Cheng-Hua Zhao

Reductase specificity and the ratio regulation of E/Z isomers in pheromone biosynthesis of the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae)

Species specificity of moth sex pheromones is in many cases achieved by means of specific blends rather than by specific components. Two pheromone strains of the European corn borer, Ostrinia nubilalis, use (E)- and (Z)-ll-tetradecenyl acetate in different ratios as their pheromone, but show the same ratio of the pheromone precursors (70:30 EIZ-11-tetradecenoic acid). The hypothesis that the ratio of the pheromone components in the two strains and their hybrids is controlled by the specificity of the reductase system, responsible for conversion of acid to the corresponding alcohol precursors, was tested. Deuterium-labeled alcohols, aldehydes and fatty acids corresponding to the two pheromone components were topically applied to the pheromone glands in different ratios and their selective incorporation into pheromone components was determined by gas chromatography with mass selective detection. Acetylation of the (E)- and (Z)-ll-tetradecenols was unselective, whereas the corresponding aldehydes and acids were selectively incorporated into the pheromone components. Z strain females selectively metabolized the Z-isomers whereas E strain females converted the E-isomers. The E strain being the most selective of the two strains. Hybrids converted both geometric isomers. The relative conversion rate of both E- and Z-isomers of all tetradecenoic acids with the double bond in positions from 7-12, was also determined. In addition to the All-isomers, the E strain females converted (E)-8-tetradecenoic acid into acetate and the Z strain females converted (E)-12-tetradecenoic acid. None of these substrates occur naturally in the pheromone gland, but (E)-12-tetradecenyl acetate is a pheromone component of the Asian corn borer O. furnacalis. Thus the possibility for conversion of (E)-12-tetradecenoic acid to acetate in the Z strain, as well as the earlier reported conversion of (Z)-ll-tetradecenoic acid to acetate in O. furnacalis, suggests that O. furnacalis is closest related to the Z strain of O. nubilalis. Pheromone biosynthesis Ratio regulation Reductase specificity Deuterium-labeled precursors Ostrinia nubilalis

Potential Chemosignals in the Anogenital Gland Secretion of Giant Pandas, Ailuropoda melanoleuca, Associated with Sex and Individual Identity

With a combination of dichloromethane extraction and analysis by gas chromatography–mass spectrometry (GC-MS), we found 39 compounds (corresponding to 38 GC peaks) in the anogenital gland secretion (AGS) of captive adult giant pandas, Ailuropoda melanoleuca, during the non-mating season. In addition to indole, squalene, and some of the straight-chain fatty acids that had been characterized previously from the AGS of giant pandas, we identified several new compounds such as decenal, two isomers of decadienal, phenylacetic acid, 5-methylhydantoin, hydroquinone, phenylpropanoic acid, and erucic acid. Quantitative comparison of the relative abundances of the 20 main GC peaks revealed that 5-methylhydantoin, indole, and erucic acid are putative female pheromones, whereas squalene and hydroquinone are putative male pheromones. In addition to the presence of a few individual-specific compounds, the relative abundances of most of the 21 constituents varied more among individuals than within individuals. This suggests that individual identity might be coded in both digital and analog form. The chemical composition of different AGS samples from the same pandas consistently displayed a minimum cluster distance, much smaller than that between samples from different individuals in a hierarchical linkage cluster (average linkage) dendrogram. Our results indicate that the AGS might contain an “odor fingerprint.” Although putative sex pheromones such as squalene and erucic acid should be assessed further by bioassay, our study suggests that synthetic chemosignals might be useful in modulating the behavior and physiology of giant pandas.

Substrate specificity of acetyltransferase and reductase enzyme systems used in pheromone biosynthesis by Asian corn borer,Ostrinia furnacalis.

The substrate specificity of the acetyltransferase and the reductase enzyme systems used byOstrinia furnacalis (Lepidoptera: Pyralidae) in pheromone biosynthesis was studied in vivo by topical application of precursors to pheromone glands. Each of the tetradecenols, varying in double bond position (from 7 to 13) and geometry of the double bond, was converted to the corresponding acetate by the acetyltransferase. The similarity in the conversion rates of all tested fatty alcohols indicated that the acetyltransferase has a low substrate specificity. Most of the corresponding tetradecenoic acids could also be converted to the respective acetates. However, very different conversion rates among the tested fatty acids demonstrated that the reductase system has a higher substrate specificity than the acetyltransferase. The conversion rates of mostE isomers were higher than those of the correspondingZ isomers, except for the (Δ)-11-tetradecenoic acids, in which much moreZ isomer was converted to the product. Saturated tetradecanoic acid was converted to the corresponding acetate at a high rate; the shorter homolog, tridecanoic acid, was converted at a lower rate (56%), and conversion to the respective acetates of the longer homolog, pentadecanoic and hexadecanoic acids, was insignificant (<5%). The results from the present study showed that specificity of pheromone production is to a large extent controlled by the pheromone gland reductase system.

