Methyl 2-(methylthio)benzoate: the unique sulfur-containing sex pheromone of Phyllophaga crinita.

The female-produced sex pheromone of Phyllophaga crinita (Burmeister) (Coleoptera: Scarabaeidae: Melolonthinae; the adult has no common name) is identified as methyl 2-(methylthio)benzoate. This is the first identification of a sulfur-containing, long-distance, female-produced sex attractant from any insect taxa. The root-feeding larvae of this species are serious pests in many crops in Texas and Mexico. In field tests, many P. crinita males were captured in traps baited with the authentic compound. Interestingly, a heteroatom analog, methyl 2-methoxybenzoate, also captured P. crinita males, but only at a dose 10,000 times higher than the lowest tested dose of the authentic pheromone.

Unusual pheromone receptor neuron responses in heliothine moth antennae derived from inter-species imaginal disc transplantation.

Single-cell electrophysiological recordings were obtained from olfactory receptor neurons housed in sensilla trichodea along the adult antennae arising from transplantation of the antennal imaginal discs between larval male Helicoverpa zea and Heliothis virescens. The olfactory receptor neurons from the majority of type C sensilla sampled on transplanted antennae displayed response characteristics consistent with those of the species that donated the antennae. However, some of the sensilla type C sampled in either transplant type contained olfactory receptor neurons that responded in a manner typical of the recipient species or other neurons that have not previously been found in the type C sensilla of either species. The single-cell data help to explain behavioral results showing that some transplant males do fly upwind to both species' pheromone blends, an outcome not expected based on known antennal sensory phenotypes. Our results suggest that host tissue can influence antennal olfactory receptor neuron development, and further that because of a common phylogenetic ancestry the donor tissue has the genetic capability to produce a variety of sensillar and receptor types.

Acyl-CoA Z9- and Z10-desaturase genes from a New Zealand leafroller moth species, Planotortrix octo.

Two cDNAs encoding acyl-CoA Z9-desaturase from the fat body and Z10-desaturase from the pheromone gland of the greenhead leafroller moth, Planotortrix octo, were obtained by RACE PCR. The Z9-desaturase (Pocto-Z9) cDNA spans 2291 nt with an ORF encoding a 352 amino-acid protein, which has 65% identity to Trichoplusia ni Delta 9 desaturase (Tni-Z9). The Z10-desaturase (Pocto-Z10) cDNA spans 2777 nt with an ORF encoding a protein with 356 amino acids. Pocto-Z10 shows lower identity to Pocto-Z9 and Tni-Z9 (48 and 46%, respectively) and relatively higher identity to the Delta 11 desaturases of T. ni and Helicoverpa zea (57 and 56%, respectively). The ORFs of these two P. octo cDNAs were constructed into an expression vector, YEpOLEX, that complemented the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient ole1 strain of Saccharomyces cerevisiae. Expression of Pocto-Z9 produced a 5:2 ratio of Z9-16 and Z9-18 acids, with minor amounts (<4%) of Z9-14, Z9-15, and Z9-17 acids. Pocto-Z10 was successfully expressed in the YEpOLEX system when complemented with Z11-18:Me, and the major desaturase product proved to be Z10-16:Acid. The results confirm the regio- and stereo-selectivity of this unusual Delta 10 desaturase.

Structural and functional conservation and divergence among acyl-CoA desaturases of two noctuid species, the corn earworm, Helicoverpa zea, and the cabbage looper, Trichoplusia ni.

In this report, we describe the structural and functional analyses of four acyl-CoA desaturase-encoding cDNAs that we isolated from RNA expressed in the pheromone gland of the corn earworm, Helicoverpa zea. We deduced the homology relationships of the encoded proteins, designated HzPGDs1, HzPGDs2, HzPGDs3 and HzFBDs, to each other and to previously described desaturases of the cabbage looper moth, Trichoplusia ni, the fly, Drosophila melanogaster, and other more distantly related organisms. We also isolated genomic DNA fragments of the four H. zea desaturase-encoding genes, determined the locations of introns present in them, and compared them to conserved intron positions in reported desaturase genes of other species. We measured the levels of the four desaturase mRNAs in H. zea pheromone glands and larval fat bodies by RT-PCR. We established the functional identities of the deduced proteins HzPGDs1 and HzPGDs2, encoded by the two desaturase mRNAs that are differentially and abundantly expressed in pheromone glands of sexually mature adult H. zea females, by functional expression of their encoding cDNAs in a desaturase-deficient mutant, ole1, of the yeast Saccharomyces cerevisiae. We compared the unique unsaturated fatty acid profiles of HzPGDs1- and HzPGDs2-expressing transformants to those of strains expressing previously described Delta11 and Delta9 desaturases of T. ni.

