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 titres 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 ( Z 9C31), 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 Z 9C31 reduced courtship and mating attempts by wild-type males. These results prove that Z 9C31 is crucial for sex discrimination in N. vitripennis ; and (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 .

Attracting and securing potential mating partners is of fundamental importance for reproduction. Therefore, signaling sexual attractiveness is expected to be tightly coordinated in communication systems synchronizing senders and receivers. Chemical signaling has permeated through all taxa of life as the earliest and most widespread form of communication and is particularly prevalent in insects. However, it has been notoriously difficult to decipher how exactly information related to sexual signaling is encoded in complex chemical profiles. Similarly, our knowledge of the genetic basis of sexual signaling is very limited and usually restricted to a few case studies with comparably simple pheromonal communication mechanisms. The present study jointly addresses these two knowledge gaps by characterizing two fatty acid synthase genes, that most likely evolved by tandem gene duplication, simultaneously impacting sexual attractiveness and complex chemical surface profiles in parasitic wasps. Gene knock-down in female wasps dramatically reduces their sexual attractiveness coinciding with a drastic decrease in male courtship and copulation behavior. Concordantly, we found a striking shift of methyl-branching patterns in the female surface pheromonal compounds, which we subsequently demonstrate to be the main cause for the greatly reduced male response. Intriguingly, this suggests a potential coding mechanism for sexual attractiveness mediated by specific methyl-branching patterns in CHC profiles. So far, the genetic underpinnings of methyl-branched CHCs are not well understood despite their high potential for encoding information. Our study sheds light on how biologically relevant information can be encoded in complex chemical profiles and on the genetic basis of sexual attractiveness.

Attracting and securing potential mating partners is of fundamental importance for successfully initiating reproduction and thus assuring the passing of genes to the next generation. Therefore, signaling sexual attractiveness is expected to be tightly coordinated in communication systems synchronizing senders and receivers. Chemical signaling has permeated through all taxa of life as the earliest and most wide-spread form of communication and is particularly prevalent in insects. However, it has been notoriously difficult to decipher how exactly information related to sexual signaling is encoded in complex chemical profiles. Similarly, our knowledge of the genetic basis of sexual signaling is very limited and usually restricted to a few case studies with comparably simple pheromonal communication mechanisms. The present study jointly addresses these two knowledge gaps by characterizing a single gene simultaneously impacting sexual attractiveness and complex chemical surface profiles in parasitic wasps. Knocking down a fatty acid synthase gene in female wasps dramatically reduces their sexual attractiveness coinciding with a drastic decrease in male courtship and copulation behavior. Concordantly, we found a striking shift of methyl-branching patterns in the female surface pheromonal compounds, which we subsequently demonstrate to be the main cause for the greatly reduced male response. Intriguingly, this suggests a potential coding mechanism for sexual attractiveness mediated by specific methyl-branching patterns, whose genetic underpinnings are not well understood despite their high potential for encoding information. Our study sheds light on how biologically relevant information can be encoded in complex chemical profiles and on the genetic basis of sexual attractiveness.

The transcription factor Doublesex (Dsx) has a conserved function in controlling sexual morphological differences in insects, but our knowledge on its role in regulating sexual behavior is widely limited to Drosophila. Here, we show in the parasitoid wasp Nasonia vitripennis that males whose Dsx gene had been silenced by RNA interference (NvDsx-i) underwent a three-level pheromonal feminization: (1) NvDsx-i males were no longer able to attract females from a distance, owing to drastically reduced titers of the abdominal long-range sex pheromone. (2) NvDsx-i males were courted by wild-type males like females which correlated with a lower abundance of alkenes in their cuticular hydrocarbon (CHC) profiles. Supplementation of NvDsx-i male CHC profiles with realistic amounts of synthetic (Z)-9-hentriacontene (Z9C31), the most significantly reduced alkene in 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 Nasonia. (3) Nvdsx-i males were hampered in eliciting female receptivity during courtship and thus experienced severely reduced mating success, suggesting that they are unable to produce the hitherto 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. Silencing Dsx by RNA interference provides a new avenue for unraveling the molecular mechanisms underlying the pheromone-mediated sexual communication in insects.