Components of honeybee royal jelly as deterrents of the parasitic Varroa mite, Varroa destructor.

The parasitic mite Varroa destructor Anderson & Trueman reproduces on the immature stage of the honeybee, Apis mellifera L. Mites are found more often on drone brood than worker brood and only infrequently on queen brood. We investigated the chemical basis for the low incidence of mites on queen brood. V. destructor mites were deterred by a crude extract of royal jelly, a glandular secretion produced by nurse bees and fed to queen larvae. Bioassay-driven fractionation of the crude extract via column chromatography resulted in one active fraction that was as active as the crude extract. Compounds in the active fraction were identified using gas chromatography (GC) and coupled gas chromatography/mass spectrometry (GC-MS). Before injection, compounds were esterified with MeOH/sulfuric acid, followed by silylation of any hydroxyl groups present. The active fraction contained at least 22 compounds, all fatty acids, several of which contained an additional hydroxyl group on the alkyl chain. Synthesis of some of these compounds that are not commercially available is described. A synthetic mixture containing most of the compounds in the active fraction was as active as the active fraction in the bioassay.

Larval Apis mellifera L. (Hymenoptera: Apidae) mortality after topical application of antibiotics and dusts.

Beekeepers apply various dusts to honey bee, Apis mellifera L., colonies to dislodge parasitic mites and control bacterial brood diseases. Anecdotal reports by beekeepers indicate that the antibiotic oxytetracycline (OTC) can be toxic when applied in powdered sugar to cells containing immature bee brood, but it was not known whether the purported toxicity is caused by the antibiotic or the sugar carrier. Additionally, the toxicity of various dusts, proposed for parasitic mite control, is poorly known. In the current studies, we tested OTC and two other antibiotics (tylosin and lincomycin, candidate compounds for use in honey bee colonies) in a powdered sugar carrier for larval toxicity. We also tested for larval toxicity, several dusts that have been proposed for mite control. OTC caused significant brood mortality of approximately 80% at the concentrations used in the hive (200 mg in 20 g sugar). In contrast, tylosin and lincomycin at the 200 mg dose were both similar to untreated controls, and only five times that concentration (1000 mg) caused significant brood mortality of approximately 65%. The addition of dusts, wheat flour, talc, and a commercially available protein supplement, BeePro, resulted in mortality levels between 65 and 80%, similar to that seen with OTC. The common antibiotic carrier, powered confectioners sugar, was nontoxic. The use of 100 unsealed brood cells was demonstrated to be a reliable means of assessing potential adverse affects of dry compounds on larval honey bees. Two new candidate antibiotics for use in honey bee colonies were less toxic to larval bees than the currently labeled antibiotic, OTC.

Identification of four additional myoinhibitory peptides (MIPs) from the ventral nerve cord of Manduca sexta.

Four new myoinhibitory peptides were isolated and identified from the ventral nerve cord of adult Manduca sexta. The new peptides are related to two previously identified myoinhibitory peptides also isolated from adult M. sexta, Mas-MIP I and Mas-MIP II. The sequences of the new peptides are APEKWAAFHGSWamide (Mas-MIP III), GWNDMSSAWamide (Mas-MIP IV), GWQDMSSAWamide (Mas-MIP V), and AWSALHGAWamide (Mas-MIP VI). Mas-MIPs III-VI were found to inhibit spontaneous peristalsis of the adult M. sexta anterior hindgut (ileum) in vitro.

Testis ecdysiotropin, an insect gonadotropin that induces synthesis of ecdysteroid.

