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Substitution of nine hydrogens by nine fluorines in the butyl terminus of (Z)-7-dodecenyl acetate, the major component of the pheromone blend of the cabbage looper,Trichoplusia ni (Lepidoptera: Noctuidae), produces a highly volatile analog that is significantly less active as a replacement for the natural pheromone component, either alone or substituted in the sixcomponent pheromone blend of this species. When 9,9,10,10,11,11,12,12, 12-nonafluoro-(Z)-7-dodecenyl acetate (nf-Z7-12: OAc) was tested in a wind tunnel as a single component or as part of the six-component blend, males exhibited preflight activation, but they did not orient in the pheromone plume and fly upwind over a five-decade range of doses at levels comparable with those of the natural component and blend. Addition of large amounts of nf-Z7-12:OAc to the optimal dose of the natural blend also did not effect flight arrestment as when increased amounts of Z7-12:OAc were added. The nonafluoro (nf) analog of (Z)-9-tetradecenyl acetate, a minor component in theT. ni blend, did not affect male behavior when substituted in the sixcomponent blend, and substitution of both nf analogs in the natural blend produced the same results as observed for blends containing nf-Z7-12:OAc. The lack of an effect with substitution of nf-Z9-14:OAc is discussed in relation to the reported redundancy in the action of minor components inT. ni.

Analogs of (Z)-9-tricosene (Z9-23: Hy) bearing methyl substituents, cyclopropyl groups, fluorine substituents, and additional double bonds were used to probe the substrate requirements for the monooxygenase system that converts Z9-23: Hy to the corresponding epoxide and ketone. Three of the seven analogs tested, 10-fluoro-(Z)-14-tricosene, 10,10-difluoro-(Z)-14-tricosene, and 14-methyl-(Z)-9-tricosene, were metabolized to the corresponding epoxide. Compounds with two methyl groups, a cyclopropane group, a hydroxy group, or an additional double bond at the 14 position were not epoxidized at the 9,10 position. This suggests that only minimal structural change at the 14-position of Z9-23: Hy is allowed with retention of metabolic activity. None of the analogs tested were hydroxylated at the position equivalent to the 14 position of Z9-23:Hy. Of the 13 analogs tested as inhibitors of Z9-23:Hy metabolism, the two compounds that were the most effective inhibitors in both male and female houseflies were (Z)-14-tricosen-10-one and 1-nonyl-1-[(Z)-4-tetradecen-1-yl]-cyclopropane. These data show that the poly substrate monooxy genase that metabolizes Z9-23: Hy in the housefly has very strict structural requirements for the substrate.

Cuticular monooxygenases in the cuticle of the female houseflyMusca domestica oxidize (Z)-9-tricosene to m-9,10-epoxytricosane and to (Z)-9-tricosen-14-one. One possible explanation for these two products is that a single monooxygenase accepts the alkene substrate in two different orientations. Eleven analogs of (Z)-9-tricosene bearing methyl substituents, cyclopropyl groups, fluorine substituents, deuterium substituents, and additional double bonds were synthesized to probe the substrate requirements of this monooxygenase system. In addition, the 11 corresponding epoxides were prepared to aid in identification of the metabolites of each modified alkene.

The role of the alkyl terminus of green leaf volatile (GLV) molecules in olfactory reception and inactivation was examined in three diverse insect species: the beet armyworm,Spodoptera exigua (Lepidoptera); the Colorado potato beetle,Leptinotarsa decemlineata (Coleoptera); and the desert locust,Schistocerca gregaria (Orthoptera), using selectively fluorinated analogs of GLVs and electroantennograms (EAGs). When only the magnitude of the depolarization of the EAG is considered (a measure of reception), the order of effectiveness was 1-hexanol (6:OH)=(Z)-3-6:OH > 5,5,6,6,6-pentafluoro-(Z)-3-6:OH =5,5-difluoro-(Z)-3-6:OH ≫ 5,5,6,6,6-pentafluoro-6: OH. Percent recovery of the EAG (a measure of inactivation) was greater for the pentafluoro-(Z)-3-6: OH analog than for the difluoro-(Z)-3-6: OH analog. Our results show that the alkyl end of GLV molecules plays an important role not only in reception, but also inactivation processes in insect olfaction. Furthermore, specificities of these two processes may differ.