A delta 9 desaturase gene with a different substrate specificity is responsible for the cuticular diene hydrocarbon polymorphism in Drosophila melanogaster.

Drosophila melanogaster cuticular pheromones consist of unsaturated hydrocarbons with at least one double bond in position 7: 7 tricosene (T) in males and 7,11 heptacosadiene (HD) in females. However, in many African populations like the Tai strain, females possess low levels of 7,11 HD and high levels of its positional isomer 5,9 HD. We have previously isolated a desaturase gene, desat1, from the Canton-S strain (CS), a 7,11 HD-2-rich morph of D. melanogaster. This desaturase is located in 87C, a locus that has been involved in the difference between 7,11 HD and 5,9 HD morphs. Therefore, we have searched for different desaturase isoforms in both strains. We first cloned desat1 in the Tai strain and report here functional expression of desat1 in CS and Tai. In both strains, the Desat1 enzymes have the same Delta9 specificity and preferentially use palmitate as a substrate, leading to the synthesis of omega7 fatty acids. Also found was a desaturase sequence, named desat2, with a homologous catalytic domain and a markedly different N-terminal domain compared with desat1. In CS genome, it lies 3.8 kb upstream of desat1 and is not transcribed in either sex. In the Tai strain, it is expressed only in females and acts preferentially on myristate, leading to the synthesis of omega5 fatty acids. We suggest, therefore, that desat2 might play a control role in the biosynthesis of 5,9 HD hydrocarbons in Tai females and could explain the dienic hydrocarbon polymorphism in D. melanogaster.

Characterizing the Hez-PBAN gene products in neuronal clusters with immunocytochemistry and MALDI MS.

Methods to characterize pheromone biosynthesis activating neuropeptide (PBAN) and other PBAN gene encoded neuropeptides (PGN) from individual subesophageal ganglion neuronal clusters of the corn earworm moth, Helicoverpa zea, were developed. Individual antisera against the N-terminal sequence to PBAN and each of the three PGNs from the Hez-PBAN prohormone were developed, and their specificity determined. In all cases, each antiserum stains the same three groups of subesophageal ganglion ventral midline neurons-the mandibular, maxillary and labial neurons-in both adult females and males. These results were confirmed using matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) of individual subesophageal ganglion neuronal clusters. Using mass spectrometry, the amidated PGN-24 was not detected but an N-terminally extended form is observed that is two amino acids longer. Other peptides resulting from the processing of the Hez-PBAN prohormone were detected. Using both the specific antisera and the cellular profiling abilities of MALDI MS, the roles of individual members of the Hez-PBAN prohormone derived peptides can now be explored.

Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA Delta11-desaturase of the cabbage looper moth, Trichoplusia ni.

Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera. The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Delta9-desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acyl-CoA Delta9-desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group. In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni, a species in which all unsaturated pheromone products are produced via a Delta11Z-desaturation mechanism. The largest ORF of the approximately 1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Delta11Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast Delta9-desaturases, suggesting conserved transmembrane topology. A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Delta9Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an approximately 1,250-nt PDesat-Tn Delta11Z mRNA that is consistent with the spatial and temporal distribution of Delta11-desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Delta11Z resulted in complementation of the strain's fatty acid auxotrophy and the production of Delta11Z-unsaturated fatty acids.

Baculovirus expression of an insect gene that encodes multiple neuropeptides.

