A butterfly egg-killing hypersensitive response in Brassica nigra is controlled by a single locus, PEK, containing a cluster of TIR-NBS-LRR receptor genes.

Knowledge of plant recognition of insects is largely limited to a few resistance (R) genes against sap-sucking insects. Hypersensitive response (HR) characterizes monogenic plant traits relying on R genes in several pathosystems. HR-like cell death can be triggered by eggs of cabbage white butterflies (Pieris spp.), pests of cabbage crops (Brassica spp.), reducing egg survival and representing an effective plant resistance trait before feeding damage occurs. Here, we performed genetic mapping of HR-like cell death induced by Pieris brassicae eggs in the black mustard Brassica nigra (B. nigra). We show that HR-like cell death segregates as a Mendelian trait and identified a single dominant locus on chromosome B3, named PEK (Pieris  egg- killing). Eleven genes are located in an approximately 50 kb region, including a cluster of genes encoding intracellular TIR-NBS-LRR (TNL) receptor proteins. The PEK locus is highly polymorphic between the parental accessions of our mapping populations and among B. nigra reference genomes. Our study is the first one to identify a single locus potentially involved in HR-like cell death induced by insect eggs in B. nigra. Further fine-mapping, comparative genomics and validation of the PEK locus will shed light on the role of these TNL receptors in egg-killing HR.

Adaptive Plasticity of Insect Eggs in Response to Environmental Challenges.

Insect eggs are exposed to a plethora of abiotic and biotic threats. Their survival depends on both an innate developmental program and genetically determined protective traits provided by the parents. In addition, there is increasing evidence that (a) parents adjust the egg phenotype to the actual needs, (b) eggs themselves respond to environmental challenges, and (c) egg-associated microbes actively shape the egg phenotype. This review focuses on the phenotypic plasticity of insect eggs and their capability to adjust themselves to their environment. We outline the ways in which the interaction between egg and environment is two-way, with the environment shaping the egg phenotype but also with insect eggs affecting their environment. Specifically, insect eggs affect plant defenses, host biology (in the case of parasitoid eggs), and insect oviposition behavior. We aim to emphasize that the insect egg, although it is a sessile life stage, actively responds to and interacts with its environment.

Genetic analysis reveals three novel QTLs underpinning a butterfly egg-induced hypersensitive response-like cell death in Brassica rapa.

BACKGROUND: Cabbage white butterflies (Pieris spp.) can be severe pests of Brassica crops such as Chinese cabbage, Pak choi (Brassica rapa) or cabbages (B. oleracea). Eggs of Pieris spp. can induce a hypersensitive response-like (HR-like) cell death which reduces egg survival in the wild black mustard (B. nigra). Unravelling the genetic basis of this egg-killing trait in Brassica crops could improve crop resistance to herbivory, reducing major crop losses and pesticides use. Here we investigated the genetic architecture of a HR-like cell death induced by P. brassicae eggs in B. rapa. RESULTS: A germplasm screening of 56 B. rapa accessions, representing the genetic and geographical diversity of a B. rapa core collection, showed phenotypic variation for cell death. An image-based phenotyping protocol was developed to accurately measure size of HR-like cell death and was then used to identify two accessions that consistently showed weak (R-o-18) or strong cell death response (L58). Screening of 160 RILs derived from these two accessions resulted in three novel QTLs for Pieris brassicae-induced cell death on chromosomes A02 (Pbc1), A03 (Pbc2), and A06 (Pbc3). The three QTLs Pbc1-3 contain cell surface receptors, intracellular receptors and other genes involved in plant immunity processes, such as ROS accumulation and cell death formation. Synteny analysis with A. thaliana suggested that Pbc1 and Pbc2 are novel QTLs associated with this trait, while Pbc3 also contains an ortholog of LecRK-I.1, a gene of A. thaliana previously associated with cell death induced by a P. brassicae egg extract. CONCLUSIONS: This study provides the first genomic regions associated with the Pieris egg-induced HR-like cell death in a Brassica crop species. It is a step closer towards unravelling the genetic basis of an egg-killing crop resistance trait, paving the way for breeders to further fine-map and validate candidate genes.

Insect egg-killing: a new front on the evolutionary arms-race between brassicaceous plants and pierid butterflies.

