Attracting a mate is critical in all species that sexually reproduce. Most animals, particularly insects, do this using chemical compounds called pheromones which can be sensed by potential mates. But how these vast range of different compounds encode and convey the information needed to secure a partner is not fully understood, and the genes that drive this complex communication mechanism are largely unknown.

To address this knowledge gap, Sun et al. studied the parasitic wasp Nasonia vitripennis. Like other insects, female N. vitripennis contain a wide range of chemical compounds on their cuticle, the outer waxy layer coating their surface. Sun et al. set out to find exactly which of these compounds, known as cuticular hydrocarbons, are involved in sexual communication.

They did this by simultaneously inactivating two related genes that they hypothesized to be responsible for synthesizing and maintaining chemical compounds on the cuticle of insects. The genetic modification altered the pattern of chemicals on the surface of the female wasps by specifically up- and down-regulating compounds with similar branching structures. The mutant females were also much less sexually attractive to male wasps.

These findings suggest that the chemical pattern identified by Sun et al. is responsible for communicating and maintaining sexual attractiveness in N. vitripennis female wasps. This is a significant stepping stone towards unravelling how sexual attractiveness can be encoded in complex mixtures of pheromones.

The results also have important implications for agriculture, as this parasitic wasp species is routinely used to exterminate particular fly populations that cause agricultural damage. The work by Sun et al. provides new insights into how these wasps sexually communicate, which may help scientists improve their rearing conditions and sustain them over multiple generations. This could contribute to a wider application of this more sustainable, eco-friendly alternative to destructive agricultural pesticides.

Tightly coordinated chemical signaling has repeatedly been shown to be of fundamental importance for successful reproduction in a wide range of animal species (Blomquist and Ginzel, 2021; Wyatt, 2014). Particularly insects have exploited this type of signaling as their primary mode of communication (Buellesbach et al., 2018a; Greenfield, 2002). Nevertheless, exactly how specific information such as mating status or attractiveness is encoded in the myriad of signaling molecules documented to be involved in sexual communication remains poorly understood (Stökl and Steiger, 2017; Allison and Carde, 2016). Cuticular hydrocarbons (CHCs), major components on the epicuticle of insects, are capable of chemically encoding and conveying a wide variety of biologically relevant information (Blomquist and Ginzel, 2021; Blomquist and Bagnères, 2010). Most prominently, CHCs have been shown to play pivotal roles in sexual communication as the main cues to attract and elicit courtship from conspecific mates (Würf et al., 2020; Finck et al., 2016b) to enable discrimination of con- from heterospecific mating partners (Shahandeh et al., 2018; Xue et al., 2018) and to signal receptivity and mating status (Berson and Simmons, 2019; Simmons, 2015).

Despite such diversified CHC-encoded signals and mediated behaviors, our knowledge on exactly how CHCs encode biologically relevant information has remained surprisingly scarce. This is particularly problematic in studies considering CHC profiles in their entirety as the main signaling entities (Berson and Simmons, 2019; Buellesbach et al., 2018b). The exact compounds or their combinations actually encoding the relevant information within CHC profiles remain largely elusive, except for a few case studies mainly involving the dipteran model organism Drosophila melanogaster, where single unsaturated CHC compounds appear to be the main mediators in sexual communication (Marcillac and Ferveur, 2004; Grillet et al., 2006). In most other cases, chemical information appears to be encoded in a much more complex manner, involving several CHC compounds in different quantitative combinations, with no deeper understanding on the actual coding patterns conveying specific information (Würf et al., 2020; Buellesbach et al., 2013).