Sex pheromone production in the corn earworm, Helicoverpa zea, is regulated by a 33-amino-acid neuropeptide named Hez-PBAN (pheromone biosynthesis activating neuropeptide). Hez-PBAN is encoded in a preprohormone that also contains four other structurally related peptides. Two recombinant baculoviruses that contain two different sequences of Hez-PBAN cDNA under the control of a strong polyhedrin promotor were constructed. The first virus, AcWT-PBAN, contains the entire prepro-Hez-PBAN coding sequence. The second virus, AcBX-PBAN, contains a synthetic chimera gene encoding a bombyxin signal peptide sequence fused to a pro-Hez-PBAN sequence. Cell extracts, culture medium of BTI-TN-5B1-4 cells, and hemolymph from 4th instar Trichoplusia ni larvae, all infected with AcBX-PBAN, showed a high level of pheromonotropic activity. Pheromonotropic activity was not detected in the cells infected with AcWT-PBAN. Results of chromatographic and immunochemical studies showed that some of the potential processing sites in the expressed pro-Hez-PBAN sequence were not used during posttranslational processing in the AcBX-PBAN-4-infected BTI-TN-5B1-4 cells and 4th instar T. ni larvae. However, the processing pattern of the recombinant pro-Hez-PBAN in AcBX-PBAN-infected 4th instar T. ni larvae was similar to that exhibited in the central nervous system of H. zea adult females, since a PBAN-like immunoreactive-peptide-band was found in the hemolymph of Ac-BX-PBAN-4-infected 4th instar T. ni larvae. In a droplet feeding assay, neonate and 3rd instar T. ni larvae infected with AcBX-PBAN-4 showed a significant reduction in survival time (26% and 19%, respectively) when compared to control larvae that were infected with a polyhedrin-deficient virus, Ac-E10.

Identification of soluble binding proteins for an insect neuropeptide.

A photoaffinity analog of Helicoverpa zea pheromone biosynthesis activating neuropeptide (Hez-PBAN) was used to identify PBAN binding proteins in various tissues of the corn earworm moth, H. zea. Synthetic Hez-PBAN was derivatized on Lys-27 with p-benzoyldihydrocinnamoyl-N-hydroxysuccinimide ester (BZDC-NHS ester). The resulting BZDC-PBAN stimulated pheromone production in H. zea isolated abdomens at levels comparable to those of the unmodified peptide. Photoaffinity labeling experiments using [3H]BZDC-PBAN with female moth tissues revealed soluble 100 and 115 kDa proteins in the brain-subesophageal ganglia complex, ventral nerve cord, and thoracic muscle that were specifically labeled with the PBAN analog.

Purification, identification, concentration and bioactivity of (Z)-7-dodecen-1-yl acetate: sex pheromone of the female Asian elephant, Elephas maximus.

In their natural ecosystems, adult male and female Asian elephants, Elephas maximus, live separately. For several weeks prior to ovulation, female elephants release a substance in their urine which elicits a high frequency of non-habituating chemosensory responses, especially flehmen responses, from male elephants. These responses occur prior to, and are an integral part of, mating. Using bioassay-guided fractionation, quantitatively dependent on these chemosensory responses, a specific sex pheromone was isolated and purified by an alternating series of organic and/or aqueous extractions, column chromatography, gas chromatography and high-performance liquid chromatography. Using primarily 1H-proton nuclear magnetic resonance (NMR) spectrometry and gas chromatography-mass spectrometry (GC-MS) of the urine-derived pheromone and its dimethyl disulfide derivative, we determined the structure of the active compound to be (Z)-7-dodecen-1-yl acetate (Z7-12:Ac). Concentrations of Z7-12:Ac in the female urine increased from non-detectable during the luteal phase to 0.48 microgram/ml (0.002 mM) early in the follicular phase and to 33.0 micrograms/ml (0.146 mM) just prior to ovulation. Bioassays with commercially available authentic synthetic Z7-12:Ac, using 10 Asian male elephants at several locations in the US, demonstrated quantitatively elevated chemosensory responses that were robust during successive tests, and several mating-associated behaviors. Bioassays with Z7-12:Ac with adult male elephants dwelling in more natural social situations in forest camps in Myanmar revealed some differing contextual pre-mating behavioral components. The remarkable convergent evolution of this compound suggests that compounds identified in mammalian exudates that are also present in pheromone blends of insects should be re-evaluated as potential mammalian chemosignals.

Biosynthetic enzymes regulating ratios of sex pheromone components in female redbanded leafroller moths.

