J. A. Klun

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.

Brain Factor Control of Sex Pheromone Production in the Female Corn Earworm Moth

Sex pheromone production in the female corn earworm moth Heliothis zea is controlled by a hormonal substance produced in the females brain. It is present in the brain in scotophase as well as photophase, but it is released into the hemolymph to stimulate pheromone production only in the scotophase. The stimulatory activity was also detected in the brains of male corn earworm moths and of other moths.

Sex pheromone chemistry of the female tobacco budworm moth, Heliothis virescens.

Abstract(Z)-11 -Hexadecenal (77–91%), (Z)-7-hexadecenal (0.1–2%), (Z)-9-hexadecenal (0.3–2%), hexadecanal (3–19%), (Z)-11-hexadecen-1-ol (1–5%), tetradecanal (1–3%), and (Z)-9-tetradecenal (1–3%) were identified from the heptane washes of the ovipositor of femaleHeliothis virescens (F.) females. In field bioassays, a 152-μg mixture of these seven compounds deployed in an insect trap exceeded the attractiveness of 4 virgin femaleH. virescens for males and was 5–6 times more attractive than a mixture of (Z)-1 1-hexadecenal and (Z)-9-tetradecenal (virelure) that was previously reported as the sex pheromone of the species. Four of the seven compounds produced byH. virescens females are also produced byH. zea (Boddie). Specificity of pheromonal signals among the two species is ostensibly dependent upon one or more of the three additional compounds [tetradecanal, (Z)-9-tetradecenal, and (Z)-1 1-hexadecen-1-ol] produced by femaleH. virescens.

Sex pheromone chemistry of female corn earworm moth, Heliothis zea.

Glass open-tubular capillary Chromatographic and combined glass open-tubular capillary chromatographic-mass spectrometric analyses of ovipositor washes of femaleHeliothis zea, chemical characterization, chemical synthesis, and laboratory and field bioassays showed that the ovipositor wash of the species is made up of 90–95% (Z)-11-hexadecenal, 1–2% (Z)-9-hexadecenal, 0.4–2% (Z)-7-hexadecenal, and 2–7% hexadecanal. Stimuli containing a binary mixture of (Z)-11-hexadecenal and (Z)-9-hexadecenal or the binary mixture in combination with any of the other aldehydes identified from the females elicited intense attraction and close-range precopulatory reactions fromH. zea males.

Trace chemicals: the essence of sexual communication systems in heliothis species.

Analysis of heptane-soluble compounds from ovipositors of Heliothis zea and Heliothis virescens shows that both species produce relatively large amounts of (Z)-11-hexadecenal, with traces of (Z)-9-hexadecenal, (Z)-7-hexadecenal, and hexadecanal. Heliothis virescens females differ from Heliothis zea in that they also produce trace amounts of tetradecanal, (Z)-9-tetradecenal, and (Z)-11-hexadecen-1-ol. In both species, trace compounds are essential to pheromonal activity and specificity of chemical signals.

Bioactivity-Guided Fractionation and GC/MS Fingerprinting of Angelica sinensis and Angelica archangelica Root Components for Antifungal and Mosquito Deterrent Activity

Bioassay-guided fractionation of the chloroform extract from the roots of Angelica sinensis led to isolation and characterization of (Z)-ligustilide using direct-bioautography with Colletotrichum species. The structure of (Z)-ligustilide was confirmed by (1)H and (13)C NMR spectroscopy and GC/MS. (Z)-Ligustilide deterred the biting of two mosquito species more effectively than DEET. Three different A. sinensis accessions and one Angelica archangelica root oil were evauated by GC and GC/MS, and the dominant component in A. sinensis was 61-69% (Z)-ligustilide. Two other prominent compounds in A. sinensis oils were 5.7-9.8% (E)-3-butylidene phthalide and 1.5-2.3% (Z)-3-butylidene phthalide. The main constituents that comprised A. archangelica oil were monoterpene hydrocarbons such as 24.5% alpha-pinene, 13.8% delta-3-carene, 10.1% beta-phellandrene, 8.8% p-cymene, 8.4% limonene, and 6.3% sabinene. Phthalides and monoterpene hydrocarbons were determined to be good systematic markers or chemical fingerprints for A. sinensis and A. archangelica root oils. Chemical fingerprinting by GC/MS of A. sinensis also confirmed the misidentification of one A. archangelica sample sold in the Chinese market.

A new in vitro bioassay system for discovery of novel human-use mosquito repellents

ABSTRACT A Klun & Debboun (K&D) test module, previously developed and used for quantitative measurement of the efficacy of mosquito repellents on human volunteers, was adapted for in vitro evaluation of repellents by coupling the module with a membrane-blood reservoir. Performance of Deet, Bayrepel®, and SS-220 insect repellents in the new in vitro system was compared with their performance on humans against mosquitoes using our standard in vivo system. For each compound, in vitro dose–response assays were conducted with compounds applied to cloth positioned over blood reservoirs covered with Baudruche membrane against Aedes aegypti. The repellents were also tested in vitro against Anopheles stephensi and Ae. aegypti at a fixed dose of 24 nmol compound/cm2 cloth over the Baudruche and Edicol collagen membranes. Concurrently, the repellents were tested at the fixed dose using the K&D module on human volunteers. The observed proportions of both mosquito species deterred from biting in the fixed doses in the in vitro assays were similar to those obtained using humans, being clearly able to distinguish controls from repellents, and differing only in the ranking of the effectiveness of some of the repellents. Dose–response relationships of the in vitro and in vivo systems were also very similar, although not directly comparable because the data were not collected concurrently. This new in vitro assay system can be used in high throughput screening of compounds to identify new repellents having potential for use as topical mosquito repellents on humans.

Genetic regulation of sex pheromone production and response

The sex pheromone communication system of the European corn borer moth varies intraspecifically. Analyses of pheromonal extracts of wild females, collected in a region where the types (each producing a different isomeric proportion ofZ- andE-11-tetradecenyl acetate) are sympatric, showed that theZ pheromone-production allele frequency was ca. 4 times greater than theE allele. The paucity ofE production and response alleles in the population indicates that moths inheriting those alleles concomitantly inherit some undefined disadvantage. The types interbreed, but the frequency of heterozygous pheromone-production types among wild females was less than predicted by Hardy-Weinberg equilibrium and was evidence of positive assortative mating. Rates of male captures in field traps baited with females of the three pheromonal types also evidenced assortative mating in the population. Progeny tests with males captured in the traps provided circumstantial evidence that pheromone response and production functions in the species are regulated by separate genetic loci and that the loci are not always complementary; i.e., a male can carry an allele coding for production of one pheromonal isomer ratio but can be genetically predisposed to respond to another.

Comparative Activity of Deet and AI3-37220 Repellents Against the Ticks Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) in Laboratory Bioassays

Abstract 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.

(E,Z)-2,13-octadecadien-1-ol acetate. A new pheromone structure for sesiid moths

Abstract (E,Z)-1,13-Octadecadien-1-ol acetate was identified from ovipositor extracts of the grape root borer and was shown to attract males of the species.