoma.

A sensitive thymocyte co-stimulator assay of IL-1 using a beta-D-galactoside specific lectin (allo A) obtained from the beetle (Allomyrina dichotoma) is reported here. Allo A stimulated [3H]thymidine uptake of mouse thymocytes in the presence of IL-1. The allo A assay was more sensitive than the PHA or PNA- thymocyte assay, especially at low doses of IL-1. Optimal conditions for the allo A assay were as follows: allo A, 2.5-5.0 micrograms/ml; whole thymocytes, 0.5-1.0 x 10(6) cells/well; incubation time, 72-96 hr. The assay is sensitive and convenient and can easily be performed in any laboratory.

Metasternal gland secretion of the locust tree borer, Megacyllene robiniae (Coleoptera: Cerambycidae).

1. The volatile components of metasternal gland extracts of male and female Megacyllene robiniae have been analyzed by gas chromatography-mass spectroscopy. 2. The major component was identified as 2-(1,3-hexadien-1-yl)-5-methyltetrahydrofuran, a new natural product. 3. 1-Phenylethanol is present only in male extracts. 4. Acetates of hexadecanol and octadecanol are also present.

Retinoid composition in the compound eyes of insects.

Retinoids in the compound eyes of insects in ten orders were extracted by the oxime method and analysed by HPLC. Four geometrical isomers (13-cis, 11-cis, 9-cis and all-trans) of syn and anti retinal oximes, and syn and anti 3-hydroxyretinal oximes were separated in a single analysis by a stepwise eluent condition. The amounts of the two isomers, syn 11-cis and syn all-trans, were quantified. 11-Cis 3-hydroxyretinal was detected in six orders: Lepidoptera, Diptera, Coleoptera, Neuroptera, Hemiptera and Odonata, and retinal and 3-hydroxyretinal were found together in the compound eyes of some species of Coleoptera and Odonata. We conclude that early in their phylogeny, insects had the ability to use 3-hydroxyretinal as the chromophore of visual pigment. Peaks corresponding to syn 9-cis and 13-cis 3-hydroxyretinal oximes were observed on the chromatogram of extracts from fly heads and compound eyes of cicadas. Retinol and 3-hydroxyretinol were also analysed and quantified relative to retinal and 3-hydroxyretinal. Larger amounts of the alcohols than the aldehydes were found in the compound eyes of butterflies, hornets, cicadas and grasshoppers, which are diurnal insects. 3-Dehydroretinal has not been detected in insects.

Changes in total and mitochondrial nucleic acid content in aging Caryedon serratus Oliver (Coleoptera).

Aging in the beetle Caryedon serratus was accompanied by a decline in total DNA content (9.28% in males and 9.79% in females). Mitochondrial DNA remained constant until the 11th day of age and then decreased gradually with advancing age in both sexes. RNA content and the RNA/DNA ratio showed similar changes in whole body as well as mitochondrial fractions. The two parameters decreased continuously and significantly (p less than 0.001) in males whereas in females an initial rise during the reproductive period was followed by decline until death. The observations suggest that there is a loss of mitochondria in the senescent period and a decline in the total nucleic acid content and protein synthetic capacity of C. serratus.

Purification and characterization of a lectin from the beetle, Allomyrina dichotoma.

A lectin was purified from the hemolymph of Allomyrina dichotoma larvae by affinity chromatography on acid-treated Sepharose 4B. The purified lectin showed two protein bands on polyacrylamide gel electrophoresis. These two lectin bands (allo A-I and -II) were separated by DEAE-Cellulofine column chromatography. By gel filtration on Sephadex G-100, the molecular weights of allo A-I and -II were estimated to be 65,000 and 66,500, respectively. On the other hand, by SDS-polyacrylamide gel electrophoresis after cross-linking of subunits with glutaraldehyde, they are estimated to be 38,000 and 39,000, respectively. On SDS-polyacrylamide gel electrophoresis, it was proved that both allo A-I and -II lectin consisted of two subunits, respectively. The molecular weights were 17,500 and 20,000 for allo A-I, and 19,000 and 20,000 for allo A-II. The isoelectric points of allo A-I and -II were estimated to be 6.4 and 5.9, respectively. On double immunodiffusion, allo A-I and -II gave single precipitin lines, which fused completely with each other, against the antibody to crude allo A. The hemagglutinating activity of allo A-I and -II was inhibited only by beta-linked D-galactose such as lactose and lactulose.

The Bushman arrow toxin, Diamphidia toxin: isolation from pupae of Diamphidia nigro-ornata.