In addition to our limited understanding on how CHC profiles encode information, our knowledge of the genetic basis of CHC biosynthesis and its impact on sexual signaling has remained comparably restricted and biased toward the Drosophila model system as well (Blomquist and Bagnères, 2010; Holze et al., 2021). In short, the CHC biosynthetic pathway consists of the elongation of fatty-acyl-Coenzyme A units to produce very long-chain fatty acids that are subsequently converted to CHCs (Blomquist and Bagnères, 2010; Nelson and Blomquist, 1995). An important early switch in CHC biosynthesis is either the incorporation of malonyl-CoA or methyl-malonyl-CoA, eventually leading to the production of straight-chain (n-) or methyl-branched (MB-) alkanes, respectively (Holze et al., 2021; Nelson and Blomquist, 1995, Figure 1). It has been hypothesized that these processes are mediated by two types of fatty-acyl-synthases (FAS), microsomal for methylmalonyl-CoA and cytosolic for malonyl-CoA (Gu et al., 1997; Juárez et al., 1992). To produce olefins (mono- and poly-unsaturated CHCs), desaturases introduce double bonds into straight-chain CHC precursors (Blomquist and Bagnères, 2010; Holze et al., 2021). In Drosophila, a couple of genes have been identified that mainly affect the biosynthesis and ratios of unsaturated CHC compounds that function in sexual signaling. For instance, two desaturases (Desat1 and DesatF) and one elongase (eloF) are involved in female diene production and consequently in their functionality as main sex pheromonal compounds (Chertemps et al., 2007; Chertemps et al., 2006). Furthermore, in the Australian congeneric species D. serrata, the male-specific D. melanogaster orthologue FASN2 has been shown to affect the biosynthesis of three MB-CHCs, among them the additional female mating stimulant 2-Me-C26 (Wicker-Thomas et al., 2015; Chung et al., 2014). Apart from these case studies limited to Drosophila, we know very little about the genetic basis linking CHC biosynthesis and sexual signaling in other insects (Holze et al., 2021).

Figure 1

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The parasitoid jewel wasp Nasonia vitripennis (Hymenoptera: Pteromalidae) has emerged as a suitable model organism to combine studies on functional genetics as well as chemical communication systems in Hymenoptera (Buellesbach et al., 2022; Niehuis et al., 2013). Female CHCs serve as sexual cues capable of eliciting male courtship and copulation behavior in N. vitripennis (Buellesbach et al., 2018b; Steiner et al., 2006). The CHC profiles of N. vitripennis females exhibit a high complexity, consisting of a mixture of n-alkanes, n-alkenes, and MB-alkanes in various quantities. Specifically, the latter fraction, which makes up more than 85% of the whole profile, displays a rich diversity in methyl-branch numbers, chain lengths, and respective relative abundances, hinting at a considerable potential for encoding differential information (Buellesbach et al., 2013; Steiner et al., 2006). However, the entire female CHC profile has long been regarded as encoding their sexual attractiveness, with no single compound, compound classes, or particular patterns being identifiable as the main conveyers of the sexual signaling function (Steiner et al., 2006; Buellesbach, 2018). Moreover, despite recent advances in unraveling the genetic architecture of CHC biosynthesis and variation in the Nasonia genus (Buellesbach et al., 2022), the effects of individual genes on CHC profiles and, more importantly, on the encoded sexual signaling function, could not be determined as of yet.

In this study, we characterize the phenotypic effects of two fatty acid synthase genes whose knockdown impacts the variation of several structurally related CHC components concordant with female sexual attractiveness. CHC profiles of knockdown females primarily showed significant up- and downregulations of MB-alkane compounds with correlated branching patterns. At the same time, these knockdown females elicited significantly less courtship and copulation attempts from conspecific males. These constitute the first hymenopteran genes with a demonstrated function in governing the variation of primarily MB-alkanes as well as communication, hinting at a chemical coding pattern for sexual attractiveness mostly conveyed by this CHC compound class.

In our study, we shed light on how sexual attractiveness can be encoded by differentially branched CHCs and unravel its genetic architecture to be based on two highly similar fatty acid synthase genes. Knocking down these two genes in N. vitripennis females leads to a consistent pattern of primarily up- and downregulated MB-alkanes with specific branching patterns. This dramatic shift is accompanied by a significant reduction of courtship and copulation behavior toward knockdown females by conspecific males which we demonstrate to be mainly determined by the altered MB-alkane fraction. This advances our understanding of how genetic information is translated into chemical information and brings us a step closer in decoding biologically relevant information from complex chemical profiles.