Evolutionary arms-races between plants and insect herbivores have long been proposed to generate key innovations such as plant toxins and detoxification mechanisms that can drive diversification of the interacting species. A novel front-line of plant defence is the killing of herbivorous insect eggs. We test whether an egg-killing plant trait has an evolutionary basis in such a plant-insect arms-race. Within the crucifer family (Brassicaceae), some species express a hypersensitive response (HR)-like necrosis underneath butterfly eggs (Pieridae) that leads to eggs desiccating or falling off the plant. We studied the phylogenetic distribution of this trait, its egg-killing effect on and elicitation by butterflies, by screening 31 Brassicales species, and nine Pieridae species. We show a clade-specific induction of strong, egg-killing HR-like necrosis mainly in species of the Brassiceae tribe including Brassica crops and close relatives. The necrosis is strongly elicited by pierid butterflies that are specialists of crucifers. Furthermore, HR-like necrosis is linked to PR1 defence gene expression, accumulation of reactive oxygen species and cell death, eventually leading to egg-killing. Our findings suggest that the plants' egg-killing trait is a new front on the evolutionary arms-race between Brassicaceae and pierid butterflies beyond the well-studied plant toxins that have evolved against their caterpillars.

Symbiotic polydnavirus and venom reveal parasitoid to its hyperparasitoids.

Symbiotic relationships may provide organisms with key innovations that aid in the establishment of new niches. For example, during oviposition, some species of parasitoid wasps, whose larvae develop inside the bodies of other insects, inject polydnaviruses into their hosts. These symbiotic viruses disrupt host immune responses, allowing the parasitoid's progeny to survive. Here we show that symbiotic polydnaviruses also have a downside to the parasitoid's progeny by initiating a multitrophic chain of interactions that reveals the parasitoid larvae to their enemies. These enemies are hyperparasitoids that use the parasitoid progeny as host for their own offspring. We found that the virus and venom injected by the parasitoid during oviposition, but not the parasitoid progeny itself, affected hyperparasitoid attraction toward plant volatiles induced by feeding of parasitized caterpillars. We identified activity of virus-related genes in the caterpillar salivary gland. Moreover, the virus affected the activity of elicitors of salivary origin that induce plant responses to caterpillar feeding. The changes in caterpillar saliva were critical in inducing plant volatiles that are used by hyperparasitoids to locate parasitized caterpillars. Our results show that symbiotic organisms may be key drivers of multitrophic ecological interactions. We anticipate that this phenomenon is widespread in nature, because of the abundance of symbiotic microorganisms across trophic levels in ecological communities. Their role should be more prominently integrated in community ecology to understand organization of natural and managed ecosystems, as well as adaptations of individual organisms that are part of these communities.

Plant volatiles induced by herbivore eggs prime defences and mediate shifts in the reproductive strategy of receiving plants.

Plants can detect cues associated with the risk of future herbivory and modify defence phenotypes accordingly; however, our current understanding is limited both with respect to the range of early warning cues to which plants respond and the nature of the responses. Here we report that exposure to volatile emissions from plant tissues infested with herbivore eggs promotes stronger defence responses to subsequent herbivory in two Brassica species. Furthermore, exposure to these volatile cues elicited an apparent shift from growth to reproduction in Brassica nigra, with exposed plants exhibiting increased flower and seed production, but reduced leaf production, relative to unexposed controls. Our results thus document plant defence priming in response to a novel environmental cue, oviposition-induced plant volatiles, while also showing that plant responses to early warning cues can include changes in both defence and life-history traits.

Legacy of a Butterfly's Parental Microbiome in Offspring Performance.

An insect's phenotype can be influenced by the experiences of the parental generation. However, the effects of the parental symbiotic microbiome and host plant use on the offspring are unclear. We addressed this gap of knowledge by studying Pieris brassicae, a multivoltine butterfly species feeding on different brassicaceous plants across generations. We investigated how disturbance of the parental bacterial community by antibiotic treatment affects F1 larval traits. We tested the effects depending on whether F1 larvae are feeding on the same plant species as their parents or on a different one. The parental treatment alone had no impact on the biomass of F1 larvae feeding on the parental plant species. However, the parental treatment had a detrimental effect on F1 larval biomass when F1 larvae had a different host plant than their parents. This effect was linked to higher larval prophenoloxidase activity and greater downregulation of the major allergen gene (MA), a glucosinolate detoxification gene of P. brassicae Bacterial abundance in untreated adult parents was high, while it was very low in F1 larvae from either parental type, and thus unlikely to directly influence larval traits. Our results suggest that transgenerational effects of the parental microbiome on the offspring's phenotype become evident when the offspring is exposed to a transgenerational host plant shift.IMPORTANCE Resident bacterial communities are almost absent in larvae of butterflies and thus are unlikely to affect their host. In contrast, adult butterflies contain conspicuous amounts of bacteria. While the host plant and immune state of adult parental butterflies are known to affect offspring traits, it has been unclear whether also the parental microbiome imposes direct effects on the offspring. Here, we show that disturbance of the bacterial community in parental butterflies by an antibiotic treatment has a detrimental effect on those offspring larvae feeding on a different host plant than their parents. Hence, the study indicates that disturbance of an insect's parental microbiome by an antibiotic treatment shapes how the offspring individuals can adjust themselves to a novel host plant.