The electrophysiological and behavioral responses of maleAgrotis segetum to fluorinated analogs of (Z)-5-decenyl acetate (Z5-10∶OAc) were investigated. The single sensillum recordings showed that 4,4-difluoro-(Z)-5-decenyl acetate (4,4-F2), 7,7-difluoro-(Z)-5-decenyl acetate (7,7-F2), 10,10,10-trifluoro-(Z)-5-decenyl acetate (10,10,10-F3) and 7,7,8,8-tetrafluoro-(Z)-5-decenyl acetate (7,7,8,8-F4) were each 100-fold less active than the natural Z5-10 ∶ OAc, whereas the 7,7,8,8,9,9,10,10,10-nonafluoro-(Z)-5-decenyl acetate (F9) analog was essentially inactive. A mixture of Z5-10 ∶ OAc, Z7-12 ∶ OAc, and Z9-14 ∶ OAc on a filter paper dispenser was as attractive as female gland extracts when tested in a flight tunnel. With Z5-10∶OAc omitted, the two-component mixture elicited a significantly lower male response. Four analogs, 7,7-F2, 10,10,10-F3, 7,7,8,8-F4, and F9, were added separately to the two-component mixture to replace Z5-10∶OAc. The responses elicited by the mixtures containing the 7,7-F2, 10,10,10-F3, and 7,7,8,8-F4 analogs did not differ significantly from that of the natural three-component mixture and the two-component mixture, whereas the mixture containing F9 elicited a significantly lower male response, as low as the response to the two-component mixture. In a field test the mixtures containing 10,10,10-F3 and 7,7,8,8-F4 were significantly more active than the two-component mixture, but still less active than the natural three-component mixture. It appears that field tests provided greater discrimination among pheromone analogs in assessing their behavioral activity than the flight-tunnel test did. Structure-activity analyses demonstrate the importance of the lipophilic interaction between the terminal alkyl chain and the receptor site for the activity of the stimulus. The lipophobicity of the fluorinated analogs impedes a productive receptor interaction.

Competitive field tests with α-fluorinated analogs of compounds III and IV (III-α-F and IV-α-F, respectively) of the boll weevil,Anthonomus grandis Boh., aggregation pheromone showed these compounds, when combined with the other pheromone components [(±)-I and II], to be as attractive as grandlure [(+)-I, II, and III+IV]. Dose-response curves constructed from electroantennograms of male boll weevils to serial stimulus loads of III, IV, III-α-F, IV-α-F, and the corresponding acyl fluorinated analogs (III-acyl-F and IV-acyl-F) showed the α-fiuorinated analogs to be as active as the pheromone components (threshold=0.1 μg), while the acyl fluorinated analogs had a 10-100 x higher threshold (=1-10 μg). Single-neuron recordings showed that IV neurons and II neurons (Dickens, 1990) responded to IV-α-F and III-α-F, respectively, while IV-acyl-F and III-acyl-F were inactive. Since a previous study showed compounds I, II, and IV to be essential for behavioral responses in the field, it seems likely that the activity of the α-fluorinated analogs observed here is due to the stimulation of IV neurons by IV-α-F as indicated in single neuron recordings.

A short synthesis of [4,5-(3)H2] (E)-9-oxo-2-decenoic acid (ODA), a high-specific-activity tritium-containing isotopomer of the queen bee pheromone, is described. Catalytic tritiation of the ketal of ethyl 9-oxo-4-decenoate introduces tritium into two positions, one of which is completely unactivated. Subsequent transformation by selenation, oxidation, and hydrolysis affords the labeled 9-ODA at60 Ci/mmol. The material is suitable for biochemical studies of binding and catabolism in ovarian, antennal, and other target tissues.

For two decades, the aggregation pheromone of the boll weevil,Anthonomus grandis Boh. (Coleoptera: Curculionidae), was thought to consist of four compounds: I [(+)-(Z)-2-isopropenyl-1-methylcyclobutane ethanol]; II [(Z)-3,3-dimethyl-Δ(I,β)-cyclohexane ethanol]; III [(Z)-3,3-dimethyl-Δ(1,α)-cyclohexane acetaldehyde); and IV [(E)-3,3-dimethyl-Δ(1,α)-cyclohexane acetaldehyde). Evidence is presented from behavioral and electrophysiological studies to show that only three of these components, I, II, and IV, are essential for attraction. Competitive field tests, in which each possible three-component blend was tested against the four-component mixture, demonstrated that omission of I, II. or IV resulted in decreased trap captures (P0.01). Trap captures by these blends lacking I, II, or IV resembled those by the hexane solvent alone in a similar experiment. However, omission of III did not significantly alter field attractiveness of the blend. Dosage-response curves constructed from electroantennogram responses of both males and females to serial dilutions of III, IV, and a 50∶50 mixture of the geometric isomers III and IV showed both sexes to be 10- to 100-fold more sensitive to IV than III. Data from the electrophysiological studies were consistent with a single acceptor type for the (E)-cyclohexylidene aldehyde, IV, for males, and possibly one or two acceptor types for III and IV for females. Possible roles for the (Z)-cyclohexylidene aldehyde, III, and implications for the pheromonal attractant currently used in boll weevil eradication/suppression programs are discussed.

