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Doublesex (Dsx) has a conserved function in controlling sexual morphological differences in insects, but our knowledge of its role in regulating sexual behaviour is primarily limited to Drosophila. Here, we show with the parasitoid wasp Nasonia vitripennis that males whose Dsx gene had been silenced (NvDsx-i) underwent a three-level pheromonal feminization: (i) NvDsx-i males were no longer able to attract females from a distance, owing to drastically reduced titers of the long-range sex pheromone. (ii) NvDsx-i males were courted by wild-type males as though they were females, which correlated with a lower abundance of alkenes in their cuticular hydrocarbon (CHC) profiles. Supplementation with realistic amounts of synthetic (Z)-9-hentriacontene (Z9C31), the most significantly reduced alkene in NvDsx-i males to NvDsx-i males interrupted courtship by wild-type conspecific males. Supplementation of female CHC profiles with Z9C31 reduced courtship and mating attempts by wild-type males. These results prove that Z9C31 is crucial for sex discrimination in N. vitripennis. (iii) Nvdsx-i males were hampered in eliciting female receptivity and thus experienced severely reduced mating success, suggesting that they are unable to produce the to-date unidentified oral aphrodisiac pheromone reported in N. vitripennis males. We conclude that Dsx is a multi-level key regulator of pheromone-mediated sexual communication in N. vitripennis.

Statistical models outcome and detailed methods for culturing the yeast, quantifying and purifying neocembrene extracts and methods for the quantification of a queen equivalent of neocembrene

The cuticle of virtually any insect is covered by a thin layer of cuticular hydrocarbons (CHCs) typically consisting of a complex mixture of n-alkanes, methyl-branched alkanes, and unsaturated hydrocarbons. Apart from their putative primary function as a desiccation barrier, CHCs are used by insects for communication. In many species of parasitoid wasps, males use CHCs to recognize females, and thus the composition of CHC profiles is typically species- and sex-specific. Sometimes, the biological activity of CHCs is synergized by more polar lipids. In most species, however, the contribution of polar lipids and the role of individual CHCs or CHC classes is unclear. Here we report a CHC-based contact sex pheromone in the pteromalid wasp Urolepis rufipes. Males of U. rufipes were arrested on and showed courtship behavior (wing fanning) and copulation attempts toward cadavers of females, whereas male cadavers and solvent-washed cadavers of either sex elicited no responses. Whole body extracts from females and the non-polar CHC fraction thereof elicited arrestment, courtship, and copulation attempts by males, whereas the fractions containing polar lipids were behaviorally inactive. Analyses of male- and female-derived CHC profiles revealed that they consisted exclusively of n-alkanes and methyl-branched alkanes. Removal of n-alkanes from female-derived CHCs with molecular sieves rendered the remaining methyl-branched fraction inactive. Synthetic n-alkanes in female-specific ratios also did not elicit any responses by males. Two 7-methylalkanes (7-MeC31 and 7-MeC33) were the only two components with a higher relative abundance in females compared to males. However, supplementation of male-derived CHCs with synthetic racemic 7-MeC31 and 7-MeC33 to levels found in females was not sufficient to render male-derived CHCs attractive to males. Enantiopure synthetic 7-MeC31 and 7-MeC33 might have caused different effects but were not available. We conclude that female-derived CHCs function as a contact sex pheromone in U. rufipes. Our data suggest that methyl-branched and n-alkanes act synergistically and that the sex-specific relative composition of the whole profile, rather than the abundances of single components, underlies the elicitation of male courtship behavior.

Biosynthesis protocol of chemical compounds, relative peak differences of cuticular compounds between reproductive classes, complete list of all identified cuticular compounds and table with p-values of the glmm.