Testes of lepidoptera synthesized ecdysteroid in a somewhat different temporal pattern than the prothoracic glands that release ecdysteroid to the hemolymph. Brain extracts from Heliothis virescens and Lymantria dispar induced testes to synthesize ecdysteroid, but did not affect prothoracic glands. The testis ecdysiotropin (LTE) was isolated from L. dispar pupal brains by a series of high-pressure chromatography steps. Its sequence was Ile-Ser-Asp-Phe-Asp-Glu-Tyr-Glu-Pro-Leu-Asn-Asp-Ala-Asp-Asn-Asn-Glu-Val-Leu-Asp-Phe-OH, of molecular mass 2,473 Daltons. The predominant signaling pathway for LTE was via G(i) protein, IP3, diacylglycerol and PKC; a modulating pathway, apparently mediated by an angiotensin II-like peptide, was controlled via G(s) protein, cAMP, and PKA. Testis ecdysteroid caused isolated testis sheaths to also synthesize a growth factor that induced development of the male genital tract. The growth factor appeared to be a glycoprotein similar to vertebrate alpha-1-glycoprotein. A polyclonal antibody to LTE indicated LTE-like peptide in L. dispar brain medial neurosecretory cells, the suboesophageal, and other ganglia, and also in its target organ, the testis sheath. LTE immunoreactivity was also seen in testis sheaths of Rhodnius prolixus. LTE-like immunoactivity was also detected in developing optic lobes, antennae, frontal ganglia, and elongating spermatids of developing L. dispar pupae. This may indicate that LTE has a role in development as well as stimulation of testis ecdysteroid synthesis. Published 2001 Wiley-Liss, Inc.

Synthesis of (Diethyl-d(10)) coumaphos and related compounds.

Two deuterated insecticides were prepared for use as internal standards for gas-liquid chromatographic-mass spectrometric analyses. Diethyl chlorothiophosphate-d(10) was prepared by reaction of ethanol-d(6) with P(2)S(5) to give labeled diethyldithiophosphoric acid, followed by chlorination. Treatment of the acid chloride with 3-chloro-4-methyl-7-hydroxycoumarin and potassium carbonate in acetone at reflux gave labeled coumaphos. An analogous reaction with 4-methyl-7-hydroxycoumarin gave labeled potasan, and the technique should be usable for synthesis of labeled forms of other dialkyl thiophosphate insecticides.

Development of a gel formulation of formic acid for control of parasitic mites of honey bees.

Formic acid has been used in various countries for the control of parasitic mites of honey bees (Apis mellifera), particularly the Varroa mite (Varroa jacobsoni) and the tracheal mite (Acarapis woodi). Its corrosivity and consequent fear of liability have precluded commercial interest in the United States, and its rapid vaporization requires frequent reapplication. We have developed a gel formulation of formic acid which provides controlled release over 2-3 weeks and improves the convenience and safety of handling of formic acid. The strong acidity of formic acid restricts the choice of gelling agents; vegetable gellants such as agar are destroyed, and bentonite clay derivatives do not gel, even with high-shear mixing. Polyacrylamides lead to viscous liquids lacking thixotropic properties. High-molecular-weight poly(acrylic acids) and fumed silicas provided gels with suitable physical characteristics. The poly(acrylic acid) gels were difficult to mix and gave slower and nonlinear release behavior, while the fumed silica gels were easy to prepare and linear in formic acid vaporization.

Structure-function analysis of Lymantria testis ecdysiotropin: a search for the active core.

A structure-function study was performed on the synthetic 21 residue neuropeptide, Lymantria testis ecdysiotropin (LTE), originally isolated from brains of Lymantria dispar pupae. The peptide induces ecdysteroid synthesis by testis sheaths of various lepidopteran species. LTE, as well as synthetic LTE 1-11, 11-21, and 11-15, stimulated synthesis in larval and pupal testes of Lymantria dispar at concentrations of 10(-9) to 10(-15) M; LTE 16-21 was weakly active, and an elongated LEU-LTE was inhibitory to synthesis at all but extremely low concentrations (10(-15) M). Since the sequence and polarity of residues in LTE 1-11, 11-15, and 11-21 are quite different, several parts of the molecule must activate receptors which initiate the cascade, resulting in ecdysiogenesis in Lepidopteran testes.

