Twelve-hour duration testing of cream formulations of three repellents against Amblyomma americanum.

The repellent efficacies of the U.S. military repellent 33% N,N-diethyl-3-methylbenzamide (deet), 10% and 20% (1S, 2'S) 2-methylpiperidinyl-3-cyclohexene-1-carboxamide (SS220) and 10% and 20% 1-methyl-propyl-2-(hydroxyethyl)-1-piperidinecarboxylate (Bayrepel) cream formulations on human volunteers against the lone star tick Amblyomma americanum (L.) were evaluated in a simulated forest floor environment over a 12-h testing period. At 2-h intervals, volunteers, with repellent applied in a 5-cm-wide band around each ankle, stood for 5 min in plastic tubs containing leaf litter and 100 host-seeking A. americanum nymphs. Ticks were allowed to remain on a volunteer's feet and ankles for an additional 5 min after the volunteer exited the tub. All repellent formulations provided high levels of protection for the entire 12 h. No ticks crossed 5-cm-wide bands of 20% SS220 and Bayrepel during any challenge, and thus 100% protection was afforded throughout the test. These formulations showed a long-lasting efficacy hitherto unknown in tick repellents intended for use on human skin.

Three dimensional structure-activity relationships (3D-QSAR) for insect repellency of diastereoisomeric compounds: a hierarchical molecular overlay approach.

2-(2-Hydroxyethyl)-1-piperidinecarboxylic acid 1-methylpropyl ester (Picaridin), and 1-(cyclohex-3-ene-1-ylcarbonyl)-2-methylpiperidine (AI3-37220; 220) are alternatives to DEET (N,N-diethyl-3-methylbenzamide), the most popular mosquito repellent. Picaridin and AI3-37220 exhibit polychiral diastereoisomerism and each has four diastereoisomers due to the presence of two asymmetric centers in their molecules. The diastereoisomers of these compounds have differing degrees of mosquito-repellent activity according to quantitative behavioral assays conducted at the United States Department of Agriculture. An insight into the stereochemical requirements for repellency is of great importance in the development of better repellents. Molecular overlay of the optimized geometries of the diastereoisomers was considered as a novel tool for Stereochemical Structure-Activity Relationship (SSAR) modeling. An earlier study using molecular mechanics (MM2) optimized geometries showed good promise. In continuation of this effort and to overcome certain defects in using MM2 geometries, a hierarchical overlay approach was developed. In this method geometry of the low energy conformer of each diastereoisomer was optimized using: the following quantum chemical methods in a graduated manner: (a) semiempirical AM1, (b) Hartree Fock (STO3G, 3-21G, 6-31G, and 6-311G), and (c) Density Functional Theory (B3LYP/6-31G, B3LYP/6-311G). The optimized geometries of different diastereoisomers were overlaid in various user defined combinations to calculate the root mean square distances (RMSD) of the overlaid structures. The RMSD with respect to the most active diastereoisomer (220SS) were found to have a strong relationship with biological potency. Common motifs in shapes and molecular surfaces that are probably critical for effective repellent activity were identified. The hierarchical approach gave valuable information on the quantum chemical level (basis set) at which optimization must be carried out to get the correct order of repellency of the diastereoisomers of Picaridin and 220.

Isolation and identification of mosquito bite deterrent terpenoids from leaves of American (Callicarpa americana) and Japanese (Callicarpa japonica) beautyberry.

Essential oil extracts from Callicarpa americana and Callicarpa japonica were investigated. Bioassay-guided fractionation of C. americana extracts using the yellow fever mosquito, Aedes aegypti, led to the isolation of alpha-humulene, humulene epoxide II, and intermedeol and a newly isolated terpenoid (callicarpenal). Similar work involving C. japonica resulted in the isolation of an additional compound, spathulenol, as well as the four compounds isolated from C. americana. Structure elucidation was performed on all isolated compounds using a combination of gas chromatography-mass spectrometry-electron ionization, high-resolution liquid chromatography-MS-electrospray ionization, and one- and two-dimensional NMR experiments. Heretofore, 13,14,15,16-tetranorclerodane, callicarpenal, has never been identified from natural sources. Complete (1)H and (13)C NMR assignment data are provided for this compound. In bite deterrent studies, spathulenol, intermedeol, and callicarpenal showed significant repellent activity against A. aegypti and Anopheles stephensi.

