Development and Competition of Three Parasitoid Wasps, Brachymeria podagrica, Dirhinus himalayanus, and Nasonia vitripennis, in Their Host, Sarcophaga dux, in Single and Mixed Infections.

Laboratory trials were carried out to investigate the development of three entomophagous parasitoid wasps in preimaginal stages of Sarcophaga dux in monoinfections and mixed infections. Laboratory-raised postfeeding S. dux third-stage larvae were exposed to Brachymeria podagrica. After pupation, 50 of these fly puparia were brought in contact with pupal parasitoid Dirhinus himalayanus and 50 with Nasonia vitripennis, and the remaining 50 puparia were left as Brachymeria monoinfection. In three further trials, each set of 50 freshly pupated host puparia from the same source was exposed to N. vitripennis and D. himalayanus, as monoinfections and mixed infections, respectively. The uninfected control group consisted of 50 S. dux larvae that were kept separately under the same conditions. The percentages of successfully developed B. podagrica and D. himalayanus in monoinfections were 56 and 86%, respectively, and progeny of N. vitripennis hatched from 88% of the exposed host puparia. In mixed infections, N. vitripennis dominated over B. podagrica and D. himalayanus with rates of successfully infected hosts of 50 and 94%, respectively. The number of Nasonia progeny in these groups ranged from 4 to 49 and 5 to 43, respectively. Dirhinus himalayanus did not develop in the simultaneous infection with N. vitripennis. Not a single S. dux eclosed in the six experimental groups, while in the uninfected control group, 46 (92%) adult flies eclosed 11 to 14 days after the start of pupation. Since the three parasitoids emerge from flesh fly pupae, these insects can become important in criminal forensic investigations when corpses are in an advanced stage of decay. More data on their preimaginal development at different temperatures are necessary.

Direct Parental (DIPA) CRISPR in the jewel wasp, Nasonia vitripennis.

While clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology has demonstrated remarkable promise as a gene-editing tool, its application in certain insects, such as the jewel wasp, Nasonia vitripennis, has been hindered by a lack of a tractable method for reagent delivery. Direct Parental (DIPA-) CRISPR recently emerged as a facile way to induce gene lesions because it involves adult injection with commercially available Cas9-sgRNA with no helper reagent. However, DIPA-CRISPR has so far been tested in only a few insects. Here, we have assessed the amenability of DIPA-CRISPR in N. vitripennis by targeting two eye pigmentation genes, cinnabar and vermilion, which function in the ommochrome pathway. Successful generation of lesions in both genes demonstrated the functionality of DIPA-CRISPR in N. vitripennis and its potential application to other genes, thereby expanding the range of insects suitable for this method. We varied two parameters, Cas9-sgRNA concentration and injection volume, to determine optimal injection conditions. We found that the larger injection volume coupled with either higher or lower reagent concentration was needed for consistent mutation production. However, DIPA-CRISPR yields an overall low mutation rate in N. vitripennis when compared to other tested insects, a characteristic that may be attributed to a proportionally low vitellogenic import efficiency in the jewel wasp. We discuss different factors that may be considered in determining when DIPA-CRISPR may be preferable over other reagent delivery methods.

Ecological determinants of prevalence of the male-killing bacterium Arsenophonus nasoniae.

Male-killing bacteria are found in a broad range of arthropods. Arsenophonus nasoniae is a male-killing bacterium, causing a 80% reduction of the male progeny in infected Nasonia vitripennis wasps. Although the discovery of A. nasoniae dates from the early 80's, knowledge about the biology and ecology of this endosymbiont is still scarce. One of these poorly studied features is the ecological factors underlying A. nasoniae incidence on its Nasonia spp. hosts in different geographical locations. Here, we studied the prevalence of A. nasoniae in Iberian wild populations of its host N. vitripennis. This wasp species is a common parasitoid of the blowfly Protocalliphora azurea pupae, which in turn is a parasite of hole-nesting birds, such as the blue tit (Cyanistes caeruleus). We also examined the effects of bird rearing conditions on the prevalence of A. nasoniae through a brood size manipulation experiment (creating enlarged, control and reduced broods). Both the wasp and bacterium presence were tested through PCR assays in blowfly pupae. We found A. nasoniae in almost half (47%) of nests containing blowflies parasitized by N. vitripennis. The prevalence of A. nasoniae was similar in the two geographical areas examined (central Portugal and southeastern Spain) and the probability of infection by A. nasoniae was independent of the number of blowfly pupae in the nest. Experimental manipulation of brood size did not affect the prevalence of A. nasoniae nor the prevalence of its host, N. vitripennis. These results suggest that the incidence of A. nasoniae in natural populations of N. vitripennis is high in the Iberian Peninsula, and the infestation frequency of nests by N. vitripennis carrying A. nasoniae is spatially stable in this geographical region independently of bird rearing conditions.

