Azasterol inhibitors in yeast. Inhibition of the 24-methylene sterol delta24(28)-reductase and delta24-sterol methyltransferase of Saccharomyces cerevisiae by 23-azacholesterol.

The effects of 23-azacholesterol on sterol biosynthesis and growth of Saccharomyces cervisiae were examined. In the presence of 0.2, 0.5, and 1 micron 23-azacholesterol, aerobically-growing yeast produced a nearly constant amount of ergosta-5,7,22,24(28)-tetraenol (approx. 36% of total sterol) and slowly accumulated zymosterol with a concommitant decline in ergosterol synthesis. Growth and total sterol content of yeast cultures treated with 0.2-1 micron 23-azacholesterol were similar to that of the control culture. Yeast cultures treated with 5 and 10 micron 23-azacholesterol produced mostly zymosterol (58-61% of total sterol), while ergosta-5,7,22,24(28)-tetraenol production declined to less than 10% of total sterol. The observed changes in the distribution of sterols in treated cultures are consistent with inhibition of 24-methylene sterol 24(28)-sterol reductase (total inhibition at 1 micron 23-azacholesterol) and of 24-sterol methyltransferase (71% inhibition at 10 micron 23-azacholesterol). Yeast cultures treated with 10 micron 23-azacholesterol were found to contain 4,4-dimethylcholesta-8,14,24-trienol and 4alpha-methylcholesta-8,14,24-trienol, which were isolated and characterized for the first time.

Identification of ergosta-8,24(28)-dien-3 beta,6 alpha-diol in A delta 8 goes to delta 7 sterol isomerase-blocked yeast mutant.

In addition to the monohydroxysterols found in the delta 8 goes to delta 7 isomerase-blocked Saccharomyces cerevisiae mutant erg 2, a noval dihydroxysterol, ergosta-8,24(38)-dien-3 beta,6 alpha-diol, was isolated. This sterol accumulated to the extent of 2.1% of the total sterol fraction when this mutant was treated with 23-azacholesterol, a known inhibitor of the 24-methylene-sterol-24(28)-reductase.

Accumulation of ergosta-8,14-dien-3beta-ol by Saccharomyces cerevisiae cultured with an azasterol antimycotic agent.

15-Aza-25-methylene-D-homocholesta-8,14-dien-3beta-ol, an antimycotic agent, at a concentration of 75 ng/ml inhibited ergosterol biosynthetis in Saccharomyces cerevisiae strain 3701B resulting in the accumulation of an unusual sterol. Experimental data presented indicate that this sterol is ergosta-8,14-dien-3beta-ol. The accumulation of the compound is supportive of current models of biosynthetic pathways for sterols in yeast and is consistent with inhibition by the azasterol of the delta14 sterol reductase.

Effect of juvenile hormone analog, fenoxycarb, on pheromone production byIps paraconfusus (Coleoptera: Scolytidae).

Topical application of the juvenile hormone analog, fenoxycarb, in acetone induced newly emerged male California five-spined ips,Ips paraconfusus Lanier, to become attractive to females, as measured by positive responses to male abdominal extracts in a laboratory bioassay. Two pheromones, ipsdienol and ipsenol, were detected by gas chromatography in the abdominal extracts of fenoxycarb-treated males. Pheromone production was minimal at a dose of 0.1 µg/insect of fenoxycarb, maximal at 10 µg, and was reduced to unmeasurable amounts at a dose of 100 µg. In comparison, peak production of pheromones was induced at a dose of 0.1 µg/insect of natural juvenile hormone (JH III). Treatment with 10 µg of fenoxycarb resulted in the occurrence of pheromones 12 hr after exposure, maximal pheromone content between 16 and 20 hr, and undetectable amounts after 36 hr. The demonstration that fenoxycarb is an active juvenile hormone analog for a bark beetle suggests that it may have practical utility in managing these insects.

Aggregation pheromones inDryocoetes affaber (Mann.) (Coleoptera: Scolytidae): stereoisomerism and species specificity.

Chemical analysis of whole body extracts and volatiles produced by feeding malesDryocoetes affaber (Mann.) disclosed (+)-exo-brevicomin and (+)-endo-brevicomin [(+)EXOB and (+)ENDOB], as the major insect-produced potential pheromones. Laboratory bioassays and field-trapping experiments demonstrated that (+)ENDOB is the main pheromone component, and (-)ENDOB has an inhibiting effect. EXOB either as (+) or (±) appears to be a multifunctional pheromone. It has a synergistic effect in blends of EXOB and ENDOB in ratios up to 1:1, and it is inhibitory at higher ratios. (-)EXOB was inactive. The most attractive blend forD. affaber was a 1:2 blend of (+)EXOB and (+)ENDOB. When this blend was compared with a 9:1 blend, the best known blend forDryocoetes confusus Swaine, the responses by beetles of each of the two species were highly specific, providing evidence for pheromonal exclusion between the two congenerics. We conclude that the combined effect of chirality and the ratio of geometrical isomers of brevicomin determines both the level of response and the species-specificity of the chemical signal inD. affaber.