Structure-activity relationships in C-terminal fragment analogs of pheromone biosynthesis activating neuropeptide in Helicoverpa zea.

A number of analogs of the C-terminal hexapeptide of PBAN were prepared and tested in vivo for pheromonotropic activity in Helicoverpa zea. Peptides prepared with longer-chain omega-aminocarboxylic acids (Tyr-6-aminocaproyl-Leu-NH2 and Tyr-7-aminoheptanoyl-NH2) were active at 25 and 2.5 nmol. Acetyl-Pro-Arg-Leu-NH2 was active at 1,000 pmol and represents a new minimum active fragment in the PBAN system. Addition of a bulky, hydrophobic tail (4-octylphenoxyacetyl) to the C-terminal hexapeptide of PBAN gave an analog that was active at all concentrations tested from 1 to 1,000 pmol when injected, had slight oral activity, but had no activity when applied topically. Glu-Tyr-Phe-Ser-Pro-Arg-Leu-NH2 was active at 1,000 but not at 100 pmol; at the latter dose it synergised the activity of 5 pmol of PBAN.

Immunocytochemical localization of testis ecdysiotropin in the pupa of the gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae).

Antiserum against testis ecdysiotropin isolated from the gypsy moth, Lymantria dispar, reacted with neurons in the protocerebrum, optic and antennal lobes, subesophageal, thoracic and abdominal ganglia, as well as in nerve tracts extending through the optic lobes, tritocerebrum, and interganglionic connectives of the pupal stage of these insects. Testis ecdysiotropin is a peptide required by immature moths to initiate production of testes ecdysteroid, which is necessary for the development of the male reproductive system and initiation of spermatogenesis. Antiserum against testis ecdysiotropin also detected an accumulation of testis ecdysiotripic-like material between the inner and outer testis sheaths of pupae. The localization of this peptide in the imaginal disks of the last larval stage, cells and nerve fibers in the optic and antennal lobes of the pupa of both sexes, as well as in the testes during development of the adult reproductive system indicates that testis ecdysiotropin has a much larger impact on adult metamorphosis than development of the reproductive system and initiation of gametogenesis. Although this peptide may have a modulatory role in the central nervous system (CNS), it may also initiate a cascade of activity required for the development of the adult nervous system, in addition to its role in reproduction.

The pheromone of the eastern tent caterpillar,Malacosoma americanum (F.) (lepidoptera, Lasiocampidae).

The pheromone system of the eastern tent caterpillar,M. americanum, has been identified as a mixture of (E,Z)-5,7-dodecadienal and the corresponding alcohol. Field data on the attractiveness of the aldehyde alone were not consistent, but mixtures of aldehyde and alcohol in varying proportions were attractive to males. Addition of small amounts ofE,Z acetate toE,Z aldehyde had no effect on male response, but larger amounts reduced trap catch. Traps baited withZ,E, E,E, orZ,Z aldehydes were not more attractive than blank traps. Pherocon IC traps fortified with extra adhesive and baited with lures consisting of 500 µg (E,Z)-5,7-dodecadienal with either 250 or 100 µg of the corresponding alcohol trapped as many as 100 males/trap/night with means of 15-20. Lures prepared from purified (94%E,Z) aldehyde and alcohol were more attractive than those prepared from unpurified (58%E,Z) materials.

Primary structure of a novel neuropeptide isolated from the corpora cardiaca of periodical cicadas having adipokinetic and hypertrehalosemic activities.

A new neuropeptide hormone was isolated from the corpora cardiaca of the periodical cicadas, Magicicada species. Primary structure of the peptide as determined by a combination of automated Edman degradation after enzymatic deblocking with pyroglutamate aminopeptidase and mass spectrometry is: pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn-NH2. Synthetic peptide assayed in the green stink bug Nezara viridula caused a 112% increase in hemolymph lipids at a dose of 0.625 pmol, and a 67% increase in hemolymph carbohydrates at a dose of 2.5 pmol. Based on these results we designate this peptide, a first from order Homoptera, as Magicicada species-adipokinetic hormone (Mcsp-AKH).