Changes in key enzymes in the biosynthetic pathways of sex pheromone components can produce differences in component ratios and structures. The sex pheromone communication system is critical to reproduction and the maintenance of a species and so changes in this system can play a major role in the speciation process. Artificial selection of female redbanded leafroller moths that produced either higher or lower ratios of 14-/12-carbon pheromone components was used to study how the biosynthetic pathways were affected in the high and low populations. The results showed that the chain shortening enzymes were selective for the (E) isomer and so left the 14-carbon acyl intermediates enriched in the (Z) isomer. Thus, the high population, which has a higher amount of 12-carbon components, also has a lower ratio of E11-/Z11-14:OAc pheromone components. The data also suggested that chain shortening occurred prior to reduction and acetylation of the 14-carbon components. These changes are not sufficient to isolate the redbanded leafroller populations, but we discuss some cases where significant changes in pheromone component ratios are affected by the chain-shortening enzymes.

Chemistry of sex attraction.

The chemical communication system used to attract mates involves not only the overt chemical signals but also indirectly a great deal of chemistry in the emitter and receiver. As an example, in emitting female moths, this includes enzymes (and cofactors, mRNA, genes) of the pheromone biosynthetic pathways, hormones (and genes) involved in controlling pheromone production, receptors and second messengers for the hormones, and host plant cues that control release of the hormone. In receiving male moths, this includes the chemistry of pheromone transportation in antennal olfactory hairs (binding proteins and sensillar esterases) and the chemistry of signal transduction, which includes specific dendritic pheromone receptors and a rapid inositol triphosphate second messenger signal. A fluctuating plume structure is an integral part of the signal since the antennal receptors need intermittent stimulation to sustain upwind flight. Input from the hundreds of thousands of sensory cells is processed and integrated with other modalities in the central nervous system, but many unknown factors modulate the information before it is fed to motor neurons for behavioral responses. An unknown brain control center for pheromone perception is discussed relative to data from behavioral-threshold studies showing modulation by biogenic amines, such as octopamine and serotonin, from genetic studies on pheromone discrimination, and from behavioral and electrophysiological studies with behavioral antagonists.

Structural organization of the Helicoverpa zea gene encoding the precursor protein for pheromone biosynthesis-activating neuropeptide and other neuropeptides.

Sex pheromone biosynthesis in a number of moth species is induced by a conserved 33-amino acid amidated neuropeptide PBAN (pheromone biosynthesis-activating neuropeptide). We have isolated and characterized the Helicoverpa zea PBAN cDNA corresponding to a 766-nucleotide mRNA that is expressed in the subesophageal ganglion of adult moths. This mRNA is encoded on a transcription unit comprising 6 exons. The longest open reading frame of the cDNA encodes a 194-amino acid precursor protein that contains the PBAN peptide sequence. Proteolytic processing of this protein, which has structural features consistent with its being a preprohormone, is predicted to generate Hez-PBAN and four additional neuropeptides having a common C-terminal pentapeptide motif, Phe-Xaa-Pro-(Arg or Lys)-Leu (Xaa = Gly, Ser, or Thr), which is also found in insect pyrokinin and myotropin peptide families.

Action of PBAN and related peptides on pheromone biosynthesis in isolated pheromone glands of the redbanded leafroller moth, Argyrotaenia velutinana.

Isolated pheromone glands from the redbanded leafroller moth, Argyrotaenia velutinana, were utilized to demonstrate the action of pheromone biosynthesis activating neuropeptide (PBAN) and bursa pheromonotropic peptide plus several other related peptides on pheromone biosynthesis. All peptides belonging to the PBAN family and the bursa peptide stimulated pheromone biosynthesis as measured by pheromone titer and incorporation of radiolabeled acetate. These peptides required the presence of extracellular Ca2+ for expression of full activity and several inorganic Ca2+ channel blockers inhibited the stimulation of pheromone biosynthesis. The Ca2+ ionophore A23187 alone stimulated pheromone biosynthesis as did a cAMP analogue. Stimulation by the cAMP analogue in the absence of extracellular Ca2+ was observed. Maximum pheromone titers were observed in 16 hr gland incubations; however, 2-6 hr incubations were required if pheromone biosynthesis was measured by incorporation of radiolabeled acetate. Radiolabeled glucose incorporation was not increased in the presence of PBAN. These results are discussed in the context of how the pheromone biosynthetic pathway is stimulated by these peptides.

Identification and synthesis of female sex pheromone of Oriental beetle,Anomala orientalis (Coleoptera: Scarabaeidae).