Investigation of polyene macrolide antibiotic-induced permeability changes in vesicles by 31P nuclear magnetic resonance.

The permeability of egg yolk lecithin (EYL) vesicles to Pr3+ has been measured by 31P nuclear magnetic resonance (nmr) spectroscopy. Measurable Pr3+ leakage into the internal aqueous compartment of EYL vesicles at ambient (21 degrees C) temperature required the presence of small (7--10 mol%) amounts of dicetyl phosphate (DCP). The permeability of DCP-containing vesicles is decreased by incorporation of sterol (cholesterol greater than ergosterol approximately 5.6-dihydroergosterol greater than zymosterol) into the lipid bilayer. Addition of the polyene macrolide antibiotic, nystatin, to DCP-containing EYL vesicles with and without sterol resulted in increased Pr3+ permeability at the three temperatures studied (21--37.5 degrees C). Permeability changes observed upon addition of nystatin to sterol-impregnated, DCP-containing vesicles varied with sterol structure: ergosterol approximately 5,6-dihydroergosterol greater than cholesterol approximately zymosterol. These results are compared with other polyene macrolide induced permeability changes on model and natural membrane systems. Permeability changes induced by nystatin in sterol-free EYL vesicles were generally greater than for comparable sterol-containing vesicles. This is attributed to a nonspecific interaction of the antibiotic with the latter vesicles.

Sex pheromone of the mullein bug,Campylomma verbasci (Meyer) (Heteroptera: Miridae).

The first known sex pheromone for the family Miridae is reported for the mullein bug,Campylomma verbasci (Meyer) (Heteroptera: Miridae). Extracts of and Porapak Q-trapped volatiles from females were attractive to males in field bioassays. Butyl butyrate and hexyl butyrate were the predominant compounds in the females' volatiles, but these compounds previously had been found to be inactive when released alone or in combination. Butyl butyrate in combination with an isolated trace chemical, (E)-crotyl butyrate (which was inactive alone), rivaled the attraction elicited by five live females. When synthetic butyl butyrate and (E)-crotyl butyrate in a 16: 1 ratio were released at 91 or 183 µg/day, they comprised an effective attractant for male bugs.

Geometrical and optical isomerism of pheromones in two sympatricDryocoetes species (Coleoptera: Scolytidae), mediates species specificity and response level.

In a field-trapping experiment, western balsam bark beetles,Dryocoetes confusus Swaine, were highly attracted to a 5∶1 mixture of (±)-exo-and (±)-endo-brevicomin. Beetles in the sympatric speciesD. affaber (Mann.), were best attracted to a 1∶1 blend of these semiochemicals [either (±)∶(±) or (±)∶(±)], suggesting that both geometrical isomers are pheromone components in these species. In laboratory bioassays and further field experiments, attraction ofD. confusus was greatest when the (+) enantiomers of both geometrical isomers of brevicomin were presented in a 9∶1 ratio. Responses by maleD. confusus to attractive mixtures were reduced in the presence of (-)-exo-brevicomin. Exploitation of the complete range of variability in pheromone structure (both geometrical and optical isomerism) would allow for optimization and regulation of response levels within a species and also could maintain reproductive isolation among sympatric congeneric species primarily through production and response to species-specific blends.

Monoterpene metabolism in female mountain pine beetles,Dendroctonus ponderosae Hopkins, attacking ponderosa pine.

Abdominal volatiles of female mountain pine beetles,Dendroctonus ponderosae Hopkins, fed in ponderosa pine,Pinus ponderosa Dougl. ex Laws, and in lodgepole pine,P. contorta var.latifolia Engelmann, were analyzed by gas chromatography and coupled gas chromatography-mass spectrometry and were found to comprise host oleoresin components and beetle-produced alliylic alcohols, aldehydes, and ketones derived from host monoterpenes. Neitherexo- andendo-brevicomin nor frontalin were detected. Three metabolic pathways are proposed to account for the distribution of beetle-produced monoterpene alcohols. The first pathway involves hydroxylation of monoterpene substrates on allylic methyl groups which areE to a methylene or vinyl group. This oxidation pathway is indiscriminate with respect to substrate and probably functions to detoxify monoterpenes. A second pathway, which hydroxylates theendo-cyclic methyleneE to a vinyl methyl group of bicyclic monoterpenes to give almost exclusively thetrans alcohol, is hypothesized to be involved in pheromone production. A third detoxification pathway involves anti-Markovnikov addition of water to theexo-cyclic double bond of ß-phellandrene to give predominantlytrans-2-p-menthen-7-ol.

