Efrapeptin production byTolypocladium fungi (Deuteromycotina: Hyphomycetes): Intra- and interspecific variation

Production of the mitochondrial ATPase inhibitory peptides efrapeptins was evaluated by HPLC analysis in 44 strains of nine species of the fungal genusTolypocladium (Deuteromycotina: Hyphomycetes). Efrapeptin identification was confirmed by mass spectral data for the first time in the speciesT. cylindrosporum. HPLC retention time data indicated thatT. nubicola andT. tundrense, two species not previously known to produce efrapeptins, also produce the peptides. No efrapeptins were detected (<0.3 mg/100 ml broth) in single strains each ofT. balanoides, T. extinguens, T. parasiticum, andT. microsporum. Five strains ofT. geodes produced detectable amounts of efrapeptins and had compound F, or F and G, as the major component(s) in the mixture with the order of abundance being F> or ∼G > H ∼ D ∼ E > C. Eleven strains ofT. niveum produced detectable amounts of efrapeptins and had efrapeptins D and E as the primary and secondary components, respectively, in the mixture with the order of abundance being D > E > F > C ∼ G. A singleT. niveum strain had an efrapeptin profile similar to that of theT. geodes strains. Ten strains ofT. cylindrosporum had detectable amounts of efrapeptins. Of these, nine had F and one had G as the major component.T. cylindrosporum had higher ratios of E to D than didT. geodes. Efrapeptins were detected in one of twoT. nubicola strains analyzed (F > G > H) and one of threeT. tundrense strains (F > G > H > E).T. niveum strains could, in most cases, be identified to species on the basis of their efrapeptin profiles. Mention of a commercial or proprietary product does not constitute an endorsement by the USDA.     

Antifungal polyoxygenated fatty acids fromAeollanthus parvifolius

Two polyoxygenated fatty acids with antifungal activity have been isolated from aerial parts ofAeollanthus parvifolius (Labiatae) using bioassay-guided isolation procedures. Spectroscopic analysis was used to identify the compounds as (Z)-4,9-diacetoxy-6,7-epoxy-5-hydroxy-8-oxododeca-2-enoic acid and (Z)-4,6,7,9-tetraacetoxy-5-hydroxy-8-oxododeca-2-enoic acid. Both of these compounds inhibited spore germination ofCladosporium cucumerinum. Minimum inhibitory doses were 1 µg and 5 µg, respectively, but only the former inhibited germination ofAspergillus niger at a dose of 5 µg. Neither compound exhibited antibacterial activity. (Z)-4,9-Diacetoxy-6,7-epoxy-5-hydroxy-8-oxododeca-2-enoic acid reduced the hyphal growth ofPythium ultimum. The mode of action was not resolved but did not involve either the disruption of the cell wall membrane or the inhibition of extracellular enzymes. Application of (Z)-4,6,7,9-tetraacetoxy-5-hydroxy-8-oxododeca-2-enoic acid to seedling plants did not give any protection against fungal attack, and indeed the compound appeared to be phytotoxic.     

Transfer of pyrrolizidine and quinolizidine alkaloids toCastilleja (Scrophulariaceae) hemiparasites from composite and legume host plants

Castilleja (Scrophulariaceae) species of the western United States contain pyrrolizidine and quinolizidine alkaloids. TheCastilleja obtain the alkaloids by root parasitism on host plants, withSenecio atratus andS. triangularis (Asteraceae) furnishing the pyrrolizidines, and quinolizidines being obtained fromLupinus species andThermopsis montana (Leguminosae). Individual plants within a givenCastilleja species population may be devoid of alkaloids while others have high alkaloid content. No populations have been found which are made up of both pyrrolizidine- and quinolizidine-containing individuals. These results have important implications forCastilleja ecology, for the study of insect herbivores which areCastilleja specialists, and in the development of systems for the investigation of the role of alkaloids as plant defenses.Paper 10 in the series Chemistry of the Scrophulariaceae. Paper 9: D.R. Gardner, J. Narum, D. Zook, and F.R. Stermitz.J. Nat. Prod. 50: In press.     