Repellency of deet and SS220 applied to skin involves olfactory sensing by two species of ticks.

Responses of host-seeking nymphs of the blacklegged tick, Ixodes scapularis Say and lone star tick, Amblyomma americanum (Linnaeus) (Acari: Ixodidae) to the repellents N,N-diethyl-3-methylbenzamide (deet) and (1S, 2'S)-2-methylpiperidinyl-3-cyclohexene-1-carboxamide (SS220) were studied using fingertip laboratory bioassays. Ethanol solutions of both compounds applied to the skin strongly repelled both species of ticks at 0.8 and 1.6 micromole of compound/cm2 skin. The ticks were also repelled when two layers of organdie cloth covered the portion of a finger treated with either deet or SS220. Gas chromatographic analyses of the outer layer of cloth that had covered skin treated with 1.6 micromole compound/cm2 skin revealed only 0.1 nmole SS220/cm2 cloth and 2.8 nmole deet/cm2 cloth. However, in bioassays in which a single layer of cloth was treated with a dose of deet or SS220 equivalent to the amount found in the outer layer of cloth, ticks were not repelled. Results unequivocally demonstrated that these ticks responded to the repellents in the vapour phase when repellent treated skin was covered with cloth to obviate tactile contact with them, and made it clear that the ticks detect the repellents by olfactory sensing. Heretofore, the mode of action of deet and SS220 was unclear.

Comparative activity of deet and AI3-37220 repellents against the ticks Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) in laboratory bioassays.

The repellents N,N-diethyl-3-methylbenzamide (deet) and racemic 2-methylpiperidinyl-3-cyclohexene-1-carboxamide (AI3-37220) were evaluated using two different laboratory bioassays to determine their relative effectiveness against host-seeking nymphs of the blacklegged tick, Ixodes scapularis Say, and the lone star tick, Amblyomma americanum (L.). In a petri dish bioassay, ticks were released within a ring of repellent on a horizontal filter paper disk. In the second bioassay, ticks were allowed to climb a vertical strip of filter paper whose central portion was treated with a repellent. Deet and AI3-37220 were more effective against I. scapularis than A. americanum nymphs. In the petri dish bioassay, none of the concentrations of deet or AI3-37220 tested confined A. americanum within the treated ring. However, in the vertical bioassay, both species exhibited avoidance of the repellents, and I. scapularis was repelled by much lower concentrations than A. americanum. I. scapularis were repelled by lower concentrations in the vertical bioassay than in the petri dish bioassay. Deet was slightly more effective against I. scapularis than AI3-37220 in both bioassays, but AI3-37220 was significantly more effective than deet against A. americanum in the vertical bioassay.

Stereochemical effects in an insect repellent.

Racemic 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine repels blood-feeding arthropods such as mosquitoes, chiggers, and ticks. The compound contains two asymmetric carbon atoms and the racemate consists of four stereoisomers. Quantitative mosquito bioassays using Aedes aegypti (L.) showed that (1S,2'S) and (1R,2'S) configurations were 2.8-3.1 and 1.6-1.8 times more effective, respectively, than the other two stereoisomers in reducing mosquito bites. (1S,2'S) was 2.5 more repellent than the racemate. Biological data show that an interaction of the (2'S)-2-methylpiperidine configuration with a repellent receptor system in A. aegypti is apparently important to repellent activity. Nuclear magnetic resonance spectra and molecular mechanics calculations for the stereoisomers provided insight into the conformation of the (2'S)-group. Results indicate that enhanced repellent effects can be realized through formulation of the most active stereoisomers of the compound.

A new module for quantitative evaluation of repellent efficacy using human subjects.