A series of 12 substituted (Z)-4-phenyl-3-buten-l-ol (PBO) derivatives were synthesized and evaluated for trail-following activity in five species of subterranean termites in the generaCoptotermes, Prorhinotermes, Reticulitermes, andSchedorhinotermes (Isoptera: Rhinotermitidae). The unsubstituted parent PBO was the most active for all species, and electron-withdrawing and electron-donating groups both reduced potency. Sensitivity to substitution in the ortho position suggests steric inhibition of binding by the 2'-substituted analogs. Different sensitivities to these pheromone analogs were found among the five species, withR.flavipes andS. lamanianus showing the highest level of trail-following activity for the PBO analogs.

Extracts of femaleRhipicephalus appendiculatus Neumann andR. pulchellus Neumann, vectors or potential vectors of East Coast fever, have yielded phenol andp-cresol. The same phenols have been found in five additional species of hard ticks, three of which also contained 2,6-dichlorophenol. Salicylaldehyde appeared as another phenolic component in four of these species. On the basis of this information- and of the rather limited literature available, it appears that ixodid ticks may generally utilize simple phenols as attractants. Behavioral observations suggest a role of the phenols in the meeting of the sexes.

Soldiers of the neotropical humivorous termiteArmitermes chagresi have large ice-tong-like mandibles for mechanical defense and a moderate length nasus from which cephalic gland defensive secretions are discharged. Soldiers do not eject secretion, but ooze droplets which are held at the nasus tip by hairs. The chemical secretion is composed of C22-C26 macrocyclic lactones, including C24 and C26 and α- and β-hydroxylated lactones, with C24 macrolides predominating. The cephalic gland secretion has no pheromonal properties and does not induce alarm or attraction in soldiers or workers. Upon application, the secretion repels and is irritating to ants, but does not appear to act as an antihealant. The defensive behavior and chemistry ofA. chagresi is discussed in light of termite ecology and evolution.

The chemistry of the defensive secretions of termite soldiers is reviewed. The structural variety of the chemical weapons, the uses of these substances in defense, and some potential biosynthetic interrelationships are discussed. Classification of the monoterpenes, sesquiterpenes, diterpenes, and various acetate-derived compounds according to termite subfamilies places the known defense substances in a phylogenetic perspective.

Soldiers of the humivorous termite generaCubitermes andCrenetermes (Isoptera: Termitidae: Termitinae) secrete a mixture of unusual diterpene hydrocarbons from the fontanellar opening of the frontal gland. The defense secretions of sixCubitermes species and oneCrenetermes species show species-specific distributions of the five chemically identified major components and 12 unidentified minor components. The secretion is also present with its characteric terpene distribution in white presoldiers and newly molted soldiers, but is completely absent in workers.

Soldiers of the East African fungus-growing termiteMacrotermes subhyalinus (Rambur) (Termitidae: Macrotermitinae) employ both mechanical and chemical defenses. Soldiers release a chemical secretion composed of long-chain saturated and monounsaturated hydrocarbons into wounds inflicted by their powerful mandibles. Chemical analysis of the secretion shows the paraffin fraction to consist primarily ofn-tricosane,n-pentacosane, 3- and 5-methylpentacosane, and 5-methylheptacosane. The major oleflns were identified as (Z)-9-heptacosene and (Z)-9-nonacosene. The secretion originates from the frontal glands of both major and minor soldiers; however, the hypertrophied gland of the major soldiers contains 500-fold more secretion than that of the minor soldiers. This secretion appears to impair the healing of wounds in test ants, and thus could represent a valuable supplement to the mechanical defense mechanism.