Novel diffusion-dilution method for release of semiochemicals: Testing pheromone component ratios on western pine beetle

Each of the pheromone components of the Western pine beetle,Dendroctonus brevicomis LeC. (Coleoptera: Scolytidae), exo-brevicomin (E) and frontalin (F), were released in the forest at various ratios 0.011, 0.11, or 11 to a constant dose of the opposite component (E or F) plus the host monoterpene myrcene (M), which were each released at 1.5 mg/day. The components were released by a new method that combines the principles of chemical diffusion through a tube with mole percentage dilution of the chemical. Both sexes ofD. brevicomis were attracted similarly at comparable ratios (and release rates) of E or F and showed similar logarithmic relationships (r ²=0.920.99). The bark beetle predator,Temnochila chlorodia (Mannerheim) (Coleoptera: Trogositidae) was apparently less sensitive to E thanD. brevicomis, being relatively less attracted to amounts of E equivalent to that released by 70 females, while none were attracted to that from seven females (while this rate still attracted significant numbers of conspecifics). The apparent insensitivity of bark beetles to extreme ratios between pheromone components in contrast to moths is discussed. The advantages of the diffusion-dilution method of releasing semiochemicals compared to previous methods of absorbents, wicks, capillary tubes, and semipermeable plastic membranes are also discussed.     

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 51 mixture of (±)-exo-and (±)-endo-brevicomin. Beetles in the sympatric speciesD. affaber (Mann.), were best attracted to a 11 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 91 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.During the course of the research, A.D. Camacho was on educational leave from the Escuela Nacional de Ciencias Biológicas. I.P.N. Depto. de Zoología. Prol. Carpio y Plan de Ayala, Mexico 11340D.F. México.     

Multichemical resistance of the coniferPodocarpus gracilior (Podocarpaceae) to insect attack

Podocarpus gracilior is resistant in nature to insect attack. Apparently, the resistance ofP. gracilior is due to a multichemical defense mechanism. Chemicals identified as potential components of the multichemical defense are four norditerpenedilactones, including nagilactones, C, D, and F, which cause insect feeding deterrent activity ultimately coupled to an insecticidal activity, and podolide, an insecticide; two nonlethal growth-inhibiting biflavones, podocarpusflavone A and 7,4-dimethylamentoflavone; and the ecdysis-inhibiting phytoecdysone, ponasterone A.     

Sexual Isolation and Cuticular Hydrocarbon Differences between <Emphasis Type="Italic">Drosophila santomea</Emphasis> and <Emphasis Type="Italic">Drosophila yakuba</Emphasis>

Drosophila santomea and Drosophila yakuba are two sister species inhabiting Saõ Tomé island. Previous studies showed that both species display strong reproductive isolation, although they can produce a few viable hybrids. Our study tried to understand the mechanism of this ethological isolation between two allopatric strains. A strong sexual isolation was confirmed, with a marked asymmetry. Comparisons of latency times to either courtship or copulation suggest that males do not discriminate females, whereas D. yakuba females, but not D. santomea females, accept their homospecifics more quickly. Cuticular hydrocarbon compositions of both species and sexes were also established with gas chromatography (GC) and GC/mass spectrometry analysis. All have (Z)-7-tricosene as their major compound. There are several quantitative differences between species for few minor compounds. The largest difference concerns n-heneicosane, which is more abundant in D. santomea than in D. yakuba flies (up to seven times more between males). A similar quantitative difference was also found in a pair of sympatric strains. Furthermore, D. yakuba males artificially perfumed with n-heneicosane were discriminated negatively by D. yakuba females, suggesting a role for this compound in the sexual isolation between these two species.     

Effect of Dehydration of VOPO4⋅2H2O on the Preparation and Reactivity of Vanadium Phosphate Catalyst for the Oxidation of n-Butane

The effect of drying of VOPO₄2H₂O on the preparation of vanadium phosphate catalysts for the selective oxidation of n-butane to maleic anhydride is described and discussed. It is found that partially dehydrated samples of the dihydrate containing small amounts of _I-VOPO₄ are formed when the material is initially dried. The presence of this impurity leads to a final catalyst containing trace amounts of _I-VOPO₄ in combination with (VO)₂P₂O₇ and this combination leads to a catalyst with a higher activity but with a lower selectivity to maleic anhydride. The drying stage is also found to influence the surface area and intrinsic activity of the activated catalyst.     