PSRs: Selfish chromosomes that manipulate reproductive development.

B chromosomes are intriguing "selfish" genetic elements, many of which exhibit higher-than-Mendelian transmission. This perspective highlights a group of B chromosomes known as Paternal Sex Ratio chromosomes (PSRs), which are found in several insects with haplo-diploid reproduction. PSRs harshly alter the organism's reproduction to facilitate their own inheritance. A manifestation of this effect is the conversion of female destined individuals into males. Key to this conversion is the mysterious ability of PSRs to cause elimination of the sperm-inherited half of the genome during zygote formation. Here we discuss how PSRs were discovered, what is known about how they alter paternal chromatin dynamics to cause sex conversion, and how PSR-induced genome elimination is different from other forms of programmed genome elimination in different insects. PSRs also stand out because their DNA sequence compositions differ in remarkable ways from their insect's essential chromosomes, a characteristic suggestive of interspecies origins. Broadly, we also highlight poorly understood aspects of PSR dynamics that need to be investigated.

Genome sequence of Staphylococcus nepalensis ZZ-2023a, isolated from Nasonia vitripennis.

We isolated a strain of Staphylococcus nepalensis from Nasonia vitripennis and presented the draft genome sequence of this strain. This research was conducted at the Institute of Zoology, Chinese Academy of Sciences (Beijing, China). The genome spans 2,910,033 bp, distributed over 144 contigs, with a G+C content of 33.33%.

Neofunctionalization of Toll Signaling in Insects: From Immunity to Dorsoventral Patterning.

Toll signaling plays a crucial role in pathogen defense throughout the animal kingdom. It was discovered, however, for its function in dorsoventral (DV) axis formation in Drosophila. In all other insects studied so far, but not outside the insects, Toll is also required for DV patterning. However, in insects more distantly related to Drosophila, Toll's patterning role is frequently reduced and substituted by an expanded influence of BMP signaling, the pathway implicated in DV axis formation in all major metazoan lineages. This suggests that Toll was integrated into an ancestral BMP-based patterning system at the base of the insects or during insect evolution. The observation that Toll signaling has an immune function in the extraembryonic serosa, an early differentiating tissue of most insect embryos, suggests a scenario of how Toll was co-opted from an ancestral immune function for its new role in axis formation.

Time measurement in insect photoperiodism: external and internal coincidence.

The identity and nature of the photoperiodic photoreceptors are now quite well known, as is the nature of the endocrine regulation of the resulting diapauses. The central problem of time measurement-how the photoperiodic clock differentiates long from short days-however, is still obscure, known only from whole-animal experiments and abstract models, although it is clearly a function of the insect circadian system. This review describes some of these experiments in terms of oscillator entrainment and two widely applicable photoperiodic clock models, external and internal coincidence, mainly using data from experiments on flesh flies (Sarcophaga spp) and the parasitic wasp, Nasonia vitripennis.

Decoding the genetic and chemical basis of sexual attractiveness in parasitic wasps.

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 and that simultaneously impact sexual attractiveness and complex chemical surface profiles in parasitic wasps. Gene knockdown 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 mating response. Intriguingly, this suggests a potential coding mechanism for sexual attractiveness mediated by specific methyl-branching patterns in complex cuticular hydrocarbon (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 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.

Sublethal Effects of Four Insecticides Targeting Cholinergic Neurons on Partner and Host Finding in the Parasitic Wasp Nasonia vitripennis.

