David A. Carlson

Sex Attractant Pheromone of the House Fly: Isolation, Identification and Synthesis

A sex pheromone isolated from the cuticle and feces of the female house fly attracts the male fly; it has been identified as (Z)-9-tricosene. Chemical and biological comparisons of the natural and synthesized compounds show that they are identical.

Elution patterns from capillary GC for methyl-branched alkanes

A common and confusing problem in analyses of insect hydrocarbons is in making sense of complicated gas chromatograms and interpreting mass spectra since branched chain compounds differing by one or two carbons in backbone or chain length may elute from the column at nearly the same time. To address this confusing situation, relative gas chromatography (GC) retention times are presented for typical mono-, di-, tri-, and tetramethylalkanes comprising most of the commonly appearing series of homologous methyl-branched alkanes up to 53 carbons that are found in insect cuticular hydrocarbons. Typical insect-derived methylalkanes with backbones of 33 carbons were characterized by Kovats indices (KI); monomethyl alkanes elute between KI 3328 and 3374, dimethylalkanes elute between KI 3340 and 3410, trimethylalkanes elute between KI 3378 and 3437, and tetramethylalkanes elute between KI 3409 and 3459, depending upon the positions of substituents. A protocol is described for identification of methyl-branched hydrocarbons eluted from nonpolar polysiloxane DB-1 capillary GC columns. In this protocol, retention indices (KI values) are assigned to peaks, then the patterns in GC peaks that probably contain homologs are marked to assist subsequent GC-mass spectrometric (GC-MS) interpretation. Use of the KI allows assignment of likely structures and the elimination of others, with demonstrative consistency, as there are no known exceptions. Interpretation of electron ionization mass spectra can then proceed within narrowed structural possibilities without the necessity of chemical ionization GC-MS analysis. Also included are specific examples of insect hydrocarbons that were assembled from 30 years of the literature, and these are intended to help with confirmation of confusing or contradictory structures.

De novo biosynthesis of juvenile hormone III and I by the accessory glands of the male mosquito

The role of the male accessory glands (MAG) in reproduction was investigated in the mosquito Aedes aegypti. MAG incubated with [14C]acetate synthesized radioactively labeled JH III, JH III bisepoxide and methyl farnesoate. MAG incubated with L-[methyl-3H]methionine synthesized [3H]JH III and a molecule that chromatographed on HPLC with JH I. Analysis of MAG and whole males extract by glass capillary combined gas-chromatography-selected ion monitoring mass spectrometry identified JH III and I as the main analogs that were synthesized by male mosquitoes. MAG of Culex nigripalpus, Anopheles rangeli and Anopheles trinkae also synthesized JH III from L-[methyl-3H]methionine, which indicates that the male mosquito has a complete JH III biosynthetic pathway. Unfed and unmated Culex quinquefasciatus do not develop their ovaries to the resting stage. Females injected with one MAG extract equivalent or implanted with A. aegypti MAG developed their ovaries to the resting previtellogenic stage, whereas females that were injected with saline did not. These results indicate that MAG synthesize and secrete JH III. The corpora allata (CA) of the male Aedes aegypti also synthesize JH III from L-[methyl-3H]methionine. This observation may suggest that JH synthesized by the males CA is used for internal regulation, whereas JH synthesized by the MAG is transferred with the sperm into the female.

Pheromones exchanged during mating: a mechanism for mate assessment in Drosophila

Abstract Many insect species are sexually dimorphic for volatile compounds associated with the cuticle. In these species, females may acquire volatile compounds from males through direct contact with males during mating. In some cases, these compounds function as antiaphrodisiacs, which inhibit subsequent male precopulatory behaviour. During mating, males may also acquire female compounds, which normally stimulate males to court. Contact during mating thus results in a mutual exchange of compounds usually found primarily, or exclusively, on one or the other sex. Drosophila melanogaster is a sexually dimorphic dipteran species in which predominant male and female cuticular hydrocarbons are mutually exchanged during mating. Using synthetic compounds, the effect of this exchange on the post-mating sexual attractiveness of both males and females was tested. The sex-predominant hydrocarbons acquired during mating decreased the attractiveness of both males and females to members of the other sex. Mated females are courted less actively than virgins, and are usually unwilling to re-mate. Similarly, recently mated males are less attractive to females than virgin males are, and may be less fertile. Thus, cuticular hydrocarbons acquired by the other sex during mating could allow both males and females to assess the immediate reproductive potential of prospective mates.

Contact sex pheromone in the tsetse flyGlossina pallidipes (Austen) Identification and Synthesis

Adult maleG. pallidipes attempted to copulate with decoys treated with a branched paraffin obtained from laboratory-reared female flies. The compound causing maximal response was isolated and identified as 13,23-dimethylpentatriacontane. The synthesized compound elicited increasing responses with increasing doses. This sex- and species-specific compound was always present in physiological amounts in females, as it increased from 2 μg at emergence to 10 μg per female at 14 days. It was present in wild-caught females from a wide geographical range.