Elucidation of the sex-pheromone biosynthesis producing 5,7-dodecadienes in Dendrolimus punctatus (Lepidoptera: Lasiocampidae) reveals Delta11- and Delta9-desaturases with unusual catalytic properties

Sex pheromones produced by female moths of the Lasiocampidae family include conjugated 5,7-dodecadiene components with various oxygenated terminal groups. Here we describe the molecular cloning, heterologous expression and functional characterization of desaturases associated with the biosynthesis of these unusual chemicals. By homology-based PCR screening we characterized five cDNAs from the female moth pheromone gland that were related to other moth desaturases, and investigated their role in the production of the (Z)-5-dodecenol and (Z5,E7)-dodecadienol, major pheromone constituents of the pine caterpillar moth, Dendrolimus punctatus. Functional expression of two desaturase cDNAs belonging to the Delta 11-subfamily, Dpu-Delta 11(1)-APSQ and Dpu-Delta 11(2)-LPAE, showed that they catalysed the formation of unsaturated fatty acyls (UFAs) that can be chain-shortened by beta-oxidation and subsequently reduced to the alcohol components. A first (Z)-11-desaturation step is performed by Dpu-Delta 11(2)-LPAE on stearic acid that leads to (Z)-11-octadecenoic acyl, which is subsequently chain shortened to the (Z)-5-dodecenoic acyl precursor. The Dpu-Delta 11(1)-APSQ desaturase had the unusual property of producing Delta 8 mono-UFA of various chain lengths, but not when transformed yeast were grown in presence of (Z)-9-hexadecenoic acyl, in which case the biosynthetic intermediate (Z9,E11)-hexadecadienoic UFA was produced. In addition to a typical Z9 activity, a third transcript, Dpu-Delta 9-KPSE produced E9 mono-UFAs of various chain lengths. When provided with the (Z)-7-tetradecenoic acyl, it formed the (Z7,E9)-tetradecadienoic UFA, another biosynthetic intermediate that can be chain-shortened to (Z5,E7)-dodecadienoic acyl. Both Dpu-Delta 11(1)-APSQ and Dpu-Delta 9-KPSE thus exhibited desaturase activities consistent with the biosynthesis of the dienoic precursor. The combined action of three desaturases in generating a dienoic sex-pheromone component emphasizes the diversity and complexity of chemical reactions that can be catalysed by pheromone biosynthetic fatty-acyl-CoA desaturases in moths.

Sex pheromone components of the sandthorn carpenterworm, Holcocerus hippophaecolus

Extracts of female sex pheromone gland of the carpenterworm moth, Holcocerus hippophaecolus Hua, a pest of sandthorn, Hippophae rhamnoides L. were found to contain (E)-3-tetradecenyl acetate (E3-14:Ac), (Z)-3-tetradecenyl acetate (Z3-14:Ac), (Z)-7-tetradecenyl acetate (Z7-14:Ac), the corresponding alcohols, E3-14:OH, Z3-14:OH, Z7-14:OH, and (E)-9-tetradecenyl acetate (E9-14:Ac). Electroantennographic (EAG) analysis of these chemicals and their analogs demonstrated that Z7-14:Ac elicited the largest male EAG response, followed by E3-14:Ac. In field trials, traps baited with either Z7-14:Ac or E3-14:Ac alone caught no male moths, whereas a combination of these two components in a 1:1 ratio caught more males than control traps. Addition of Z7-14:OH and Z3-14:OH or the alcohols plus E9-14:Ac did not enhance trap catches. We conclude that the sex pheromone of H. hippophaecolusis composed of Z7-14:Ac and E3-14:Ac. Optimal ratios and doses of these two components, and the possible role of other minor components, remain to be determined.

Responses of Tea Tussock Moth, Euproctis pseudoconspersa, to Its Pheromone, (R)-10,14-Dimethylpentadecyl Isobutyrate, and to the S-Enantiomer of Its Pheromone

Field trials were conducted with each synthetic enantiomer (>98% ee) and blends of the two synthetic enantiomers of the female-produced sex pheromone (10,14-dimethylpentadecyl isobutyrate) of the tea tussock moth, Euproctis pseudoconspersa. Male moths were attracted to each enantiomer alone and to various blends of them. Short syntheses of both enantiomers of the pheromone from commercially available (R)- and (S)-citronellyl bromide and a method of checking the enantiomeric purity of the citronellyl bromide enantiomers are described.