Lethal and sublethal effects of pesticides on nontarget organisms are one of the causes of the current decline of many insect species. However, research in the past decades has focused primarily on pollinators, although other beneficial nontarget organisms such as parasitic wasps may also be affected. We studied the sublethal effects of the four insecticides acetamiprid, dimethoate, flupyradifurone, and sulfoxaflor on pheromone-mediated sexual communication and olfactory host finding of the parasitic wasp Nasonia vitripennis. All agents target cholinergic neurons, which are involved in the processing of chemical information by insects. We applied insecticide doses topically and tested the response of treated wasps to sex pheromones and host-associated chemical cues. In addition, we investigated the mating rate of insecticide-treated wasps. The pheromone response of females surviving insecticide treatment was disrupted by acetamiprid (≥0.63 ng), dimethoate (≥0.105 ng), and flupyradifurone (≥21 ng), whereas sulfoxaflor had no significant effects at the tested doses. Olfactory host finding was affected by all insecticides (acetamiprid ≥1.05 ng, dimethoate ≥0.105 ng, flupyradifurone ≥5.25 ng, sulfoxaflor ≥0.52 ng). Remarkably, females treated with ≥0.21 ng dimethoate even avoided host odor. The mating rate of treated N. vitripennis couples was decreased by acetamiprid (6.3 ng), flupyradifurone (≥2.63 ng), and sulfoxaflor (2.63 ng), whereas dimethoate showed only minor effects. Finally, we determined the amount of artificial nectar consumed by N. vitripennis females within 48 h. Considering this amount (∼2 µL) and the maximum concentrations of the insecticides reported in nectar, tested doses can be considered field-realistic. Our results suggest that exposure of parasitic wasps to field-realistic doses of insecticides targeting the cholinergic system reduces their effectiveness as natural enemies by impairing the olfactory sense. Environ Toxicol Chem 2023;42:2400-2411. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The son-killer microbe Arsenophonus nasoniae is a widespread associate of the parasitic wasp Nasonia vitripennis in Europe.

Heritable microbes that exhibit reproductive parasitism are common in insects. One class of these are the male-killing bacteria, which are found in a broad range of insect hosts. Commonly, our knowledge of the incidence of these microbes is based on one or a few sampling sites, and the degree and causes of spatial variation are unclear. In this paper, we examine the incidence of the son-killer microbe Arsenophonus nasoniae across European populations of its wasp host, Nasonia vitripennis. In preliminary work, we noticed two female N. vitripennis producing highly female biased sex ratios in a field study from the Netherlands and Germany. When tested, the brood from Germany was revealed to be infected with A. nasoniae. We then completed a broad survey in 2012, in which fly pupal hosts of N. vitripennis were collected from vacated birds' nests from four European populations, N. vitripennis wasps allowed to emerge and then tested for A. nasoniae presence through PCR assay. We then developed a new screening methodology based on direct PCR assays of fly pupae and applied this to ethanol-preserved material collected from great tit (Parus major) nests in Portugal. These data show A. nasoniae is found widely in European N. vitripennis, being present in Germany, the UK, Finland, Switzerland and Portugal. Samples varied in the frequency with which they carry A. nasoniae, from being rare to being present in 50% of the pupae parasitised by N. vitripennis. Direct screening of ethanol-preserved fly pupae was an effective method for revealing both wasp and A. nasoniae infection, and will facilitate sample transport across national boundaries. Future research should examine the causes of variation in frequency, in particular testing the hypothesis that N. vitripennis superparasitism rates drive the variation in A. nasoniae frequency through providing opportunities for infectious transmission.

Laboratory Evaluation of Pupal Parasitoids for Control of the Cornsilk Fly Species, Chaetopsis massyla and Euxesta eluta.

Cornsilk flies are serious pests of sweet corn through damage to cobs and secondary fungal establishment. As pupae are generally outside the infested cob on the ground, there can be potential for use of pupal parasitoids for control. Two species of gregarious parasitoids, Muscidifurax raptorellus and Nasonia vitripennis, and three species of solitary parasitoids, Spalangia endius, Spalangia cameroni and Muscidifurax raptor, were evaluated against pupae of the two cornsilk fly species, Euxesta eluta and Chaetopsis massyla. House fly pupae, the most common host for most of the parasitoids, were included for comparison. All of the parasitoids killed and successfully parasitized pupae of the two cornsilk fly species at rates that were similar to house fly pupae. Adult parasitoids that emerged from cornsilk fly hosts were somewhat smaller than parasitoids reared from house flies and had proportionally fewer females. These parasitoids, which are widely and commercially available for filth fly control, warrant further consideration for their potential against cornsilk flies in the field.

Bacterial supergroup-specific "cost" of Wolbachia infections in Nasonia vitripennis.