Influence of pheromone chirality on response byOryzaephilus surinamensis andOryzaephilus mercator (Coleoptera: Cucujidae).

The response of the sawtoothed grain beetle,Oryzaephilus surinamensis (L.), and the merchant grain beetle,O. mercator (Fauvel), to synthetic racemic and chiral macrolide aggregation pheromones was assessed in pitfall olfactometers.O.mercator utilizes theR enantiomers of (Z)-3-dodecen-11-olide and (Z,Z)-3,6-dodecadien-11-olide.O. surinamensis utilizes theR enantiomers of (Z,Z)-3,6-dodecadien-11-olide and the Synergist (Z,Z)-5,8-tetradecadien-13-olide in combination with achiral (Z,Z)-3,6-dodecadienolide. For both species, the racemates of the respective chiral pheromones were effective attractants. The respectiveS enantiomers were inactive for both species and had no effect on the biological activity of the active antipodes. No diel periodicity in responsiveness to pheromones was detected inOryzaephilus spp. reared either on a 12∶12 light-dark photoperiod or in darkness. Nonpheromone macrolides, naturally released in trace amounts byOryzaephilus spp., did not affect the aggregation response of either species to its pheromones when these additional macrolides were combined with the pheromone mixtures.

Sites of production and occurrence of volatiles in Douglas-fir beetle,Dendroctonus pseudotsugae hopkins.

Volatiles were found to be distributed throughout adult Douglasfir beetles,Dendroctonus pseudotsugae Hopkins, suggesting that the hindgut may not be the sole site of production. The volatile content of individual beetles increased dramatically with feeding; qualitative changes reflected the different bark habitats of newly emerged and established beetles. All detected volatiles were also found in beetle frass. Myrcene was the predominant monoterpene of emergent beetles and α-pinene of fed beetles. Linalool and bornyl acetate occurred in significant amounts after feeding, and diacetone alcohol and sulcatol were also detected for the first time. The presence of sulcatol explains the reported cross-attraction withGnathotrichus spp.cis-Verbenol was also found to occur in addition totrans-verbenol. The volatile content of male beetles was similar to that of females but amounts of individual compounds were less. Acetic acid was found in both emerged and fed beetles and in lesser amounts in frass, suggesting that microbial metabolism could be a potential source of volatile production.

Chemical communication in cucujid grain beetles.

Males of five sympatric species of economically damaging cucujid grain beetles,Cryptolestes ferrugineus (Stephens),C. pusillus (Schönhen),C. turcicus (Grouvelle),Oryzaephilus mercator (Fauvel), andO. surinamensis (L.), produce macrolide aggregation pheromones especially in the presence of food. Work leading to the isolation, identification, and establishment of biological activity of these semiochemicals is reviewed. The trivial name "cucujolide" is proposed and used to identify these compounds that are characteristic of the Cucujidae. The twoOryzaephilus share species share a common cucujolide pheromone, whileCryptolestes species use cucujolides that are either enantiomeric, unique to the genus, or released in trace quantities byOryzaephilus spp. and not used as pheromones by the latter species. The major mechanisms for species specificity in chemical communication are: (1) presence of a unique pheromone (C. ferrugineus andC. pusillus); (2) use of pheromones that are inactive alone but synergize response to cucujolides unique to a species (C. pusillus, C. turcicus, andO. surinamensis); (3) response to only one enantiomer of a pheromone (C. ferrugineus, O. surinamensis, andO. mercator); and (4) synergism between enantiomers of a pheromone (C. turcicus). The only species for which cross-attraction was evident wasO. mercator toO. surinamensis. Both sexes ofOryzaephilus spp. produce (R)-1-octen-3-ol, which highly synergizes response to the cucujolide pheromones. Similar synergism occurs between hexanal, octanal, and nonanal and the cucujolide pheromones ofOryzaephilus spp. The males of a sixth cucujid species,Cathartus quadricollis (Guér) produce a different aggregation pheromone, (3R,6E)-7-methyl-6-nonen-3-yl acetate. Trapping ofCryptolestes andOryzaephilus spp. in cardboard traps baited with pheromones is efficient in environments mimicking food-storage areas. Pheromone-baited plastic probe traps are the most efficient at capturing these species in infested grain.