Many species of cerambycid beetles in the subfamily Cerambycinae are known to use male-produced pheromones composed of one or a few components such as 3-hydroxyalkan-2-ones and the related 2,3-alkanediols. Here, we show that this pheromone structure is characteristic of the cerambycine genus Neoclytus Thomson, based on laboratory and field studies of 10 species and subspecies. Males of seven taxa produced pheromones composed of (R)-3-hydroxyhexan-2-one as a single component, and the synthetic pheromone attracted adults of both sexes in field bioassays, including the eastern North American taxa Neoclytus caprea (Say), Neoclytus mucronatus mucronatus (F.), and Neoclytus scutellaris (Olivier), and the western taxa Neoclytus conjunctus (LeConte), Neoclytus irroratus (LeConte), and Neoclytus modestus modestus Fall. Males of the eastern Neoclytus acuminatus acuminatus (F.) and the western Neoclytus tenuiscriptus Fall produced (2S,3S)-2,3-hexanediol as their dominant or sole pheromone component. Preliminary data also revealed that males of the western Neoclytus balteatus LeConte produced a blend of (R)-3-hydroxyhexan-2-one and (2S,3S)-2,3-hexanediol but also (2S,3S)-2,3-octanediol as a minor component. The fact that the hydroxyketone-hexanediol structural motif is consistent among these North American species provides further evidence of the high degree of conservation of pheromone structures among species in the subfamily Cerambycinae.

The promethea moth Callosamia promethea is one of three species of silkmoths from the genus Callosamia that occur in North America. Cross attraction of males to heterospecific calling females has been observed in the field, and hybrid progeny have been produced by pairing heterospecifics in captivity. These observations suggest that all three species share or have considerable overlap in the sex attractant pheromones produced by females, so that other prezygotic isolating mechanisms, such as diel differences in reproductive activity, limit hybridization in the field. Coupled gas chromatography-electroantennogram detection and gas chromatography- mass-spectrometry analyses of extracts of volatiles collected from female promethea moths supported the identification of (4E,6E,11Z,13Z)-hexadeca-4,6,11,13-tetraenal [(4E,6E,11Z,13Z)-16:Ald] as the compound in extracts that elicited the largest responses from antennae of males. The identification was confirmed by non-selective synthesis of several isomers as analytical standards, and stereoselective synthesis of (4E,6E,11Z,13Z)-16:Ald for testing in field trials. Male moths were strongly attracted to synthetic (4E,6E,11Z,13Z)-16:Ald, suggesting that this compound is the major and possibly the only component of the sex pheromone of these large saturniid moths. Based on the cross-attraction of heterospecifics, it is likely that this is also a major pheromone component of the other two North American Callosamia species as well., Ministry of Science and Innovation LSU AgCenter Kentucky Agricultural Experiment Station CICYT

Bagrada hilaris Burmeister, is a stink bug native to Asia and Africa and invasive in the United States, Mexico, and more recently, South America. This species can cause serious damage to various vegetable crops in the genus Brassica, with seedlings being particularly susceptible to B. hilaris feeding activity. In this study, the role of volatile organic compounds (VOCs) emitted by seedlings of three Brassica species on the host preference of B. hilaris was evaluated. In dual choice arena and olfactometer bioassays, adult painted bugs preferred B. oleracea var. botrytis and B. napus over B. carinata. Volatiles from B. oleracea seedlings were collected and bioassayed with B. hilaris adults and late stage nymphs, using electroantennographic (EAG) and behavioral (olfactometer) techniques. When crude extracts of the VOCs from B. oleracea var. botrytis seedlings and liquid chromatography fractions thereof were bioassayed, B. hilaris adults and nymphs were attracted to the crude extract, and to a non-polar fraction containing hydrocarbons, whereas there were no responses to the more polar fractions. GC-MS analysis indicated that the main constituents of the non-polar fraction was an as yet unidentified diterpene hydrocarbon, with trace amounts of several other diterpene hydrocarbons. The major diterpene occurred in VOCs from both of the preferred host plants B. oleracea and B. napus, but not in VOCs of B. carinata. Our results suggest that this diterpene, alone or in combination with one or more of the minor compounds, is a key mediator in this insect-plant interaction, and could be a good candidate for use in lures for monitoring B. hilaris in the field.