The maternally inherited endosymbiont, Wolbachia, is known to alter the reproductive biology of its arthropod hosts for its own benefit and can induce both positive and negative fitness effects in many hosts. Here, we describe the effects of the maintenance of two distinct Wolbachia infections, one each from supergroups A and B, on the parasitoid host Nasonia vitripennis. We compare the effect of Wolbachia infections on various traits between the uninfected, single A-infected, single B-infected, and double-infected lines with their cured versions. Contrary to some previous reports, our results suggest that there is a significant cost associated with the maintenance of Wolbachia infections where traits such as family size, fecundity, longevity, and rates of male copulation are compromised in Wolbachia-infected lines. The double Wolbachia infection has the most detrimental impact on the host as compared to single infections. Moreover, there is a supergroup-specific negative impact on these wasps as the supergroup B infection elicits the most pronounced negative effects. These negative effects can be attributed to a higher Wolbachia titer seen in the double and the single supergroup B infection lines when compared to supergroup A. Our findings raise important questions on the mechanism of survival and maintenance of these reproductive parasites in arthropod hosts.

Genetic and genomic architecture of species-specific cuticular hydrocarbon variation in parasitoid wasps.

Cuticular hydrocarbons (CHCs) serve two fundamental functions in insects: protection against desiccation and chemical signalling. How the interaction of genes shapes CHC profiles, which are essential for insect survival, adaptation and reproductive success, is still poorly understood. Here we investigate the genetic and genomic basis of CHC biosynthesis and variation in parasitoid wasps of the genus Nasonia. We mapped 91 quantitative trait loci (QTL) explaining the variation of a total of 43 CHCs in F2 hybrid males from interspecific crosses between three Nasonia species. To identify candidate genes, we localized orthologues of CHC biosynthesis-related genes in the Nasonia genomes. We discovered multiple genomic regions where the location of QTL coincides with the location of CHC biosynthesis-related candidate genes. Most conspicuously, on a region close to the centromere of chromosome 1, multiple CHC biosynthesis-related candidate genes co-localize with several QTL explaining variation in methyl-branched alkanes. The genetic underpinnings behind this compound class are not well understood so far, despite their high potential for encoding chemical information as well as their prevalence in hymenopteran CHC profiles. Our study considerably extends our knowledge on the genetic architecture governing this important compound class, establishing a model for methyl-branched alkane genetics in the Hymenoptera in general.

The biological significance of lipogenesis in Nasonia vitripennis.

Parasitic wasps have long been thought to be unable to synthesize fatty acids de novo, but recent 13C-labelling studies have challenged this view. It remained unclear, however, whether the reported biosynthesis rates are of biological relevance. Here, we show in Nasonia vitripennis that ageing females with partly depleted lipid reserves produce biologically relevant amounts of fatty acids de novo. Females with varying oviposition history (0-48 h) prior to feeding 20% 13C-labelled glucose solution showed 13C-incorporation rates of (mean ± SEM) 30 ± 2%, 50 ± 2%, 49 ± 3% and 21 ± 2% in palmitic, stearic, oleic and linoleic acid, respectively. The absolute amounts of fatty acids synthesized de novo across treatments corresponded to 28 ± 3 egg lipid equivalents. Females incorporated de novo synthesized fatty acids into their eggs, and glucose-fed females laid more eggs than water-fed control females. The number of eggs laid prior to glucose feeding did not correlate with the degree of lipogenesis, but the amounts of de novo synthesized fatty acids correlated with constitutive (not synthesized de novo) fatty acids. Hence, glucose feeding has a twofold effect on the fatty acid status of N. vitripennis females by decelerating the catabolism of existing fat reserves and partially replenishing ebbing fat reserves by lipogenesis.

Juvenile hormone as a causal factor for maternal regulation of diapause in a wasp.

Most temperate multivoltine insects enter diapause, a hormonally controlled developmental suspension, in response to seasonal photoperiodic and/or thermal cues. Some insect species exhibit maternal regulation of diapause in which developmental trajectories of the offspring are determined by mothers in response to environmental cues that the mother received. Although maternally regulated diapause is common among insects, the maternal endocrinological mechanisms are largely veiled. To approach this issue, we used the jewel wasp Nasonia vitripennis, which produces non-diapause-destined offspring under long days and diapause-destined offspring under short days or low temperatures. Comparative transcriptomics of these wasps revealed possible involvement of the juvenile hormone (JH) biosynthetic cascade in maternal diapause regulation. The expression of juvenile hormone acid O-methyltransferase (jhamt) was typically downregulated in short-day wasps, and this was reflected by a reduction in haemolymph JH concentrations. RNAi targeted at jhamt reduced haemolymph JH concentration and induced wasps to produce diapause-destined offspring even under long days. In addition, topical application of JH suppressed the production of diapause-destined offspring under short days or low temperatures. These results indicate that diapause in N. vitripennis is determined by maternal jhamt expression and haemolymph JH concentration in response to day length. We therefore report a novel role for JH in insect seasonality.