Fate of quinolizidine alkaloids through three trophic levels:Laburnum anagyroides (Leguminosae) and associated organisms

The quinolizidine alkaloids (QA) of golden rain,Laburnum anagyroides, and those of phytophagous insects associated with the plant, as well as of parasitoids of the latter, were analyzed by capillary GLC and GLC-MS. The alkaloid content in samples of vegetative plant parts was high at the beginning of the season, then decreased, while that of reproductive organs was high throughout flowering, pod formation, and maturation. The analyses showed that the QA of the plant passed through two higher trophic levels (herbivorous insects and their parasitoids) and that the alkaloid pattern changed little during the passage. The alkaloids were present in two phytophagous insect species associated with golden rain: the predispersal seed predator,Bruchidius villosus [5–13g/g fresh weight (fw)], andAphis cytisorum (182–1012g/g fw), an aphid that feeds on shoots, leaves, and inflorescences. Braconid and chalcidoid parasitoids emerging from the bruchid host also contained alkaloids (1.3–3g/g fw), as did three foraging ant species,Lasius niger, Formica rufibarbis, andF. cunicularia (45g/g fw), that visited the aphid colonies or honeydew-covered leaves of aphid-infested plants. The hypothesis that developing bruchid larvae and/or the plant manipulate QA supply to infested seeds was not supported, because QA content of leftover endosperm in seeds after bruchid development was similar to that of uninfested seeds. The frass of developing bruchid larvae was rich in QA (31g/ g dry weight). While aphids sequestered, the bruchid larvae took up and eliminated QA with the frass without chemical transformation.     

Effect of Volatile Constituents from <Emphasis Type="Italic">Securidaca</Emphasis><Emphasis Type="Italic">Longepedunculata</Emphasis> on Insect pests Of Stored Grain

Securidaca longepedunculata Fers (Polygalaceae) is commonly used as a traditional medicine in many parts of Africa as well as against a number of invertebrate pests, including insects infesting stored grain. The present study showed that S. longepedunculata root powder, its methanol extract, and the main volatile component, methyl salicylate, exhibit repellent and toxic properties to Sitophilus zeamais adults. Adult S. zeamais that were given a choice between untreated maize and maize treated with root powder, extract, or synthetic methyl salicylate in a four-way choice olfactometer significantly preferred the control maize. Methyl salicylate vapor also had a dose-dependant fumigant effect against S. zeamais, Rhyzopertha dominica, and Prostephanus truncates, with a LD₁₀₀ achieved with a 60 l dose in a 1-l container against all three insect species after 24 hr of exposure. Probit analyses estimated LD₅₀ values between 34 and 36 l (95% CI) for all insect species. Furthermore, prolonged exposure for 6 days showed that lower amounts (30 l) of methyl salicylate vapor were able to induce 100% adult mortality of the three insect species. The implications are discussed in the context of improving stored product pest control by small-scale subsistence farmers in Africa.     

Role of secondary metabolites in feeding associations between a predatory nudibranch,two grazing nudibranchs,and a bryozoan

The carnivorous nudibranchRoboastra tigris preys preferentially upon two nudibranchs,Tambja abdere andT. eliora, that in turn feed upon the bryozoanSessibugula translucens. All four organisms contain tambjamines A–D (I–IV) that were shown to be fish feeding inhibitors. When attacked byRoboastra, T. abdere secretes a distasteful mucus containing a total of 3 mg of the tambjamines that sometimes causes theRoboastra to break off the attack. Under similar circumstancesT. eliora attempts to swim away; it presumably contains insufficient of the tambjamines to deterRoboastra. Roboastra follows the slime trail of nudibranchs using contact chemoreception and reverses direction when the trail is broken. The slime trail ofT. abdere contains low concentrations of the tambjamines. In Y-maze experiments,T. eliora was attracted towards seawater containingS. translucens and seawater containing 10^–10 M tambjamines A and B (11) but was repelled by seawater containing > 10^–8 M tambjamines A and B. At higher concentrations the mixture of tambjamines may be recognized as an alarm pheromone.     