Cohabitation impaired physiology, fitness and sex-related chemosignals in golden hamsters

This study investigated the impact of long-term paternal presence (cohabitation) on several physiological parameters such as body weight, adrenal weight, cortisol of parents, and the survival of pups compared with brief daily encounters (isolation) of male-female pairs in golden hamsters (Mesocricetus auratus). We showed that females were affected more by cohabitation as evidenced by increased body and adrenal weights, elevated cortisol concentrations, and heavier uteri and spleens as compared with cohabiting male and isolated females. Furthermore, we found that tetradecanoic and hexadecanoic acids of the flank glands were sexually dimorphic, for which they were putative female pheromones. These two compounds were suppressed in females and elevated in males by cohabitation, suggesting that cohabitation impaired sex chemosignals. Overall, we concluded that housing females and males together had deleterious effects on adults and the survival of their pups in the golden hamster.

Genetic basis of sex pheromone blend difference between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae)

The two closely related moth species, Helicoverpa armigera and H. assulta, are sympatric in China. Both species use a mixture of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald) as their sex pheromones but in widely different ratios. Hybridization and backcrossing experiments between H. armigera and H. assulta were conducted and sex pheromone compositions of the parent species, their F(1) hybrids and backcrosses were compared to study the genetic basis of the production of their sex pheromone blend composition. Results show that the difference in sex pheromone blend ratios of these Helicoverpa species is mainly controlled by an autosomal locus with two alleles, with the allele from H. armigera being almost completely dominant over that derived from H. assulta.

Identification of Components of the Female Sex Pheromone of the Simao Pine Caterpillar Moth, Dendrolimus kikuchii Matsumura

The pine caterpillar moth, Dendrolimus kikuchii Matsumura (Lepidoptera: Lasiocampidae), is a pest of economic importance on pine in southwest China. Three active compounds were detected during analyses of solvent extracts and effluvia sampled by solid phase microextraction (SPME) from virgin female D. kikuchii using gas chromatography (GC) coupled with electroantennographic (EAG) recording with antennae from a male moth. The compounds were identified as (5Z,7E)-5,7-dodecadien-1-yl acetate (Z5,E7-12:OAc), (5Z,7E)-5,7-dodecadien-1-ol (Z5,E7-12:OH), and (5Z)-5-dodecenyl acetate (Z5-12:OAc) by comparison of their GC retention indices, mass spectra, and EAG activities with those of synthetic standards. Microchemical reactions of gland extracts provided further information confirming the identifications of the three components. Solvent extractions and SPME samples of pheromone effluvia from virgin calling females provided 100:18:0.6 and 100:7:1 ratios of Z5,E7-12:OAc:Z5,E7-12:OH:Z5-12:OAc, respectively. Field behavioral assays showed that Z5,E7-12:OAc and Z5,E7-12:OH were essential for attraction of male D. kikuchii moths. However, the most attractive blend contained these three components in a 100:20:25 ratio in a gray rubber septa. Our results demonstrated that the blend of Z5,E7-12:OAc, Z5,E7-12:OH, and Z5-12:OAc comprise the sex pheromone of D. kikuchii. The optimized three-component lure blend is recommended for monitoring D. kikuchii infestations.

Biosynthesis and PBAN-Regulated Transport of Pheromone Polyenes in the Winter Moth, Operophtera brumata

The trienoic and tetraenoic polyenes, (3Z,6Z,9Z)-3,6,9-nonadecatriene, (3Z,6Z,9Z)-3,6,9-henicosatriene, and (3Z,6Z,9Z)-1,3,6,9-henicosatetraene were found in the abdominal cuticle and pheromone gland of the winter moth Operophtera brumata L. (Lepidoptera: Geometridae), in addition to the previously identified single component sex pheromone (3Z,6Z,9Z)-1,3,6,9-nonadecatetraene. The pheromone biosynthesis activating neuropeptide (PBAN) is involved in the regulation of polyene transport from abdominal cuticle to the pheromone gland. In vivo deuterium labeling experiments showed that (11Z,14Z,17Z)-11,14,17-icosatrienoic acid, the malonate elongation product of linolenic acid, (9Z,12Z,15Z)-9,12,15-octadecatrienoic acid, is used to produce (3Z,6Z,9Z)-3,6,9-nonadecatriene and (3Z,6Z,9Z)-1,3,6,9-nonadecatetraene.