Most conspicuously in females, quantities of CHCs with their first methyl groups at the 7th and 9th (and higher) C-atom positions are dramatically downregulated, whereas the ones with their first methyl groups at the respective 3rd and 5th positions appear mainly upregulated, in most cases by several orders of magnitude (Figure 2 and Supplementary file 3). Intriguingly, overall CHC quantities do not differ between knockdown and control individuals (Figure 2—figure supplement 3A), suggesting a very specific regulatory function for fas5/fas6 in governing these opposing and potentially compensatory branching patterns. Concordantly, this emphasizes the pivotal role of both these particular genes and the wild type MB-alkane branching patterns in encoding and maintaining the attractiveness of female N. vitripennis CHC profiles. Moreover, there appear to be a number of sex-specific differences in the knockdown effects on the different CHC compound classes, most notably apparent in significant upregulations of both total and n-alkane CHC quantities in knockdown males as opposed to females and partially differentially affected MB-alkane quantities (compare Figure 2 to Figure 2—figure supplement 4). Curiously, GFP dsRNAi appears to have a generally upregulating effect as opposed to WT controls despite for n-alkene quantities in males, which has not been reported before. However, a recent study hints at potential off-target effects of GFP dsRNAi on a small subset of N. vitripennis genes mainly involved in microtubule and sperm development (Rougeot et al., 2021). Interestingly, this appears to be also the case for the expressions of fas1 and fas2 gene transcripts, which are significantly upregulated in the GFP dsRNAi controls, again exclusively in males (Figure 2—figure supplement 1B). Though no off-target effects on CHC profile biosynthesis and variation have been reported so far for GFP dsRNAi, we cannot exclude this possibility, particularly since a trend toward higher CHC quantities in GFP dsRNAi controls as opposed to WT controls is also discernible in some cases for the females (Figure 2E–G). Therefore, as Rougeot et al. already hinted at , we strongly suggest alternative non-target controls in future studies on the genetics of CHC biosynthesis and variation. Concerning the partially different effects of fas5 on CHC quantities in females and males, as no functionality in chemical communication could be attributed to the latters’ CHC profiles so far, these could not be investigated any further. However, opposing sex-specific effects of CHC biosynthesis genes on the quantity of different compound classes appear to be a rather common occurrence, particularly in the insect model system D. melanogaster (Holze et al., 2021). Based mainly on research on the latter, CHC-based sexual signaling mechanisms have long been assumed to be comparatively simple, mainly mediated by two doubly unsaturated dienes in females and a mono-unsaturated n-alkene in males (Marcillac and Ferveur, 2004; Grillet et al., 2006). However, experimental evidence has accumulated that other CHC compounds can also complement sexual signaling in Drosophila in various ways (Chung et al., 2014; Everaerts et al., 2010). When regarding CHC profiles in their entirety as opposed to single compounds, direct causal links between sexual attractiveness and CHC profiles properties have rarely been experimentally demonstrated and have most often defied clear patterns (Würf et al., 2020; Xue et al., 2018). In the genus Nasonia and other related parasitoid wasp species, it has so far been assumed that sexual attractiveness is a trait attributed to their entire CHC profile as either present or absent depending on the studied species and also potentially reinforced by other factors such as polar cuticular compounds (Buellesbach et al., 2013; Mair et al., 2017). Our study clearly shows that CHC-mediated sexual attractiveness is primarily conveyed through a relatively complex chemical pattern with a comparatively simple genetic basis.