The Nasonia pair-rule gene regulatory network retains its function over 300 million years of evolution.

Insect segmentation is a well-studied and tractable system with which to investigate the genetic regulation of development. Though insects segment their germband using a variety of methods, modelling work implies that a single gene regulatory network can underpin the two main types of insect segmentation. This means limited genetic changes are required to explain significant differences in segmentation mode between different insects. This idea needs to be tested in a wider variety of species, and the nature of the gene regulatory network (GRN) underlying this model has not been tested. Some insects, e.g. Nasonia vitripennis and Apis mellifera segment progressively, a pattern not examined in previous studies of this segmentation model, producing stripes at different times progressively through the embryo, but not from a segment addition zone. Here, we aim to understand the GRNs patterning Nasonia using a simulation-based approach. We found that an existing model of Drosophila segmentation ( Clark, 2017) can be used to recapitulate the progressive segmentation of Nasonia, if provided with altered inputs in the form of expression of the timer genes Nv-caudal and Nv-odd paired. We predict limited topological changes to the pair-rule network and show, by RNAi knockdown, that Nv-odd paired is required for morphological segmentation. Together this implies that very limited changes to the Drosophila network are required to simulate Nasonia segmentation, despite significant differences in segmentation modes, implying that Nasonia use a very similar version of an ancestral GRN used by Drosophila, which must therefore have been conserved for at least 300 million years.

Expression and Function of Toll Pathway Components in the Early Development of the Wasp Nasonia vitripennis.

The Toll signaling pathway is the main source of embryonic DV polarity in the fly Drosophila melanogaster. This pathway appears to have been co-opted from an ancestral innate immunity system within the insects and has been deployed in different ways among insect taxa. Here we report the expression and function of homologs of the important components of the D. melanogaster Toll pathway in the wasp Nasonia vitripennis. We found homologs for all the components; many components had one or more additional paralogs in the wasp relative the fly. We also found significant deviations in expression patterns of N. vitripennis homologs. Finally, we provide some preliminary functional analyses of the N. vitripennis homologs, where we find a mixture of conservation and divergence of function.

Males of the parasitoid wasp, Nasonia vitripennis, can identify which fly hosts contain females.

The reproductive success of a male is limited by the number of females it can mate with. Thus, males deploy elaborate strategies to maximize access to females. In Nasonia, which are parasitoids of cyclorrhaphous flies, such reproductive strategies are thought to be restricted to competition among males for access to females in the natal patch. This study investigates whether additional strategies are present, especially the capability to identify which fly hosts contain adult females inside. Behavioural assays revealed that only one out of the four species, N. vitripennis, can distinguish which hosts specifically have adult female wasps, indicating a species-specific reproductive strategy. Results of gas chromatography-mass spectrometry analyses and behavioural data suggest that female-signature cuticular hydrocarbons (CHCs) are used as chemical cues, possibly emanating from within the host puparium. Further assays indicated that N. vitripennis males can also detect differences in the intensities of female-signature CHCs, giving them the capability to seek out hosts with maximum number of females. This study uncovers a previously unknown reproductive strategy in one of the most widely studied parasitoid wasps.

Doublesex regulates male-specific differentiation during distinct developmental time windows in a parasitoid wasp.

Sexually dimorphic traits in insects are subject to sexual selection, but our knowledge of the underlying molecular mechanisms is still scarce. Here we investigate how the highly conserved gene, Doublesex (Dsx), is involved in shaping sexual dimorphism in the model parasitoid wasp Nasonia vitripennis (Hymenoptera: Pteromalidae). First, we present the revised Dsx gene structure including an alternative transcription start, and two additional male NvDsx transcript isoforms. We show sex-specific NvDsx expression and splicing throughout development, and demonstrate that transient NvDsx silencing in different male developmental stages shifts two sexually dimorphic traits from male to female morphology, with the effect being dependent on the timing of silencing. In addition, we determined the effect of NvDsx on the development of reproductive organs. Transient silencing of NvDsx in early male larvae affects the growth and differentiation of the internal and external reproductive tissues. We did not observe phenotypic changes in females after NvDsx silencing. Our results indicate that male NvDsx is required to suppress female-specific traits and/or to promote male-specific traits during distinct developmental windows. This provides new insights into the regulatory activity of Dsx during male wasp development in the Hymenoptera.

Silencing Doublesex expression triggers three-level pheromonal feminization in Nasonia vitripennis males.

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 (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; 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.