Degradation of pheromone biosynthesis-activating neuropeptide (PBAN) by hemolymph enzymes of the tobacco hornworm, Manduca sexta, and the corn earworm, Helicoverpa zea.

The tritium-labeled bis-norleucine analog of Helicoverpa zea pheromone biosynthesis-activating neuropeptide ([3H]NLPBAN) was incubated in vitro with hemolymph from Manduca sexta or H. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [3H]NLPBAN concentration of 0.9 microM the degradation proceeded at a very slow but physiologically plausible rate (2-10 fmol/min/microliters hemolymph). The primary [3H]NLPBAN degradation reaction in M. sexta hemolymph was not inhibited by 20 microM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10-phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio-HPLC profile of the degradation products. It is concluded that hemolymph of M. sexta and H. zea contains peptidase(s) capable of inactivating circulating PBAN.

Characterization of the substrate specificity of sucrose-phosphate synthase protein kinase.

Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated by reversible protein phosphorylation. When the enzyme is phosphorylated it is inactivated and can be reactivated by removal of phosphate. The major regulatory phosphorylation site is known to be Ser158 in the spinach-leaf enzyme, and two protein kinase activities have been resolved chromatographically which phosphorylate SPS at this site in vitro. In this report, we use a set of synthetic peptide substrate analogs based on the phosphorylation site sequence, and a set of Escherichia coli-expressed 26-kDa fragments of spinach SPS which contain the site, to identify the recognition elements that target the two protein kinases to Ser158. The major recognition element consists of basic residues at P-3 and P-6 relative to the phosphorylated serine. Comparison of the spinach enzyme amino-acid sequence with two other plant species show conservation of these amino acids and implies that these signals are also conserved. We also present evidence that glucose-6-phosphate is not only an allosteric activator of SPS but also an inhibitor of SPS-protein kinase per se, thereby allowing it to act at both levels of SPS regulation.

The identification of two myoinhibitory peptides, with sequence similarities to the galanins, isolated from the ventral nerve cord of Manduca sexta.

Two new myoinhibitory peptides, Mas-MIP I and Mas-MIP II, were identified from the ventral nerve cord of the adult tobacco hornworm, Manduca sexta. Sequences obtained by a combination of automated Edman degradation and electrospray mass spectrometry were, respectively, AWQDLNSAW and GWQDLNSAW. The native peptides were found to co-elute with synthetic C-terminal amides on a reverse phase HPLC system. When applied to isolated ilea (anterior hindgut) of adult M. sexta, both peptides were found to significantly reduce the rate of peristalsis, or abolish peristalsis entirely, at concentrations of 1 x 10(-9) M. Both peptides share sequence similarities with Lom-MIP, a previously identified myoinhibitory peptide from Locusta migratoria, and with the N-terminal portion of vertebrate peptides in the galanin family.

The glycosylceramides of the nematode Caenorhabditis elegans contain an unusual, branched-chain sphingoid base.

Caenorhabditis elegans was cultured in semi-defined medium containing yeast extract, soy peptone, glucose, hemoglobin, Tween 80, and sitosterol. Monoglycosylceramides were chromatographically purified from nematode extracts. Their structures were elucidated with mass spectrometry, nuclear magnetic resonance spectroscopy, and analysis of methanolysis products of the parent cerebrosides. The glycosylceramides were unusual in that the only long-chain sphingoid base detected was an iso-branched compound with a C-4 double bond (i.e., 15-methyl-2-aminohexadec-4-en-1,3-diol). Glucose was the only sugar moiety detected. The fatty acids consisted of a series of primarily straight-chain, saturated, 2-hydroxylated C20-C26 acids; some iso-branched analogs also occurred. The sphingomyelins of C. elegans were also hydrolyzed, and the same iso-branched C17 compound was the only sphingoid base detected. This is the first structural analysis of a nematode glycosphingolipid and the first report of an organism in which the long-chain sphingoid bases are entirely iso-branched.