Plant responses to insect eggs are not induced by egg-associated microbes, but by a secretion attached to the eggs.

Plants can enhance their defence against herbivorous insects by responding to insect egg depositions preceding larval feeding. The similarity of plant responses to insect eggs with those to phytopathogens gave rise to the hypothesis that egg-associated microbes might act as elicitors. We tested this hypothesis by investigating first if elimination of microbes in the butterfly Pieris brassicae changes the responses of Brassica nigra and Arabidopsis thaliana to eggs and larvae of this insect species. An antibiotic treatment of butterflies mitigated the plant transcriptional response to the eggs and the egg-mediated enhancement of the plant's defence against larvae. However, application of cultivated microbial isolates from the eggs onto Arabidopsis thaliana did not enhance the plant's anti-herbivore defence. Instead, application of an egg-associated glandular secretion, which is attaching the eggs to the leaves, elicited the enhancing effect on the plant's defence against larvae. However, this effect was only achieved when the secretion was applied in similar quantities as released by control butterflies, but not when applied in the reduced quantity as released by antibiotic-treated butterflies. We conclude that glandular secretions rather than egg-associated microbes act in a dose-dependent manner as elicitor of the egg-mediated enhancement of the plant's defence against insect larvae.

Plant responses to butterfly oviposition partly explain preference-performance relationships on different brassicaceous species.

The preference-performance hypothesis (PPH) states that herbivorous female insects prefer to oviposit on those host plants that are best for their offspring. Yet, past attempts to show the adaptiveness of host selection decisions by herbivores often failed. Here, we tested the PPH by including often neglected oviposition-induced plant responses, and how they may affect both egg survival and larval weight. We used seven Brassicaceae species of which most are common hosts of two cabbage white butterfly species, the solitary Pieris rapae and gregarious P. brassicae. Brassicaceous species can respond to Pieris eggs with leaf necrosis, which can lower egg survival. Moreover, plant-mediated responses to eggs can affect larval performance. We show a positive correlation between P. brassicae preference and performance only when including the egg phase: 7-day-old caterpillars gained higher weight on those plant species which had received most eggs. Pieris eggs frequently induced necrosis in the tested plant species. Survival of clustered P. brassicae eggs was unaffected by the necrosis in most tested species and no relationship between P. brassicae egg survival and oviposition preference was found. Pieris rapae preferred to oviposit on plant species most frequently expressing necrosis although egg survival was lower on those plants. In contrast to the lower egg survival on plants expressing necrosis, larval biomass on these plants was higher than on plants without a necrosis. We conclude that egg survival is not a crucial factor for oviposition choices but rather egg-mediated responses affecting larval performance explained the preference-performance relationship of the two butterfly species.

Plant responses to insect egg deposition.

Plants can respond to insect egg deposition and thus resist attack by herbivorous insects from the beginning of the attack, egg deposition. We review ecological effects of plant responses to insect eggs and differentiate between egg-induced plant defenses that directly harm the eggs and indirect defenses that involve egg parasitoids. Furthermore, we discuss the ability of plants to take insect eggs as warning signals; the eggs indicate future larval feeding damage and trigger plant changes that either directly impair larval performance or attract enemies of the larvae. We address the questions of how egg-associated cues elicit plant defenses, how the information that eggs have been laid is transmitted within a plant, and which molecular and chemical plant responses are induced by egg deposition. Finally, we highlight evolutionary aspects of the interactions between plants and insect eggs and ask how the herbivorous insect copes with egg-induced plant defenses and may avoid them by counteradaptations.

Early herbivore alert matters: plant-mediated effects of egg deposition on higher trophic levels benefit plant fitness.