A series of mono-, di-, and trihalogenated acetate analogs of Zl 1–16: Ac were prepared and examined for electrophysiological activity in antennae of males of the diamondback moth,Plutella xylostella. In addition, two potential affinity labels, a diazoacetate (Dza) and a trifluoromethyl ketone (Tfp), were evaluated for EAG activity. The Z11–16∶Ac showed the highest activity in EAG assays, followed by the fluorinated acetates, but other halo-acetates were essentially inactive. The polar diazoacetate and the trifluoromethyl ketone were also very weak EAG stimulants. The effects of these analogs on the hydrolysis of [3H]Z11–16∶Ac to [3H]Z11–16∶OH by antennal esterases was also examined. The three fluorinated acetates showed the greatest activity as inhibitors in competition assays, with rank order F2Ac > F3Ac > FAc > Ac > Cl2Ac > ClAc > Dza > Br2Ac > BrAc > Tfp > I > Cl3Ac > Br3Ac > OH. The relative polarities of the haloacetates, as determined by TLC mobility, are in the order mono- > di- > trihalo, but F, Cl, Br, and I all confer similar polarities within a substitution group. Thus, the steric size appears to be the predominant parameter affecting the interactions of the haloacetate analogs with both receptor and catabolic proteins inP. xylostella males.

The chemical defense secretions of major and minor soldiers of over 18 colonies of the primitive glue-squirting nasute termiteLongipeditermes longipes (Isoptera, Termitidae, Nasutitermitinae) were analyzed chormatographically. The colonies, collected from four rainforest sites in peninsular Malaysia, showed monoterpene patterns rich in pinenes and limonene but with few quantitative differences between colonies. In marked contrast, the diterpene chemistry is high variable, and includes tricyclic (trinervitane), tetracyclic (rippertane), bicyclic (secotrinervitane), and a new spirotetracyclic (longipane) skeleton. Three new natural products are included in this remarkable and unprecedented example of structural diversity among different colonies of a single species.

Aldehyde components of lepidopterous pheromones are converted to carboxylic acids by aldehyde oxidizing enzymes (AOEs) that are present at high levels in antennal tissues of adult moths. The AOEs may include O2-requiring aldehyde oxidases as well as nucleotide-cofactor-requiring aldehyde dehydrogenases. Three classes of inhibitors were synthesized and examined in vitro for AOE inhibition usingHeliothis virescens antennae: (1) cyclopropanols, (2) α-fluorinated aldehydes, and (3) α,β-unsaturated carbonyls. First, cyclopropanated analogs of (Z)-11-hexadecenal (Z11-16∶A1), a common unsaturated aldehyde component of many species' pheromone blends, were synthesized as isosteric pheromone analogs and as potential inhibitors of the moth AOEs. NMR assignments are reported for thecis- andtrans-cyclopropanols. Cyclopropanols appear to act as oxidase-activated AOE inhibitors, perhaps via the unstable cyclopropanones. Second, α-fluoro and α,α-difluoro substituted analogs ofZ9-14∶A1 were synthesized and shown to be modest inhibitors of both the alcohol oxidase and AOE activities. Finally, the most potent inhibitors were α,β-unsaturated carbonyl mimics of theZ11-16∶A1. The α-methylene aldehyde was 1000-fold less effective than the vinyl ketoneZ1,11-16∶3-oxo. This inhibition appears irreversible and is postulated to involve electrophilic modification of an active site sulfur nucleophile.

Perfluoroalkyl analogs of pheromone constituents were synthesized and responses from male antennal olfactory receptor neurons from three moth species were recorded during stimulation by these analogs. In each analog, the hydrophobic terminus, either a butyl or hexyl substituent on the (Z)-alkenyl chain, was replaced with a perfluorobutyl (Pfb, C4F9) or perfluorohexyl (Pfh, C6F13) moiety. Perfluoroalkyl analogs were more volatile than their hydrocarbon analogs, showing a decrease in gas chromatographic retention time by two to four methylene equivalents (Kovats retention indices). Specialist neurons of maleHeliothis zea responded to a 0.02-μg dose of (Z)-11-hexadecenal (Z11–16∶Al) and a dose of 200 μg of Pfb-Z11–16∶Al with similar spike discharge rates. The HS(a) neurons ofTrichoplusia ni responded to a dose of 0.02 μg ofZ7–14∶OAc and a dose of 10 μg of Pfb-Z7–12∶Ac with similar discharge rates. The same difference in sensitivity to Pfb-Z7–12∶OH andZ7–12∶OH was observed for the responses of the HS(b) neuron and for the responses of theNS(a) neurons to Pfb-Z9–14∶Ac andZ9–14∶Ac. Sensilla ofDiatraea grandiosella similarly showed 100- to 1000-fold greater sensitivity toZ9–16—Al andZ11–16∶Al than to Pfh-Z9–16∶Al and Pfb-Z11–16∶Al. Thus, replacement of terminal alkyl groups with perfluoroalkyl groups in pheromone components produced biologically active compounds with increased volatility and displaced electrophysiological response profiles. Because of the diminished receptor cell sensitivity, we suggest that the binding of the fluorinated analogs to a putative receptor is reduced as a result of less favorable interaction between the hydrophobic protein binding site and the more rigid and more polar perfluoroalkyl moiety.