Synthesis of a selenomethionine peptide and a preliminary study of transport into Escherichia coli monitored by high-performance liquid chromatography.

The tripeptide Gly-SeMet-Gly has been synthesized by a combination of solution and solid-phase methods. Increase in weight of the resin was very nearly theoretical, and purification was straightforward. Its absorption was compared to that of the corresponding peptide, Gly-Met-Gly, in E. coli using HPLC ion-exchange separation and fluorometric determination of the disappearance of peptides in the culture medium and the appearance of methionine and selenomethionine in the same culture medium. As E. coli are not known to possess extracellular peptidases, and in fact have been shown to possess transport systems for peptides, this absorption is interpreted as transport of the peptide through the cell wall and membrane into the cytoplasm, endohydrolysis of the peptide, and efflux of the peptides' amino acids. Uptake of both peptides was approximately equal, but was slowed when both peptides were present simultaneously.

Isolation and identification of a pheromonotropic neuropeptide from the brain-suboesophageal ganglion complex of Lymantria dispar: a new member of the PBAN family.

A pheromonotropic peptide was isolated from brain-suboesophageal ganglion complexes of the adult female gypsy moth, Lymantria dispar, using a 5-step HPLC purification protocol and an in vivo bioassay in Helicoverpa zea. The intact peptide was sequenced by automated Edman degradation. The L. dispar pheromone biosynthesis activating neuropeptide (Lyd-PBAN) is a C-terminally amidated 33-amino acid peptide with a molecular weight of 3881. The peptide was synthesized using Fmoc procedures. Lyd-PBAN has sequence homology with Hez-PBAN (81.8%) and Bom-PBAN-I (66.7%). All three PBANs share the C-terminal hexapeptide sequence, Tyr-Phe-Ser-Pro-Arg-Leu-NH2. In addition, the C-terminal pentapeptide sequences of Pseudaletia pheromonotropin (Pss-PT), Bombyx diapause hormone (Bom-DH), the locustamyotropins (Lom-MT) and leucopyrokinin (Lem-PK) are identical or have a high degree of homology to the C-terminus of PBANs.

Identification of male-specific volatiles from nearctic and neotropical stink bugs (Heteroptera: Pentatomidae).

Males of the Central American stink bug species,Euschistus obscurus, produce an attractant pheromone composed of a blend of compounds characteristic of North AmericanEuschistus spp. and the South American soybean pest,E. heros. The range ofE. obscurus extends into the southern United States, the species is easy to rear, and males produce an exceptionally large quantity of pheromone (>0.5µg/day/male). These factors madeE. obscurus useful for characterizing the novel pheromone components ofE. heros without importing this pest species into the United States.Euschistus obscurus males produce methyl (2E,4Z)-decadienoate (61 %) in abundance, which is characteristic of North American species, and methyl 2,6,10-trimethyltridecanoate (27%), the main male-specific ester ofE. heros. The chirality ofEuschistus spp. methyl-branched esters, and field activity of synthetic formulations, remain to be determined.

Isolation and identification of a novel peptide from the accessory sex gland of the female house fly, Musca domestica.

A peptide of novel structure was isolated from accessory glands of the female house fly, Musca domestica by molecular-weight limit centrifugal ultrafiltration and reversed-phase HPLC. After amino acid and protein sequence analyses, the structure of this peptide was confirmed by chemical synthesis and plasma desorption mass spectral analysis to be Leu-Leu-Asn-Ala-Leu-Pro-Leu-Asp-Ala-Leu-Ser-Ser-Leu-Thr-Gly-NH2. The accessory gland peptide stimulated contraction of the house fly oviduct at concentrations of 10(-7) M and above.