Induction of plant defences, specifically in response to herbivore attack, can save costs that would otherwise be needed to maintain defences even in the absence of herbivores. However, plants may suffer considerable damage during the time required to mount these defences against an attacker. This could be resolved if plants could respond to early cues, such as egg deposition, that reliably indicate future herbivory. We tested this hypothesis in a field experiment and found that egg deposition by the butterfly Pieris brassicae on black mustard (Brassica nigra) induced a plant response that negatively affected feeding caterpillars. The effect cascaded up to the third and fourth trophic levels (larval parasitoids and hyperparasitoids) by affecting the parasitisation rate and parasitoid performance. Overall, the defences induced by egg deposition had a positive effect on plant seed production and may therefore play an important role in the evolution of plant resistance to herbivores.

Attraction of egg-killing parasitoids toward induced plant volatiles in a multi-herbivore context.

In response to insect herbivory, plants emit volatile organic compounds which may act as indirect plant defenses by attracting natural enemies of the attacking herbivore. In nature, plants are often attacked by multiple herbivores, but the majority of studies which have investigated indirect plant defenses to date have focused on the recruitment of different parasitoid species in a single-herbivore context. Here, we report our investigation on the attraction of egg parasitoids of lepidopteran hosts (Trichogramma brassicae and T. evanescens) toward plant volatiles induced by different insect herbivores in olfactometer bioassays. We used a system consisting of a native crucifer, Brassica nigra, two naturally associated herbivores [the butterfly Pieris brassicae (eggs and caterpillars) and the aphid Brevicoryne brassicae] and an alien invasive herbivore (eggs and caterpillars of the moth Spodoptera exigua). We found that Trichogramma wasps were attracted by volatiles induced in the plants by P. brassicae eggs, but not by those induced in the plants by S. exigua eggs, indicating the specificity of the plant responses toward lepidopteran herbivores. The results of the chemical analysis revealed significant differences between the volatile blends emitted by plants in response to attack by P. brassicae and S. exigua eggs which were in agreement with the behavioural observations. We investigated the attraction of Trichogramma wasps toward P. brassicae egg-induced volatiles in plants simultaneously attacked by larvae and nymphs of different non-hosts. The two chewing caterpillars P. brassicae and S. exigua, but not the phloem-feeding aphid B. brassicae, can disrupt the attraction of Trichogramma species toward P. brassicae egg-induced volatiles. Indirect plant defenses are discussed in the context of multiple herbivory by evaluating the importance of origin, dietary specialization and feeding guild of different attackers on the recruitment of egg-killing parasitoids.

To be in time: egg deposition enhances plant-mediated detection of young caterpillars by parasitoids.

Animals use information from their environment while foraging for food or prey. When parasitic wasps forage for hosts, they use plant volatiles induced by herbivore activities such as feeding and oviposition. Little information is available on how wasps exploit specific plant volatiles over time, and which compounds indicate changes in host quality. In experiments investigating the role of herbivore-induced plant volatiles in wasp foraging, induction of plant response is usually achieved by placing larvae on clean plants instead of allowing the natural sequence of events: to let eggs deposited by the herbivore develop into larvae. We compared the attraction of the parasitoid Cotesia glomerata to volatiles emitted by black mustard (Brassica nigra) plants induced by eggs and successive larval stages of the Large Cabbage White butterfly (Pieris brassicae) to the attraction of this parasitoid to black mustard plant volatiles induced only by larval feeding in a wind tunnel setup. We show that wasps are attracted to plants infested with eggs just before and shortly after larval hatching. However, wasp preference changed at later time points towards plants induced only by larval feeding. These temporal changes in parasitoid attraction matched with changes in the chemical compositions of the blends of plant volatiles. Previous studies have shown that host quality/suitability decreases with caterpillar age and that P. brassicae oviposition induces plant defences that negatively affect subsequently feeding caterpillars. We investigated parasitoid performance in hosts of different ages. Wasp performance was positively correlated with preference. Moreover, parasitism success decreased with time and host stage. In conclusion, the behaviour of Cotesia glomerata is fine-tuned to exploit volatiles induced by eggs and early host stages that benefit parasitoid fitness.

Synergistic effects of direct and indirect defences on herbivore egg survival in a wild crucifer.

Evolutionary theory of plant defences against herbivores predicts a trade-off between direct (anti-herbivore traits) and indirect defences (attraction of carnivores) when carnivore fitness is reduced. Such a trade-off is expected in plant species that kill herbivore eggs by exhibiting a hypersensitive response (HR)-like necrosis, which should then negatively affect carnivores. We used the black mustard (Brassica nigra) to investigate how this potentially lethal direct trait affects preferences and/or performances of specialist cabbage white butterflies (Pieris spp.), and their natural enemies, tiny egg parasitoid wasps (Trichogramma spp.). Both within and between black mustard populations, we observed variation in the expression of Pieris egg-induced HR. Butterfly eggs on plants with HR-like necrosis suffered lower hatching rates and higher parasitism than eggs that did not induce the trait. In addition, Trichogramma wasps were attracted to volatiles of egg-induced plants that also expressed HR, and this attraction depended on the Trichogramma strain used. Consequently, HR did not have a negative effect on egg parasitoid survival. We conclude that even within a system where plants deploy lethal direct defences, such defences may still act with indirect defences in a synergistic manner to reduce herbivore pressure.