Aggregation pheromone ofDrosophila mauritiana,Drosophila yakuba,andDrosophila rajasekari

(Z)-11-Octadecenyl acetate (Z11–18Ac) was identified as the aggregation pheromone ofDrosophila mauritiana, D. yakuba, andD. rajasekari. The amount of pheromone in the ejaculatory bulb of male flies increased with age, reaching plateau levels of ca. 240, 800, and 1100 ng/fly forD. mauritiana, D. yakuba, andD. rajasekari, respectively. Thirty to 50% of the pheromone in the ejaculatory bulb was transferred to the female during mating, with the majority transferred to the female's reproductive tract. In the subsequent 6-hr period, over half the pheromone in the female's reproductive tract was transferred to the surroundings. In a wind-tunnel olfactometer,Z11–18Ac was attractive toD. yakuba andD. mauritiana; however,D. rajasekari required food or food odors in combination withZ11–18Ac to demonstrate aggregation activity.Z11–16Ac andZ11–20Ac were not attractive forD. mauritiana, D. yakuba, andD. rajasekari. For all three species, food was synergistic withZ11–18Ac and both sexes were attracted.     

Differential metabolism of 1,8-cineole in insects

In order to compare the metabolism of 1,8-cineole in the pyrgo beetle, Paropsisterna tigrina, three other herbivorous insect species, Faex nigroconspersa, Chrysophtharta bimaculata, and Oxyops vitiosa, were fed 1,8-cineole leaf diets. F. nigroconspersa adults excreted predominantly 9-hydroxy-1,8-cineole (36.2% of the volatile constituents) with some 2-hydroxy-1,8-cineole (11.4%). In contrast, larvae excreted predominantly 2-hydroxy-1,8-cineole (27.4%) and smaller proportions of 9-hydroxy-1,8-cineole (5.2%) and 3-hydroxy-1,8-cineole (4.3%). C. bimaculata adults excreted predominantly 3-hydroxy-1,8-cineole (16.5%). Oxyops vitiosa adults, on a lower 1,8-cineole diet, excreted predominantly 2,9-dihydroxy-1,8-cineole (4.2%) and 2-hydroxy-1,8-cineole (3.5%), with smaller proportions of 3-hydroxy-1,8-cineole (1.1%) and 9-hydroxy-1,8-cineole (0.5%). This is the first reported occurrence of a dihydroxycineole as an insect metabolite. Gas chromatographic and mass spectral data for hydroxycineoles are recorded and interspecific metabolite variation discussed.     

Aggregation pheromones inDrosophila borealis andDrosophila littoralis

Mature males ofDrosophila borealis andD. littoralis (Diptera: Drosophilidae) produce pheromones that attract both males and females in a wind-tunnel bioassay. Ethyl tiglate is a major pheromone component in both species. Isopropyl tiglate is a minor component, as active as ethyl tiglate on an equal-weight basis, but less abundant in the flies. Both species respond to (Z)-9-heneicosene, a compound they do not possess, but which is a pheromone component in closely related species.D. borealis andD. littoralis are also able to discriminate among various heneicosene isomers. These responses to hydrocarbons may represent artifacts of evolution in this group. For both species, the pheromone was strongly synergized by an extract of fermenting aspen bark, a host material ofD. borealis. Benzyl alcohol was identified as one active component, although it did not account for all the activity of the extract. In nature, the flies probably respond to mixtures of fly-derived and host-derived volatiles.     