Sensory hair proteins from antennae of males of the wild silk moth,Antheraea polyphemus (Lepidoptera, Saturniidae) were incubated with radiolabeled 6E,11Z-hexadecadienyl acetate in the presence of unlabeled pheromone analogs as competitive inhibitors. The two extracellular proteins of importance, a highly active sensillar esterase and an abundant 15,000 mol wt binding protein, interact to degrade labeled pheromone less efficiently in the presence of certain unsaturated acetate analogs of the natural pheromone.Enzymatic hydrolysis of the acetate (or diazoacetate) was also examined for three pheromone analogs: [11,12-(3)H2]-6E,11Z-hexadecadienyl diazoacetate, [11,12-(3)H2]-hexadecyl acetate, and [9,10-(3)H2]-9Z-tetradecenyl acetate. The former two are poor substrates at concentrations over four orders of magnitude. The 9Z-14:Ac, however, is the best alternative substrate for this in vitro pheromone metabolism system. Unlabeled 9Z-14: Ac is also the best competitive inhibitor of the hydrolysis of labeled 6E, 11Z-16: Ac. Whereas the tritiated natural pheromone shows a flat response (ca. 40% conversion) to increasing concentrations from 3 × 10(-9) to 3 × 10(-6) M, tritiated 9Z-14: Ac is degraded more rapidly at higher concentrations.

Unsaturated aliphatic pheromones ofH. VIRESCENS were prepared at high specific activity ((3)H, 58 Ci/mmol) and were employed to study tissue specificity of acetate esterase, alcohol oxidase, and aldehyde dehydrogenase in male and femaleHeliothis virescens. Thus, [9,10-(3)H2]Z9-14:Ac was synthesized by partial tritiation of the corresponding alkyne and was converted to the labeledZ9-14∶OH andZ9-14∶Al for metabolic studies. Soluble and membrane-associated enzyme activities were determined by radio-TLC assays. Esterase activity is highest in legs of both sexes, but also occurs in antennal and glandular tissues. Oxidase activity requires O2 and is highest in female pheromone gland tissues, but it is also high in the male hairpencils. Aldehyde dehydrogenase activity was uniformly high in all tissues, but highest in antennal tissues of both males and females.

The soldier defense secretions of major and minor soldiers of the East African nasute termiteTrinervitermes bettonianus were analyzed chromatographically and spectrometrically, and the diterpene and monoterpene constituents were determined for three allopatric populations. Incipient colonies from one population raised on food from a second population produced their population-specific terpene pattern independent of food source. Secretion compositions are thus genetically rather than nutritionally determined, in agreement with biosynthetic studies in other nasutes. A minor volatile constitutent, previously proposed to be 3-ethyl-2-octanol was re-identified as (R)-2-decanol.

The defense secretions of advanced “nasutoid” rhinotermitine soldiers from the New World contain enolic β-ketoaldehydes as the major components. The secretions of minor soldiers ofRhinotermes hispidus (Emerson) andR. marginalis (Emerson) consist primarily of 3-keto-13-tetradecenal and 3-ketotetradecanal, but possess in addition C13, C14, C15, and C17 saturated and unsaturated ketones. Major soldiers lacked these compounds and in fact had virtually no frontal gland secretion. The defense secretion of the monomorphic soldiers ofAcorhinotermes subfusciceps (Emerson) contains mostly 3-keto-(Z)-9-hexadecenal and (Z)-8-pentadecen-2-one. Biosynthetic origins and interrelationships are postulated for these compounds, and the concomitant evolution of chemical weaponry and the modified labral brush is discussed.

Analogs of the two geometrical isomers of the dimethylcyclohexylidene acetaldehyde component of the pheromone of the boll weevilAnthonomus grandis were synthesized in which the α-vinylic proton or the aldehydic proton were replaced by fluorine. These isosteric substitutions substantially alter charge distribution and reactivity of the enal system, as documented by spectroscopic changes and changes in reactivity. The electrophysiological activity of the (E)- and (Z)-acyl fluorides is two orders of magnitude lower than that of the natural aldehyde. In contrast, the EAG response of female antennae to the (E)- and (Z)-α-fluoro compounds show that the thresholds are quite similar to (and in one isomer lower than) those of the natural aldehyde isomers.