Oviposition strategies in Pieridae butterflies and the role of an egg-killing plant trait therein.

Most herbivorous insects are host-plant specialists that evolved detoxification mechanisms to overcome their host plant's toxins. In the evolutionary arms-races between Pieridae butterflies and Brassicaceae plants, some plant species have evolved another defence against the pierids: egg-killing. Underneath the eggs, leaves develop a so-called hypersensitive response (HR)-like cell death. Whether some butterflies have evolved oviposition strategies to counter-adapt against egg-killing remains to be studied. In this study, we assessed the oviposition site location of Pieridae butterflies on their natural host plants. We described the plant tissue on which we located the eggs of the most common Pieridae in the Netherlands: Gonepteryx rhamni, Anthocharis cardamines, Pieris rapae, P. napi, P. brassicae and P. mannii. Additionally, we assessed expression of HR-like cell death in response to the deposited butterfly eggs. We found that both A. cardamines and G. rhamni mainly oviposited on the floral stem and the branch, respectively, and oviposited on host plants from lineages not expected to kill pierid eggs. Accordingly, no HR-like cell death was seen. All Pieris eggs found were located on leaves of their host, the only tissue found to express HR-like cell death. Furthermore, each Pieris species was found to at least occasionally oviposit on Brassica nigra. This was the only plant species in this survey that expressed HR-like cell death in response to the eggs of P. rapae, P. napi and P. brassicae. Our observations demonstrate that HR-like cell death remains an effective defence strategy against these Pieris species and as such did not find evidence for the hypothesized counterstrategies. Surveying certain key species and disentangling the micro-evolution of oviposition strategies within a species would allow us to further investigate potential counter-adaptations that evolved against HR-like cell death. This study provides the basis for further investigation of potential counter-adaptations to egg-killing defences.

Bridging biotremology and chemical ecology: a new terminology.

Living organisms use both chemical and mechanical stimuli to survive in their environment. Substrate-borne vibrations play a significant role in mediating behaviors in animals and inducing physiological responses in plants, leading to the emergence of the discipline of biotremology. Biotremology is experiencing rapid growth both in fundamental research and in applications like pest control, drawing attention from diverse audiences. As parallels with concepts and approaches in chemical ecology emerge, there is a pressing need for a shared standardized vocabulary in the area of overlap for mutual understanding. In this article, we propose an updated set of terms in biotremology rooted in chemical ecology, using the suffix '-done' derived from the classic Greek word 'δονέω' (pronounced 'doneo'), meaning 'to shake'.

Phenotypic plasticity of plant response to herbivore eggs: effects on resistance to caterpillars and plant development.

Herbivory induces direct resistance responses in plants that negatively affect subsequently colonizing herbivores. Moreover, eggs of herbivorous insects can also activate plant resistance, which in some cases prevents hatching larvae from feeding. Until now, plant-mediated effects of eggs on subsequent herbivory, and the specificity of such responses, have remained poorly understood. We studied the specificity and effects of plant resistance induced by herbivore egg deposition against lepidopteran larvae of species with different dietary breadths, feeding on a wild annual plant, the crucifer Brassica nigra. We examined whether this plant-mediated response affects the growth of caterpillars of a specialist (Pieris brassicae) that feeds on B. nigra leaves and flowers, and a generalist (Mamestra brassicae) that rarely attacks this wild crucifer. We measured growth rates of neonate larvae to the end of their second instar after the larvae had hatched on plants exposed to eggs vs. plants without eggs, under laboratory and semi-field conditions. Moreover, we studied the effects of egg deposition by the two herbivore species on plant height and flowering rate before and after larval hatching. Larvae of both herbivore species that developed on plants previously infested with eggs of the specialist butterfly P. brassicae gained less mass compared with larvae that developed on egg-free plants. Plants exposed to butterfly eggs showed accelerated plant growth and flowering compared to egg-free plants. Egg deposition by the generalist moth M. brassicae, in contrast, had no effect on subsequent performance by either herbivore species, or on plant development. Our results demonstrate that B. nigra plants respond differently to eggs of two herbivore species in terms of plant development and induced resistance to caterpillar attack. For this annual crucifer, the retardation of caterpillar growth in response to deposition of eggs by P. brassicae in combination with enhanced growth and flowering likely result in reproductive assurance, after being exposed to eggs from an herbivore whose larvae rapidly reduce the plant's reproductive potential through florivory.