Interestingly, knockdown of fas5 also upregulated n-alkene quantities (Figure 2—figure supplement 3C), which have recently been shown to have a repellent effect on N. vitripennis males, preventing them from engaging in homosexual courtship behavior (Wang et al., 2022). However, our CHC compound class separation greatly reduced the increased proportion of n-alkenes in fas5 knockdown females to levels almost equivalent to those found in wild type females (0.94% and 0.91 %, respectively, in the extract of fas5 and WT MB fractions, compare Figure 4—source data 1 to Supplementary file 3). This renders the contribution of n-alkenes to the sharp reduction in sexual attractiveness in fas5 knockdown females unlikely and strongly suggests that the female sexual signaling function is mainly mediated by MB-alkanes (Figure 4). We argue that this CHC compound class indeed possesses the highest potential for encoding a wide variety of chemical information through the myriad of possible positions and numbers of methyl branches. In fact, a couple of studies have already hinted at MB-alkanes as the main carriers for chemical information in insect CHC profiles, providing evidence for the involvement of MB-alkanes in chemical communication processes (Spikes et al., 2010; Holman et al., 2016). This might be of particular importance in Hymenoptera, an insect order with CHC profiles largely dominated by MB-alkanes (Kather and Martin, 2015; Martin and Drijfhout, 2009) and in which both theoretical considerations and empirical evidence have accumulated for the increased complexity and high sophistication of their chemical communication systems (Saad et al., 2018; Obiero et al., 2021). Contrary to this, it has been argued that olefins (mainly n-alkenes and dienes) have a higher potential for encoding chemical information than MB-alkanes (Chung and Carroll, 2015). However, this view might have been biased and mainly informed by findings from Drosophila, where unsaturated compounds appear to be, in fact, the main mediators of sexual communication (Marcillac and Ferveur, 2004; Ferveur, 2005). In direct comparison, MB-alkanes constitute the dominant fraction in Nasonia CHC profiles (>85%) (Buellesbach et al., 2022) as opposed to Drosophila profiles (16–24%) (Dembeck et al., 2015). Since the split between Hymenoptera and other holometabolous insects including Diptera has been estimated to have occurred 327 mya (Misof et al., 2014), fundamental shifts in basic properties of both surface profile compositions and chemical signaling functionalities might be expected. Therefore, the promising role of MB-alkanes in conveying chemical information should be investigated more prominently in future studies. Through a wider evolutionary lens, it will be interesting to investigate how the present findings compare to other CHC-based communication systems in the vastly diverse insect order Hymenoptera. Notably, whereas in solitary Hymenoptera CHCs can function as contact sex pheromones, they are prominent and fundamental nestmate and caste recognition cues in eusocial Hymenoptera (Buellesbach et al., 2018b; Vereecken et al., 2007; Leonhardt et al., 2016). However, the main encoding mechanisms as well as the underlying genetic basis still remain largely elusive in most taxa, and our study constitutes an important stepping stone for investigating potential similarities in these important communication modalities in a larger evolutionary context. Moreover, since CHC profiles can be highly species-specific, their potential involvement in species recognition and assortative mating warrants further investigation to elucidate the exact underlying chemical and genetic differentiation mechanisms (Buellesbach et al., 2018b; Wurdack et al., 2015; Weiss et al., 2015).