Mass spectrometry and characterization of Aedes aegypti trypsin modulating oostatic factor (TMOF) and its analogs

Trypsin modulating oostatic factor (TMOF), a decapeptide that directly inhibits the biosynthesis of trypsin- and chymotrypsin-like enzymes in epithelial cells of mosquito midgut and indirectly inhibits vitellogenesis in anautogenous females, has been sequenced by Fourier transform mass spectrometry analysis. The peptide has a primary amino acid sequence of NH2-Tyr-Asp-Pro-Ala-(Pro)6-COOH and probably exhibits left-handed helical conformation as was shown by computer stereoview simulation. The factor is metabolized very rapidly (half-life of 1.6 h) in intact mosquitoes when injected after the blood meal. Inhibition of trypsin biosynthesis was followed in ligated abdomens, which synthesize trypsin but do not metabolise TMOF. At concentrations of 3 x 10(-9) M and 6.8 x 10(-6) M, TMOF inhibited 50 and 90% of trypsin-like enzyme biosynthesis, respectively. Several analogs of varying chain lengths were synthesized and evaluated for biological activity using dose-response curves. Switching the positions of Tyr and Asp at the N-terminus reduced the activity of the hormone, indicating that the N-terminus is important for biological activity. Removal of two to five prolines at the C-terminus also reduced activity, indicating that both the N- and C-termini are important. Synthesis of trypsin-like isozyme was followed in several insect species using [1,3-3H]diisopropyl-fluorophosphate (DFP) in the presence of tosylamide-2-phenylethyl chloromethyl ketone. Marked reduction of [1,3-3H]diisopropyl-phosphoryl-trypsin-like derivatives was noted after TMOF treatment, as assessed by polyacrylamide gel electrophoresis. These results indicate that the biosynthesis of trypsin-like enzyme in mosquitoes and other insects may be regulated by sequence-related TMOFs.

A material isolated from human hands that attracts female mosquitoes.

The residue left on glass surfaces by human hands was found to be attractive to femaleAedes aegypti (L.) andAnopheles quadrimaculatus Say mosquitoes. The material lost half of its activity in 1 hr. A solvent wash technique was developed to recover and concentrate the residuum from handled glass beads. The residuum could be recovered effectively with absolute ethanol and less effectively with several other solvents. More mosquitoes were attracted to heated than to unheated residuum, an indication of its volatility. Also, attraction of the residuum decreased with decreasing concentration or dose. Concentrated residuum collections, stored under refrigeration and tested for longevity, showed no appreciable loss of attractiveness up to 60 days of storage.

Gas chromatography/mass spectrometry analysis of the cuticular hydrocarbons from parasitic wasps of the genus Muscidifurax

Parasitic Hymenoptera can be difficult to identify by conventional taxonomic techniques. Examination of the cuticular hydrocarbons (CHCs) provides a basis for chemotaxonomic differentiation, which may lead to the discovery of pheromones, and can be a means of examining colonies for species cross-contamination. The parasitic wasps examined were Muscidifurax raptor, M. zaraptor, M. uniraptor, and the gregarious form of M. raptorellus. Species within the genus Muscidifurax, as well as the sex, can clearly be differentiated by examining the gas chromatograms of the CHCs. Identification of the alkanes by mass spectrometry shows uncommon dimethylalkanes and trimethylalkanes for members of the genus. The methyl branched cuticular hydrocarbons of these insects are rare compared to those found on insects reported in the literature, but are present in significant amounts on these insects. Additionally, sexual dimorphism is observed in long chain alkanes (C21–C39) present on male and female cuticular surfaces for these species. Females tend to have cuticular hydrocarbons with methyl branches located externally on the carbon backbone chain for dimethyl-, trimethyl-, and tetramethylalkanes, whereas males tend to have dimethyl- and trimethylalkanes located internally on the hydrocarbon backbone chains. Mass spectra of novel and rare methyl branched compounds identified on these parasitoids are presented.

Stable fly sex attractant and mating pheromones found in female body hydrocarbons

Investigation into the nature and function of stable fly pheromones indicated that both male sex attractant and mating pheromones are present. Polyolefin fraction of female body hydrocarbons attracted virgin males while thetrans andcis olefins were found to be responsible for mating stimulation. Characterization of the attractive components is under way.

Unusual polymethyl alkenes in tsetse flies acting as abstinon inGlossina morsitans

The major alkene of the male tsetse fly,Glossina morsitans morsitans, was isolated for characterization by thin-layer and gas chromatography (GC). The mass spectra of the alkene and the alkene DMDS derivative indicated one isomer, 19,23-dimethyltritriacont-1-ene. The material is present at 1–2μg/male fly and is partially transferred to the female preparatory to or during mating. A dose-dependent antiaphrodisiac effect was seen with exposed male flies using the isolated natural product, with 2 and 4μg causing 80% loss of copulatory attempts, and 10μg extinguishing the attempts. This effect was increased by addition of male-produced alkane. This compound and a 31-carbon homolog also appear inG. m. submorsitans. Similar quantities of alkenes that are species-specific appear in all tsetse males. Structures of male-produced trimethylalkenes that appear in two other species,G. palpalis palpalis andG. fuscipes fuscipes, were investigated.