Azasterol inhibition of delta 24-sterol methyltransferase in Saccharomyces cerevisiae.

The inhibition of the delta 24-sterol methyltransferase (24-SMT) of Saccharomyces cerevisiae by side-chain azasterols is related to their nuclear skeleton and side chain nitrogen position. Inhibitory power [I50 (microM)] was found to be in the order of 25-azacholesterol hydrochloride salt (0.05) greater than 25-aza-24,25-dihydrozymosterol (0.08) greater than 25-azacholesterol approximately equal to 25-azacholestanol (0.14) greater than (20R)- and (20S)-22,25-diazacholesterol (0.18) greater than 24-azacholesterol (0.22) greater than 25-aza-24,25-dihydrolanosterol (1.14) greater than 23-azacholesterol (4.8). In the presence of azasterols, S. cerevisiae produces increased amounts of zymosterol, decreased amounts of ergosterol and ergostatetraenol, and the new metabolites cholesta-7,24-dienol, cholesta-5,7,24-trienol, and cholesta-5,7,22,24-tetraenol. Kinetic inhibition studies with partially purified 24-SMT and several azasterols suggest the azasterols act uncompetitively with respect to zymosterol and are competitive inhibitors with respect to S-adenosyl-L-methionine (SAM). These results are consistent with at least two kinetic mechanisms. One excludes competition of azasterol and zymosterol for the same site, whereas a second could involve a ping-pong mechanism in which 24-SMT is methylated by SAM and the methylated enzyme reacts with sterol substrate.

Volatile compounds released by disturbed and calm adults of the tarnished plant bug, Lygus lineolaris.

Volatile compounds released by disturbed and calm female and male Lygus lineolaris were collected and analyzed. Six major compounds were present in samples from disturbed bugs and from calm females: (E)-2-hexenal, 1-hexanol, (E)-2-hexenol, hexyl butyrate, (E)-2-hexenyl butyrate, and (E)-2,4-oxohexenal. (E)-2-hexenal was lacking in volatiles collected from calm males. Hexyl butyrate accounted for approximately 68% and 66% of volatiles released by agitated and calm females, and 87% and 88% of volatiles released by agitated and calm males, respectively. Blends released by disturbed insects differed quantitatively from blends released by calm insects, with amounts of compounds increasing 75-350 times in samples from disturbed insects. In static air bioassays, both females and males were repelled by natural volatiles collected from females and by five-component [(E)-2,4-oxohexenal excluded] and six-component synthetic blends at doses of 1 and 10 bug-hours, indicating that these volatiles may serve an alarm or epideictic function, as well as a possible role as defensive allomones. Adults also avoided hexyl butyrate, (E)-2-hexenyl butyrate, (E)-2-hexenol, and (E)-2,4-oxohexenal, but not 1-hexanol and (E)-2-hexenal when compounds were assayed individually in static air bioassays at doses equal to 1 bug-hour. When tested over 1 day in two-choice cage trials, adults did not prefer untreated bean plants over bean plants surrounded by vials releasing up to 8.1 mg/hr (= 234 bug-hours) of the five-component synthetic blend. Therefore, the volatiles produced by disturbed adults would not be useful as a repellent for L. lineolaris.

Dendroctonus pseudotsugae hopkins (Coleoptera: Scolytidae): Production of and response to enantiomers of 1-methylcyclohex-2-en-1-ol.

Female Douglas-fir beetles,Dendroctonus pseudotsugae, produced an average 45∶55 mixture of the (S)-(-)- and (R)-(+)-enantiomers of 1-methylcyclohex-2-en-1-ol (MCOL). The (S)-(-)∶(R)-(+) ratio of 3-methylcyclohex-2-en-1-ol (seudenol) produced by this population of females was 34∶66. Lindgren funnel traps baited with (R)-(+)-MCOL attracted significantly more males and females than (S)-(-)-MCOL-baited traps, which captured significantly more beetles than unbaited controls. The combined effect of the enantiomers was additive, rather than synergistic. Either enantiomer of MCOL increased catches by frontalin-baited traps. Racemic MCOL can be used for trapping Douglas-fir beetles in south-central British Columbia.

Insect feeding and oviposition deterrents from western red cedar foliage.