Concerning gene orthology, fas5 has been annotated as a homolog to FASN3 in D. melanogaster (Buellesbach et al., 2022; Lammers et al., 2019). Interestingly, knockdowns of FASN3 alone do not induce any compound changes in D. melanogaster CHC profiles but increase the flies’ sensitivity to desiccation (Wicker-Thomas et al., 2015). A FASN3 ortholog expressed in the kissing bug Rhodinius prolixus also contributes to desiccation resistance, but simultaneously downregulates MB- while upregulating straight-chain alkanes (Moriconi et al., 2019). Similarly, in the migratory locust Locusta migratoria, silencing two FAS genes decreased insect survival under desiccation stress while altering the amounts of both MB- and straight-chain alkanes (Yang et al., 2020). In contrast to these studies, we were not able confirm any functionality in desiccation resistance for fas5 in both N. vitripennis males and females (Figure 5), nor did the previous studies report any impact on CHC-based chemical signaling. There are two additional FAS genes characterized in D. melanogaster: FASN1, which is responsible for overall CHC production with no specific effects on any particular compound classes (Wicker-Thomas et al., 2015) and FASN2, which exclusively regulates MB-alkane production in males (Chung et al., 2014). Although not a direct homolog, the main impact on MB-alkane variations of FASN2 in D. melanogaster is comparable to that of fas5 in N. vitripennis, with the exception that we were able to document effects on this compound class for both sexes (compare Figure 2 and Figure 2—figure supplement 4). Such diversified functionalities of the FAS genes characterized so far indicate their high versatility as early mediators in the CHC biosynthesis pathway (Figure 1). FAS genes have been implied as instrumental for generating the huge diversity of different CHC profiles across insects, with high evolutionary turnover rates and differences in the specific functional recruitments of FAS gene family members (Finck et al., 2016a; Moriconi et al., 2019). However, FAS genes are far from restricted to impact CHC biosynthesis alone and have been documented to be involved in a wide variety of other physiological processes, ranging from lipogenesis to diapause induction (Tan et al., 2017; Lammers et al., 2019; Moriconi et al., 2019). Therefore, specifically predicting functionalities of FAS genes and unambiguously associating them with CHC biosynthesis and variation has been notoriously difficult (Holze et al., 2021). Our study suggests a very specific affinity of fas5 for particular methyl-branching patterns, potentially originating in an enzymatic preference for methyl-malonyl-CoA predecessors with pre-existing branching patterns (Blomquist and Bagnères, 2010; Nelson and Blomquist, 1995). To clarify this, future studies should determine whether the fas5 gene product constitutes a soluble cytosolic or membrane-bound microsomal FAS enzyme, the latter of which has been postulated to be specific for the incorporation of methyl-malonyl-CoA subunits (Gu et al., 1997; Juárez et al., 1992, Figure 1).

The previously uncharacterized fas gene fas6 not only shows high sequence similarity to fas5,but is also its physical neighbor (Figure 2—figure supplement 2). Moreover, both of these genes show similarly high expression patterns in wild type N. vitripennis wasps (Figure 2H and Figure 2—figure supplement 4I). This basically implies that these two genes originated from a tandem gene duplication event and therefore constitute paralogs of the D. melanogaster FASN3. Hence, we cannot state at this point whether either one of these two genes alone or both are responsible for the observed phenotypic changes. Interestingly, this mirrors a similar finding in N. vitripennis males concerning the biosynthesis of a lactone functioning in a long-range pheromonal blend attractive to virgin females (Niehuis et al., 2013). The production of this compound was unambiguously attributed to three short-chain dehydrogenase/reductase genes with high sequence similarity, which could also not be targeted independently by dsRNAi knockdowns. This demonstrates the difficulty to narrow down specific genes responsible for the biosynthesis of pheromonally active compounds when their sequences show particularly high degrees of similarity and simultaneously high expression patterns.

In conclusion, our study demonstrates the considerable impact of two highly similar fatty acid synthase genes on female sexual attractiveness in a parasitoid wasp, thereby immediately suggesting how this trait can be encoded through specific patterns of MB-alkanes. To the best of our knowledge, these are the first identified hymenopteran genes with a specific effect on MB-alkane ratios that simultaneously impact sexual attractiveness. Transcending the demonstrated impact on sexual signaling and elicited mating behavior, the present findings also substantially advance our general knowledge on the so far little investigated genetic underpinnings of MB-alkane variation and production. This particular compound class dominates the surface profiles of many insects, most notably in the ecologically and economically important insect order Hymenoptera, and harbors a considerable potential for encoding chemical information, inviting a stronger emphasis on these compounds in future studies on chemical signaling and its behavioral impact.

The datasets generated or analyzed during this study are available at the figshare data repository under https://doi.org/10.6084/m9.figshare.20411958.

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Author details

1. Weizhao Sun

   Institute for Evolution & Biodiversity, University of Münster, Hüfferstr, Münster, Germany

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2781-1228

2. Michelle Ina Lange

   Institute for Evolution & Biodiversity, University of Münster, Hüfferstr, Münster, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Jürgen Gadau

   Institute for Evolution & Biodiversity, University of Münster, Hüfferstr, Münster, Germany

   Contribution

   Resources, Supervision, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

4. Jan Buellesbach

   Institute for Evolution & Biodiversity, University of Münster, Hüfferstr, Münster, Germany

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   buellesb@uni-muenster.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8493-692X

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