A new module for quantitative evaluation of arthropod repellents in human subjects was designed, constructed, and protocols for use of the module were developed. Doses of 3 arthropod repellents, 1-[3-cyclohexen-1-ylcarbonyl] piperidine (AI3-35765), 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine (AI3-37220), and N,N-diethyl-3-methylbenzamide (deet), were evaluated using the mosquito, Anopheles stephensi Liston. Biting responses to varied doses of the repellents on human skin were quantified, and the effectiveness of the 3 repellents was compared. The new module consists of 6 test cells and permits the simultaneous comparison of up to 5 repellent doses or chemical types and a control using a complete randomized block design with minimal treatment interaction and with > or = 6 replicates per human subject.

Optically active arthropod repellents for use against disease vectors.

Optically inactive 1-[3-cyclohexen-1-ylcarbonyl] piperidine and 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine are repellents against blood-feeding arthropods. Pure stereoisomers of these compounds were synthesized and characterized for use in bioassays. Initial laboratory tests with the malaria vector Anopheles stephensi Liston showed that this species was repelled differentially by the stereoisomers of 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine. Two stereoisomers were twice as repellent as the other stereoisomers. These results indicate that stereoisomerism influences repellent efficacy in this class of compounds.

Structure-activity relationship observations for the bagworm moth pheromone.

Structure-activity relationship (SAR) observations were made for the bagworm moth pheromone, (R)-2-pentyl decanoate, and a series of analogs with modifications in the alcohol portion of the molecule. Observed attractiveness of these analogs was related to molecular structure and their physical attributes using computational chemistry. Electrostatic potential and Van der Waals (VdW) electrostatic coded surface three-dimensional (3D) maps of the molecular mechanics (MM) minimized lowest energy conformation of the pheromone show that size, shape, charge distribution, and chirality of the molecule are related to attractiveness.

Evidence for kairomonal influence on selection of host-ambushing sites by adult Ixodes scapularis (Acari: Ixodidae).

Unfed adult blacklegged ticks Ixodes scapularis Say, were tested under laboratory conditions for behavioral responses to substances rubbed from external glands on legs of white-tailed deer, Odocoileus virginianus (Zimmermann). In replicated trials, significantly more female ticks became stationary on portions of vertical glass tubing rubbed with the tarsal gland substances of both bucks and does than on nontreated tubing. This behavior, apparently an arrestant response, was infrequent among males. White-tailed deer urinate upon their tarsal glands, and doe urine also produced an arrestant response among female ticks. Metatarsal gland secretions of bucks elicited no arrestant response in either male or female I. scapularis, but doe metatarsal gland secretions elicited low-level positive responses from both sexes. A low-level arrestant response also was exhibited by females to secretions from the interdigital glands of bucks. In a second bioassay, three times as many females came to rest on the terminal ends of vertical glass rods whose bases were treated with secretions from buck interdigital glands than on rods with nontreated bases. This suggests that interdigital gland secretion influences blacklegged tick climbing behavior. Substances produced by or associated with the leg glands of O. virginianus may therefore serve as kairomones for host-seeking adult I. scapularis; interdigital gland secretions on soil or leaf litter may signal active deer trails, and tarsal gland substances may denote portions of vegetation contacted previously by white-tailed deer. In nature, use of these chemical cues by host-seeking adult I. scapularis may increase the probability of acquiring a suitable host.

Structure-activity relationship observations for European corn borer moth pheromone and fluoro analogs via computer molecular modeling.

Structure-activity relationship (SAR) observations were made for theZ-type European corn borer moth pheromone, (Z)-11-tetradecen-1-ol acetate, and a series of analogs with fluorination in the alcohol portion of the molecule. The attractiveness of these analogs and the pheromone was compared to the electrostatic potential map of the molecular mechanics (MM) minimized lowest energy conformation for each compound. A critical range of electrostatic potential on the protons of the double-bond appears to be essential for optimal acceptor fit and attractiveness.

Moth responses to selectively fluorinated sex pheromone analogs.