Finding an egg in a haystack: variation in chemical cue use by egg parasitoids of herbivorous insects.

Egg parasitoids of herbivorous insects use an interplay of short- and long-range chemical cues emitted by hosts and host plants to find eggs to parasitize. Volatile compounds that attract egg parasitoids can be identified via behavioral assays and used to manipulate parasitoid behavior in the field for biological control of herbivorous pests. However, how and when a particular cue will be used varies over the life of an individual, as well as at and below species level. Future research should expand taxonomic coverage to explore variation in chemical cue use in more natural, dynamic settings. More nuanced understanding of the variability of egg parasitoid host-finding strategies will aid in disentangling the underlying genetics and further enhancing biological control.

Insect egg deposition induces indirect defense and epicuticular wax changes in Arabidopsis thaliana.

Egg deposition by the Large Cabbage White butterfly Pieris brassicae on Brussels sprouts plants induces indirect defense by changing the leaf surface, which arrests the egg parasitoid Trichogramma brassicae. Previous studies revealed that this indirect defense response is elicited by benzyl cyanide (BC), which is present in the female accessory reproductive gland (ARG) secretion and is released to the leaf during egg deposition. Here, we aimed (1) to elucidate whether P. brassicae eggs induce parasitoid-arresting leaf surface changes in another Brassicacean plant, i.e., Arabidopsis thaliana, and, if so, (2) to chemically characterize the egg-induced leaf surface changes. Egg deposition by P. brassicae on A. thaliana leaves had similar effects to egg deposition on Brussels sprouts with respect to the following: (a) Egg deposition induced leaf surface changes that arrested T. brassicae egg parasitoids. (b) Application of ARG secretion of mated female butterflies or of BC to leaves had the same inductive effects as egg deposition. Based on these results, we conducted GC-MS analysis of leaf surface compounds from egg- or ARG-induced A. thaliana leaves. We found significant quantitative differences in epicuticular waxes compared to control leaves. A discriminant analysis separated surface extracts of egg-laden, ARG-treated, untreated control and Ringer solution-treated control leaves according to their quantitative chemical composition. Quantities of the fatty acid tetratriacontanoic acid (C34) were significantly higher in extracts of leaf surfaces arresting the parasitoids (egg-laden or ARG-treated) than in respective controls. In contrast, the level of tetracosanoic acid (C24) was lower in extracts of egg-laden leaves compared to controls. Our study shows that insect egg deposition on a plant can significantly affect the quantitative leaf epicuticular wax composition. The ecological relevance of this finding is discussed with respect to its impact on the behavior of egg parasitoids.

Hitch-hiking parasitic wasp learns to exploit butterfly antiaphrodisiac.

Many insects possess a sexual communication system that is vulnerable to chemical espionage by parasitic wasps. We recently discovered that a hitch-hiking (H) egg parasitoid exploits the antiaphrodisiac pheromone benzyl cyanide (BC) of the Large Cabbage White butterfly Pieris brassicae. This pheromone is passed from male butterflies to females during mating to render them less attractive to conspecific males. When the tiny parasitic wasp Trichogramma brassicae detects the antiaphrodisiac, it rides on a mated female butterfly to a host plant and then parasitizes her freshly laid eggs. The present study demonstrates that a closely related generalist wasp, Trichogramma evanescens, exploits BC in a similar way, but only after learning. Interestingly, the wasp learns to associate an H response to the odors of a mated female P. brassicae butterfly with reinforcement by parasitizing freshly laid butterfly eggs. Behavioral assays, before which we specifically inhibited long-term memory (LTM) formation with a translation inhibitor, reveal that the wasp has formed protein synthesis-dependent LTM at 24 h after learning. To our knowledge, the combination of associatively learning to exploit the sexual communication system of a host and the formation of protein synthesis-dependent LTM after a single learning event has not been documented before. We expect it to be widespread in nature, because it is highly adaptive in many species of egg parasitoids. Our finding of the exploitation of an antiaphrodisiac by multiple species of parasitic wasps suggests its use by Pieris butterflies to be under strong selective pressure.