Attraction of scolytids and associated beetles by different absolute amounts and proportions of α-pinene and ethanol

The attraction of bark and ambrosia beetles as well as associated beetles to -pinene and ethanol was studied in field experiments with flight barrier traps. -Pinene and ethanol were released individually and as combinations in approximately 11 or 110 ratios and at four different release rates. Ethanol attractedTomicus piniperda (L.),Hylurgops palliatus (Gyll.),Trypodendron lineatum (Oliv.),Hylastes cunicularius Er.,H. brunneus Er.,H. opacus Er., andAnisandrus dispar (F.) (Scolytidae);Glischrochilus quadripunctatus (L.) andEpuraea spp. (Nitidulidae);Thanasimus formicarius (L.) (Cleridae); andRhizophagus depressus (F.) (Rhizophagidae). -Pinene attracted all these species with the exception ofT. lineatum, H. cunicularius, andA. dispar. Combinations of a-pinene and ethanol resulted in synergistically increased attraction of all species with the exception ofH. opacus andA. dispar. A. dispar, the only hardwood-associated species in the study, was repelled by -pinene. Both the release rates and the ratio at which the two substances were released influenced the response of the beetles. The differences in response between the beetle species seem to reflect dissimilarities in the release of the two substances among the various types of breeding material to which the species are adapted.     

Kairomonal Effects of Sawfly Sex Pheromones on Egg Parasitoids

The response of the two eulophid egg parasitoid species Chrysonotomyia ruforum and Dipriocampe diprioni to sex pheromones of their sawfly hosts Diprion pini and Neodiprion sertifer was studied in olfactometer bioassays. Females of C. ruforum were arrested when exposed to odor of the tested major sex pheromone components of Diprion pini [acetate and propionate of (2S,3R,7R)-3,7–dimethyl-2–tridecanol] or Neodiprion sertifer [(2S,3S,7S)-3,7–dimethyl-2–pentadecyl acetate]. This behavioral response of C. ruforum was observed whether (1) parasitoid females had oviposition experience with D. pini eggs or not, (2) parasitoid females emerged from D. pini eggs of a French population or from N. sertifer eggs of a Finnish population, and (3) the tested sex pheromone concentration was low (1 ng/l hexane) or high (100 ng/l hexane). However, C. ruforum did not behaviorally respond to a stereoisomer of the major sex pheromone component of N. sertifer, which is known to act as a sex pheromone antagonist [antagonist = (2S,3R,7R)-3,7–dimethyl-2–pentadecyl acetate]. Thus, C. ruforum responded stereospecifically to the S,S,S configured pheromone of N. sertifer, but not to the S,R,R configuration. Females of the parasitoid D. diprioni were also arrested by the tested major sex pheromone components of the host D. pini. The kairomonal effects of diprionid host sex pheromones on these egg parasitoids are compared to known responses of egg parasitoids to lepidopteran sex pheromones.     

Factors influencing distribution ofDiabrotica spp. in blossoms of cultivatedCucurbita spp.

Cultivars representing three species ofCucurbita were examined for blossom preference byDiabrotica spp.C. maxima cultivars were found to be preferred byD. undecimpunctata howardi over those ofC. pepo andC. moschata.D. virgifera virgifera preferredC. maxima and the Connecticut Field cultivar ofC. pepo.C. moschata and other cultivars ofC. pepo were not preferred. Cultivars were examined for differences in floral volatile release, blossom cucurbitacin content, and pollen content of male blossoms.C. maxima male blossoms released a larger quantity of volatiles thanC. pepo orC. moschata. Also, onlyC. maxima male blossoms contained cucurbitacins. Cultivars ofC. moschata contained the largest quantities of pollen, but all three species contained relatively large quantities. The data indicate a correspondence ofD. u. howardi distribution in the field with high volatile release rates and high cucurbitacin levels that are found inC. maxima blossoms.D. v. virgifera distribution appears to be somewhat independent of these factors since this species was abundant in blossoms of aC. pepo cultivar as well as cultivars ofC. maxima.Coleoptera: Chyrsomelidae.     