Partially fluorinated analogs of the European corn borer (Ostrinia nubilalis) female sex pheromone, 11-tetradecenyl acetate (97:3Z:E), having mono- and trifluorsubstitutions at the terminal carbon of the pheromone chain, mimicked the biological activity of the pheromone, while analogs with fluorine at either side of the double bond and a pentafluoro analog were essentially inactive. Comparison of the pheromonal activity of these analogs with the previously reported activity of similarly fluorinated pheromones in five other species of moths revealed an unpredictable relationship between fluorine substitution pattern and pheromone-mimicking activity. Fluorine substitution patterns that rendered pheromonal analogs biologically inactive in the European corn borer had no detrimental influence upon pheromonal activity in other species and the converse was also true. This is evidence that the relative importance of electronic qualities of sites within a pheromone molecule differ from species to species. Furthermore, it indicates that the biochemical components (pheromone receptor proteins, binding proteins, and enzymes) that make up moth olfactory chemosensory systems must also vary structurally from species to species, despite the fact that they are involved in olfactory sensing of compounds having very similar chemical structure.

Sex pheromone catabolism in the redbanded leafroller moth.

Tritium-labeled components of the red-banded leaf-roller female sex pheromone, (Z)- and (E)-[11,12-(3)H2]-11-tetradecenyl acetate (57 Ci/mmol), applied to antennae of males and females were degraded causing formation of tritiated 11-tetradecenol, 11-tetradecenoic acid, and water. Results indicate that the catabolic pathway involves acetate hydrolysis, oxidation of alcohol to fatty acid, and degradation of the acid viaß-oxidation. Both geometric isomers were degraded equally well by males but degradation proceeded comparatively less rapidly with female antennae. It is surmised that under natural conditions of olfactory sensing, sex pheromone impinging upon the moth's antennae is probably subject to a similar catabolic fate.

Biological activity and in vivo degradation of tritiated female sex pheromone in the male European corn borer.

Isomers of [11,12-(3)H2]-11-tetradecenyl acetate (57 Ci/mM) were synthesized. Behavioral assay of the two compounds using Z- and E-type European corn borer (ECB) males showed that introduction of tritons into the double bond of the pheromone caused a significant isotope effect in the E-type ECB but not in the Z-type ECB. Measurements of tritium associated with the male antennae after a 3-min exposure showed that radioactivity equivalent to 10(-7) mol pheromone was adsorbed onto male antennae. Time-course in vivo metabolic studies with picogram amounts of compound applied topically to antennae of E- and Z-type males and Z-type females showed that they metabolized pheromone similarly but females degraded pheromone more slowly than males. Pheromone was hydrolyzed, and the only other major radiolabeled metabolite observed by combined high-pressure liquid chromatography-radiodetection was tritiated water. Capillary gas chromatography and radiomonitoring permitted detection of a trace amount of 11-tetradecenoic acid, which indicated alcohol oxidase activity is associated with the antennae. Evidence shows that clearing of pheromone from the ECB male antennae involves hydrolysis and oxidation of the alcohol to fatty acid, which in turn is degraded, probably via ß-oxidation, to carbon dioxide and water.

European corn borer: Pheromonal catabolism and behavioral response to sex pheromone.

When physiologically excessive amounts of the female sex pheromone of the European corn borer (ECB) or esters analogous to the pheromone were applied to the antennae of males, their behavioral responsiveness to pheromone in a flight tunnel was significantly impaired for 2 hr. Concurrent quantitative analyses of heptane extracts of the male antennae by gasliquid chromatography showed that the compounds applied to antennae were hydrolyzed and, at 2 and 4 hr posttreatment, little or none of the compound applied or hydrolysis product was detectable in the antennal extracts. After 4 hr of in vivo incubation, male responsiveness to pheromone was restored among moths treated with the analogs but not among moths treated with pheromone. Esterase activity on the antennae was moderately inhibited in vivo by a pheromone analog that is a so-called transition-state esterase inhibitor, 1,1, 1-trifluoro-14-heptadecen-2-one. However, the analog did not inhibit male behavior when it was coevaporated with pheromone in a flight-tunnel assay. Therefore, in the presence of pheromone, the analog did not compete well for esterase or the pheromone receptor. Treating the antennae of intact males with tetrahydrofuran obliterated sex pheromone response capability in males, but the treatment did not significantly attenuate esterase and other catabolic activity of the antennae. Indications are that degradation of esters on the ECB antennae involves substrate-nonspecific esterase activity and other metabolic processes that in turn remove hydrolysis products from the antennae. Maintenance of a male's ability to respond to pheromone is linked to these processes.