The Bushmen of the Kalahari Desert in Botswana use the pupae of the beetle Diamphidia nigro-ornata Ståhl to poison their arrows. Sequential aqueous extraction, ammonium sulfate precipitation, ultrafiltration and chromatofocusing have given an apparently homogeneous active protein from these pupae with an approximate mol. wt of 54,000, an isoelectric point of about 8.0 pH and a lethal potency (minimum lethal dose, MLD) between 5 and 20 micrograms/kg (i.p. mouse). Preliminary pharmacological studies on less purified material show that, after a delay, this Diamphidia toxin causes sustained contraction of isolated intestinal smooth muscle. This contraction is not blocked by atropine or mepyramine and, therefore, is not due to release of acetylcholine or histamine. Results on the phrenic nerve - hemidiaphragm preparation demonstrate that in the presence of the toxin, contraction in response to indirect stimulation gradually fails and is accompanied by contracture. Since direct stimulation of the muscle still elicits a contraction, the toxin apparently does not affect the contractile mechanism itself. We conclude that Diamphidia pupae contain a protein toxin that is responsible for its lethality. Although this toxin appears to differ in some properties from the toxins reported by Mebs et al., de la Harpe et al. and Kündig, these protein preparations undoubtedly correspond to each other. We did not find any evidence of the low molecular weight toxic component reported by Mebs et al.

Presence of 5-hydroxy-indolacetic acid in Diloboderus abderus larvae without monoamine-oxidase-like activity.

The authors confirmed the presence of monoamines and 5-hydroxy-indolacetic acid (5-HIAA) in the cephalic region of Diloboderus abderus larvae employing a high pressure liquid chromatography (HPLC) with electrochemical detector. An anticholinesterase insecticide produced an increase in the serotonin and 5-HIAA endogenous levels, and did not modify the catecholamines. Monoamine-oxidase activity was undetectable with a radioactive method using 3H-tyramine as substrate.

Preliminary studies on the chemical properties of the toxic principle from Diamphidia nigroornata larvae, a source of Bushman arrow poison.

The Bushmen of southern Africa use the expressed contents of beetle larvae (Diamphidia, Lebistina and Polyclada species) as arrow poison. an aqueous extract of Diamphidia nigroornata larvae was fractionated by gel filtration on Sephadex G-50. Two fractions were obtained: one (I) of high molecular weight which contains a protein of 60 000 daltons, and a low molecular weight fraction (II) of non-protein nature. Both fractions proved to be lethal to mice: an LD50 of 0.5 - 0.95 (I) and 3.2 - 3.5 (II) mg/kg (intraperitoneal injection), respectively, was determined. The toxic principle of fraction I could be partly separated from the protein by ammonium sulfate precipitation followed by gel filtration. That of fraction II was further resolved into several subfractions by gel filtration of Sephadex G-10; however, the lethal activity was completely lost during purification. In thin-layer chromatography the low molecular weight toxin(s) did not react with reagents for steroids, alkaloids, sugars or terpenes, but showed a positive ninhydrin reaction. It is concluded that the toxic principle of the Bushman arrow poison is a highly labile, low molecular weight compound which is closely attached or bound to a protein protecting it from inactivation.

Electron impact, chemical ionization and field desorption mass spectrometry of substituted dihydroxyphenyl-benzodioxins isolated from insect cuticle. Occurrence of thermal decomposition and oligomerization reactions in the mass spectrometer.

The electron impact, field desorption and chemical ionization mass spectra of seven dihydroxyphenyl-benzodioxins isolated from insect cuticle are discussed. Reproducible electron impact and chemical ionization spectra are obtained as a result of thermal decomposition and either electron impact or chemical ionization. In some of the chemical ionization spectra dimerization of the thermal degradation products is observed. Structure determination is possible based upon the electron impact and field desorption spectra.

High pressure liquid chromatographic determination of cantharidin, using a derivatization method in specimens from animals acutely poisoned by ingestion of blister beetles, Epicauta lemniscata.

Experimental animals (rabbit, rat, goat, sheep, and pony) were given cantharidin or dried preparations of blister beetles (Epicauta lemniscata) to stimulate naturally occurring toxicosis in which beetles were ingested with alfalfa hay. A sensitive high-pressure liquid chromatographic method, involving derivatization of cantharidin with p-nitrobenzyloxyamine, was developed to detect the toxin extracts of ingesta, fluids, and tissues from these severely poisoned animals. Urine and ingesta from the upper portion of the gastrointestinal tract, containing from 1 to 20 ppm of cantharidin, were the most satisfactory samples for diagnosing toxicosis. Beetle preparations also were assayed and found to contain widely varying amounts of cantharidin (0.89% to 5.40% of dry weight). Blood chemical analyses on sera and urine samples from the sheep and pony indicated renal dysfunction.