Differences in attraction to semiochemicals present in sympatric pine shoot beetles,Tomicus minor andT. piniperda

The chemical ecology of host- and mate-finding in the pine shoot beetles,Tomicus minor andT. piniperda, was studied in southern Sweden. Beetles were collected in the field from defined attack phases on Scots pine. Using gas chromatography-mass spectroscopy, a number of oxygen-containing monoterpenes, e.g., 3-carene-10-ol, myrtenol,trans-verbenol, and verbenone, were identified from hindgut extracts of both sexes of both species. Compared toT. minor,T. piniperda contained additional compounds and in larger amounts. The amounts were highest in both species at the time when the beetles had bored into contact with the resin-producing xylem-phloem tissue. The synthesis of (1S,6R)-3-carene-10-ol by photooxidatipn of (+)-(1S,6R)-3-carene is described. In comparative electroantennogram (EAG) measurements on males and females of both species, the most active of the tested compounds wastrans-verbenol. Laboratory bioassays of walking beetles showed thatT. piniperda was attracted to uninfestèd pine logs.T. minor was more strongly attracted to pine logs infested with females than to uninfested pine logs, indicating a female-produced aggregation pheromone. Field tests confirmed thatT. piniperda was strongly attracted to pine logs. The attraction ofT. minor to logs was significant only when logs were combined with racemictrans-verbenol and (1S,6R)-3-carene-10-ol.T. minor was also attracted to a combination of these monoterpene alcohols alone. We suggest that host and mate location inT. piniperda is achieved by means of a kairomone composed of host monoterpenes, whileT. minor utilizes a primitive pheromone synergized by host odors. Evolution of host colonization strategies of the two beetles are discussed.Coleoptera: Scolytidae.This study was made within the project Odour signals for control of pest insects.     

(E4,E10)-dodecadienyl acetate: Novel sex pheromone component of tentiform leafminer,Phyllonorycter mespilella (Hübner) (Lepidoptera: Gracillariidae)

(E4,E10)-dodecadienyl acetate (E4,E10-12OAc) is a newly discovered sex pheromone component of the tentiform leafminer,Phyllonorycter mespilella (Hübner). In apple orchards, traps baited with 1g ofE4,E1012OAc attractedP. mespilella in British Columbia andP. blancardella (F.) in Massachusetts and Nova Scotia. The compound was identified inP. mespilella by gas chromatographic-electroantennographic analysis (GC-EAD) of pheromone gland extracts, retention index calculations, EAD profiles toE3 toE10 dodecenyl acetates, and synthesis of candidate pheromone components. Even thoughE4,E10-12OAc was not detected in gland extracts by GC-mass spectroscopy, several factors indicate that it is female-produced. Antennal responses to gland extracts coincided with authenticE4,E10-12OAc on four GC columns with different retention characteristics.E4,E10-12OAc andE10-12OAc, a known female-produced pheromone component, elicited equally strong EAD responses. In field tests,E4,E10-12OAc was two to four times more attractive thanE10-12OAc. There was no additive or synergistic effect between the two components.     

Electrophysiological and behavioral responses of female Helicoverpa armigera to compounds identified in flowers of African marigold, Tagetes erecta

Seven electrophysiologically active compounds were detected in air-entrained headspace samples of live flowers of Tagetes erectaanalyzed by gas chromatography (GC) linked to a female Helicoverpa armigeraelectroantennograph (EAG) using polar and nonpolar capillary columns. These compounds were subsequently identified using GC linked to mass spectrometry as benzaldehyde, (S)-(–)-limonene, (R,S)-(±)-linalool, (E)-myroxide, (Z)--ocimene, phenylacetaldehyde, and (R)-(–)-piperitone. Electrophysiological activity was confirmed by EAG with a 1-g dose of each compound on filter paper eliciting EAG responses that were significantly greater than the solvent control response from female moths. Wind-tunnel bioassays with T. erectaheadspace samples, equivalent to 0.4 flower/hr emission from a live flower, elicited a significant increase in the number of upwind approaches from female H. armigerarelative to a solvent control. Similarly, a seven-component synthetic blend of EAG-active compounds identified from T. erectapresented in the same ratio (1.0 : 1.6 : 0.7 : 1.4 : 0.4 : 5.0 : 2.7, respectively) and concentration (7.2 g) as found in the natural sample elicited a significant increase in the number of upwind approaches relative to a solvent control during a 12-min bioassay that was equivalent to that elicited by the natural T. erectafloral volatiles.