European corn borer sex pheromone : Inhibition and elicitation of behavioral response by analogs.

The male sexual behavior-stimulating and inhibiting properties of a series of analogs of the European corn borer sex pheromone were determined in a flight tunnel. The structural requirements for inhibition of pheromonal response were far less restrictive than those for elicitation of that response. Analogs that by themselves elicited upwind flight response from males at a low dose were generally less inhibitory to male response than many of the analogs that had no pheromonal activity. These findings suggest that many pheromone analogs bind to pheromone receptors without provoking behavioral response and possibly undergo slower degradation on the antenna than pheromonally active compounds. The disparity of response to analogs by two pheromonal types of the European corn borer indicates that the pheromone receptor and pheromone catabolic systems are biochemically very different in the two types.

Identification of a neuropeptide hormone that regulates sex pheromone production in female moths.

A pheromone biosynthesis activating neuropeptide (PBAN) hormone that controls sex pheromone production in female moths was identified from the brain-subesophageal ganglion complexes of the adult corn earworm, Heliothis zea. PBAN has 33 amino acid residues and a molecular weight of 3900. Its amino acid sequence has no significant homology with any of the fully characterized peptide hormones. The synthetic peptide, at a dose of between 2 and 4 picomoles, induced production of a normal quantity of sex pheromone in ligated H. zea females. The peptide also induced pheromone production in six other species of moths, thus indicating that this or similar peptides may be responsible for the regulation of pheromone production in moths.

European corn borer sex pheromone : Structure-activity relationships.

The biological activity of analogs of the pheromone components of the European corn borer,Ostrinia nubilalis, (Z)- and (E)-11-tetradecen1-ol acetate, in which modifications were made in the terminal alkyl portion were studied in the three pheromonal types of the insect. European corn borer males respond to pheromonal stimuli at three levels of behavioral activity, i.e., short-range sexual stimulation, activation in the flight tunnel, and response in the field. Structural requirements for elicitation of response at these levels were found to be increasingly restrictive, respectively. Flighttunnel activity was induced only by compounds that had a total chain length of 13 or 14 carbons and in which branching at carbon 13 was limited to one methyl group or a cyclopropyl group. Three new analogs were active in the flight tunnel, viz., (E and (Z)-13-methyl-11-tetradecen-1-ol acetate and (Z)-12-cyclopropyl-11-dodecen-1-ol acetate. The cyclopropyl analog was the most active analog against theZZ type of the European corn borer. TheE isomer, however, was pheromonally inactive in theEE type and was shown to be a pheromone antagonist. This dissimilarity is most likely due to differences in structure of the receptors in the European corn borer strains. Analogs that were biologically active against the European corn borer were tested against the redbanded leafroller,Argyrotaenia velutinana, which also uses (Z)- and (E)-11-tetradecen-1-ol acetate as part of its pheromone. Results showed that the redbanded leafroller pheromone acceptor system is different from that of the European com borer; marginal behavioral response was elicited by only one of the new analogs. Thus, although both species use 11-tetradecen-1-ol acetate isomers as their pheromone, the mechanisms by which they are perceived are different.

Female sex pheromone of iris borer,Macronoctua onusta (Lepidoptera: Noctuidae).

Chromatographic and mass spectrometry studies of heptane extracts of the ovipositors of the iris borer,Macronoctua onusta, showed that the females produce several compounds that are the same as those produced by females of the tobacco budworm,Heliothis virescens. In trapping experiments, a mixture of (Z)-11-hexadecenal, (Z)-11-hexadecen-1-ol, and (Z)-9-tetradecenal (94∶4∶2) proved to be the minimum set of compounds required to cause effective capture of iris borer males in the field.