Females of the Oriental beetle,Anomala orientalis (Waterhouse), release a sex pheromone composed of a 9:1 blend of (Z)- and (E)-7-tetradecen-2-one. The double-bond position of the pheromone was determined by DMDS derivatization and interpretation of the fragmentation patterns produced by monounsaturated ketones. In a sustained-flight tunnel, males responded by flying toward female beetles and attempting to copulate with them. Both effluvium and whole-body extracts of OB females were analyzed, and the activity was found only in the airborne extracts. Flight-tunnel bioassays also showed that a synthetic 90:10Z/E blend on a rubber septum was attractive and that the responses of males to this blend were equivalent toZ isomer alone, but much better than to the singleE isomer.

Sex pheromone for the brownbanded cockroach is an unusual dialkyl-substituted alpha-pyrone.

Female brownbanded cockroaches, Supella longipalpa, emit a sex pheromone that attracts males from a distance. This pheromone was isolated and identified as 5-(2,4-dimethylheptanyl)-3-methyl-2H-pyran-2-one (which we refer to as supellapyrone), and its structure was confirmed by synthesis. A racemic blend of the synthetic compound elicited behavioral and electrophysiological responses comparable to the natural pheromone across a range of doses. This compound is not only a very different type of cockroach pheromone but also makes up an additional class of natural products--namely, 3,5-dialkyl-substituted alpha-pyrones.

Levels of biogenic amines and peptides in individual corn earworm moths, Helicoverpa zea, using high performance liquid chromatography with electrochemical detection.

Quantitative levels of octopamine, serotonin, and dopamine were measured from brain and corpus cardiacum in individual male and female Helicoverpa zea, using high performance liquid chromatography (HPLC) with electrochemical detection. By increasing the proportion of organic modifiers in the mobile phase we were also able to quantify two peptides, adipokinetic hormone and hypertrehalosemic hormone. Levels of amines in both tissues were similar in males and females, but with greater quantities in the brain than the corpus cardiacum. In contrast, the two hormones were found predominantly in the corpus cardiacum, with the level of adipokinetic hormone three times greater than that of the hypertrehalosemic hormone, a finding in agreement with other studies. The described methodology demonstrates the power of HPLC with electrochemical detection for analysis of amines and peptides found in small quantities within the nervous tissue of individual insects.

Stimulation of pheromone biosynthesis in the moth Helicoverpa zea: action of a brain hormone on pheromone glands involves Ca2+ and cAMP as second messengers.

Isolated abdomen and pheromone gland bioassays were utilized to determine the physiological action of the pheromone-biosynthesis-activating neuropeptide (PBAN) in the corn earworm moth Helicoverpa (= Heliothis) zea. An isolated pheromone gland bioassay showed that synthetic PBAN was active at 0.02 pmol, with maximal activity occurring at 0.5 pmol and 60 min of incubation. Second-messenger studies demonstrated that extracellular Ca2+ is necessary for PBAN activity on isolated pheromone glands. The Ca2+ ionophore A23187 stimulated pheromone biosynthesis alone, whereas the Ca2+ channel blockers La3+ and Mn2+ inhibited PBAN activity. However, the organic Ca2+ channel blockers verapamil and nifedipine did not inhibit PBAN activity. Both forskolin and two cAMP analogues stimulated pheromone biosynthesis in the absence of extracellular Ca2+, indicating that Ca2+ may activate an adenylate cyclase. The biogenic amine octopamine did not elicit pheromone production in isolated gland or abdomen bioassays or when injected into intact female moths. Removal of the ventral nerve chord, including the terminal abdominal ganglia in isolated abdomens, did not affect PBAN stimulation of pheromone production. Similar levels of stimulation were found when isolated abdomens were treated with PBAN in scotophase or photophase.

Identification of sex pheromone of tomato pinworm,Keiferia lycopersicella (Wals.).

A sex pheromone produced by femaleKeiferia lycopersicella (Walsingham) was isolated and identified as (E)-4-tridecenyl acetate, based on chemical analyses, electroantennogram assays, and field trapping in California and Florida. Males were captured equally well in traps baited with (E)-4-tridecenyl acetate alone or a variety of (Z)- and (E)-4-tridecenyl acetate blends, although theZ isomer was not detected in extracts of female glands.