The feeding deterrent activity of fractions from the foliage of western red cedar,Thujaplicata Donn, was studied in laboratory bioassays using the white pine weevil,Pissodes strobi Peck, as a test insect. The most active fraction was the volatile mixture that comprises the leaf oil of this tree species. Further fractionation of the leaf oil indicated feeding deterrent activity in the monoterpene hydrocarbon, thujone, and terpene alcohol fractions. When tested alone, both (-)-3-isothujone and (+)-3-thujone, which made up 75-88% and 5-10% of the leaf oil, respectively, deterred feeding by the weevils. Western red cedar leaf oil also showed antifeedant activity with the alder flea beetle,Altica ambiens (Le Conte), and served as an oviposition deterrent for the onion root maggot,Hylemya antiqua Meigen. The leaf oil, however, had no inhibitory effect on the feeding of the leaf roller,Epinotia solandriana L., and the red-backed sawfly,Eriocampa ovata L.

Attraction ofOryzaephilus surinamensis (L.) andOryzaephilus mercator (Fauvel) (Coleoptera: Cucujidae) to some common volatiles of food.

Responses by adultOryzaephilus surinamensis (L.) andOryzaephilus mercator (Fauvel) to various food volatiles were assessed by means of a two-choice, pitfall olfactometer. The individual experimental stimuli, all potential products of lipid oxidation, had a range of attractive doses of ≤ 1000-fold over the test dose ranges of 0.001-100 gmg, or 0.01-1000 gmg. Of 13 aliphatic C3-C14 aldehydes and benzaldehyde tested forOryzaephilus spp., 10 C3-C10 aliphatic aldehydes and benzaldehyde showed some attractiveness for both species. ForO. mercator, nonanal had the lowest lower threshold for positive response at 0.01 µg. The addition of small amounts of nonanal or of a 1∶1∶1 mixture of hexanal, octanal, and nonanal to small amounts of cucujolide aggregation pheromones enhanced response by mixed-sexO. mercator to the pheromones. Eleven aliphatic C2-C9 free fatty acids showed some attractiveness for bothOryzaephilus spp. Isovaleric acid and valeric acid had the lowest lower thresholds for positive response at 0.1 µg forO. mercator andO. surinamensis, respectively. Four olefinic oat volatiles were found to possess various degrees of attractiveness for bothOryzaephilus spp. The data suggest that food volatiles in this study might be used byOryzaephilus spp. as host-finding kairomones in nature.

3-methyl-3-buten-1-ol: An aggregation pheromone of the four-eyed spruce bark beetle,Polygraphus rufipennis (Kirby) (Coleoptera: Scolytidae).

Porapak Q-captured volatiles of malePolygraphus rufipennis (Kirby) boring in black spruce and white spruce from Newfoundland and British Columbia, respectively, contained 3-methyl-3-buten-1-ol. Volatiles from logs in which the males had been joined by females contained the compound in reduced quantities. Hindgut extracts from male and femaleP. rufipennis disclosed no male-specific volatiles, but 3-methyl-3-buten-1-ol was detected in extracts of male-produced frass. The compound was not present in extracts from fresh phloem tissue.P. rufipennis of both sexes responded strongly in the field to traps baited with 3-methyl-3-buten-1-ol released at 4390 µg/day. There was little response to unbaited traps, fresh uninfested black spruce logs, or to 3-methyl-3-buten-1-ol released at lower rates. Combination of 3-methyl-3-buten-1-ol with either one of five terpenes prevalent in black spruce did not enhance beetle catch. Approximately half of 20 black spruce trees baited with 3-methyl-3-buten-1-ol were attacked, compared to 20.0% of 10 unbaited control trees. This new aggregation pheromone could be used to monitor or manageP. rufipennis populations.

1-Octen-3-ol, attractive semiochemical for foreign grain beetle,Ahasverus advena (Waltl) (Coleoptera: Cucujidae).

Volatiles were captured on Porapak Q from foreign grain beetles,Ahasverus advena (Waltl), feeding on rolled oats at various population densities. At low population density, males, females, and mixed-sex beetles four to six weeks posteclosion and older produced 1-octen-3-ol. Mixed-sex beetles emitted almost pure (R)-(-) enantiomer. Weekly production rates of 1-octen-3-ol by males were at least four times greater than those of females. Production of 1-octen-3-ol was barely detectable in volatiles from mixed-sex adults maintained at the highest population density. Laboratory bioassays in a two-choice, pitfall olfactometer modified to retain responding beetles revealed that 1-octen-3-ol serves as an aggregation pheromone forA. advena. Both racemic and chiral 1-octen-3-ols were good attractants for mixed-sex adults in the pitfall olfactometer.