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The studies of the long-term effects of insect infestations on plant anti-herbivore defences tend to focus on feeding-induced damage. Infestations by an entire insect generation, including egg depositions as well as the feeding insects, are often neglected. Whilst there is increasing evidence that the presence of insect eggs can intensify plants’ anti-herbivore defences against hatching larvae in the short term, little is known about how insect infestations, including insect egg depositions, affect plant defences in the long term. We addressed this knowledge gap by investigating long-term effects of insect infestation on elm’s (Ulmus minor Mill. cv. ‘Dahlem’) defences against subsequent infestation. In greenhouse experiments, elms were exposed to elm leaf beetle (ELB, Xanthogaleruca luteola) infestation (adults, eggs and larvae). Thereafter, the trees cast their leaves under simulated winter conditions and were re-infested with ELB after the regrowth of their leaves under simulated summer conditions. Elm leaf beetles performed moderately worse on previously infested elms with respect to several developmental parameters. The concentrations of the phenylpropanoids kaempferol and quercetin, which are involved in egg-mediated, short-term effects on elm defences, were slightly higher in the ELB-challenged leaves of previously infested trees than in the challenged leaves of naïve trees. The expression of several genes involved in the phenylpropanoid pathway, jasmonic acid signalling, and DNA and histone modifications appeared to be affected by ELB infestation; however, prior infestation did not alter the expression intensities of these genes. The concentrations of several phytohormones were similarly affected in the currently challenged leaves of previously infested trees and naïve trees. Our study shows that prior infestation of elms by a specialised insect leads to moderately improved defences against subsequent infestation in the following growing season. Prior infestation adds a long-term effect to the short-term enhancer effect that plants show in response to egg depositions when defending against hatching larvae.

While traits of plant resistance to herbivory often change during ontogeny, it is unknown whether the primability of this resistance depends on the plant’s developmental stage. Resistance in non-flowering Arabidopsis thaliana against Pieris brassicae larvae is known to be primable by prior egg deposition on leaves. We investigated whether this priming effect is maintained in plants at the flowering stage. Larval performance assays revealed that flowering plants’ resistance to herbivory was not primable by egg deposition. Accordingly, transcriptomes of flowering plants showed almost no response to eggs. In contrast, egg deposition on non-flowering plants enhanced the expression of genes induced by subsequent larval feeding. Strikingly, flowering plants showed constitutively high expression levels of these genes. Larvae performed generally worse on flowering than on non-flowering plants, indicating that flowering plants constitutively resist herbivory. Furthermore, we determined the seed weight in regrown plants that had been exposed to eggs and larvae during the non-flowering or flowering stage. Non-flowering plants benefitted from egg priming with a smaller loss in seed yield. The seed yield of flowering plants was unaffected by the treatments, indicating tolerance towards the larvae. Our results show that the primability of anti-herbivore defences in Arabidopsis depends on the plant’s developmental stage.

Known elicitors of plant defenses against eggs of herbivorous insects are low-molecular-weight organic compounds associated with the eggs. However, previous studies provided evidence that also proteinaceous compounds present in secretion associated with eggs of the herbivorous sawfly Diprion pini can elicit defensive responses in Pinus sylvestris. Pine responses induced by the proteinaceous secretion are known to result in enhanced emission of (E)-β-farnesene, which attracts egg parasitoids killing the eggs. Here, we aimed to identify the defense-eliciting protein and elucidate its function. After isolating the defense-eliciting protein from D. pini egg-associated secretion by ultrafiltration and gel electrophoresis, we identified it by MALDI-TOF mass spectrometry as an annexin-like protein, which we named 'diprionin'. Further GC-MS analyses showed that pine needles treated with heterologously expressed diprionin released enhanced quantities of (E)-β-farnesene. Our bioassays confirmed attractiveness of diprionin-treated pine to egg parasitoids. Expression of several pine candidate genes involved in terpene biosynthesis and regulation of ROS homeostasis was similarly affected by diprionin and natural sawfly egg deposition. However, the two treatments had different effects on expression of pathogenesis-related genes (PR1, PR5). Diprionin is the first egg-associated proteinaceous elicitor of indirect plant defense against insect eggs described so far.

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.

Aims We aimed to elucidate whether the DNA extraction kit and bacteria therein affect the characterization of bacterial communities associated with butterfly samples harbouring different bacterial abundancies. Methods and Results We analysed bacteria associated with eggs of Pieris brassicae and with adults of this butterfly, which were either untreated or treated with antibiotics (ABs). Three DNA extraction kits were used. Regardless of the extraction kit used, PCR amplification of the bacterial 16S rRNA gene detected very low bacterial presence in eggs and AB-treated butterflies. In untreated butterflies, bacterial signal intensity varied according to the kit and primers used. Sequencing (MiSeq) of the bacterial communities in untreated and AB-treated butterflies revealed a low alpha diversity in untreated butterflies because of the dominance of few bacteria genera, which were detectable regardless of the kit. However, a significantly greater alpha diversity was found in AB-treated butterflies, evidencing a true bias of the results due to bacterial contaminants in the kit. Conclusions The so-called ‘kitome’ can impact the profiling of Lepidoptera-associated bacteria in samples with low bacterial biomass. Significance and Impact of the Study Our study highlights the necessity of method testing and analysis of negative controls when investigating Lepidoptera-associated bacterial communities.

Several studies have shown that insectivorous birds are attracted to herbivore‐damaged trees even when they cannot see or smell the actual herbivores or their feces. However, it often remained an open question whether birds are attracted by herbivore‐induced changes in leaf odor or in leaf light reflectance or by both types of changes. Our study addressed this question by investigating the response of great tits (Parus major) and blue tits (Cyanistes caeruleus) to Scots pine (Pinus sylvestris) damaged by pine sawfly larvae (Diprion pini). We released the birds individually to a study booth, where they were simultaneously offered a systemically herbivore‐induced and a noninfested control pine branch. In the first experiment, the birds could see the branches, but could not smell them, because each branch was kept inside a transparent, airtight cylinder. In the second experiment, the birds could smell the branches, but could not see them, because each branch was placed inside a nontransparent cylinder with a mesh lid. The results show that the birds were more attracted to the herbivore‐induced branch in both experiments. Hence, either type of the tested cues, the herbivore‐induced visual plant cue alone as well as the olfactory cues per se, is attractive to the birds., Both great tits and blue tits were attracted to systemically herbivore‐damaged pines when they could only see or only smell the experimental branches. This is the first ever study to show that both vision and olfaction alone are enough for attraction of insectivorous birds to herbivore‐damaged trees.

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.

According to the preference-performance hypothesis (PPH), also known as ‘mother-knows-best hypothesis’, herbivorous insects prefer those plants for oviposition, which yield the best offspring performance. Yet, most studies testing the PPH neglect the possibility that plant responses to insect eggs may affect both egg survival and larval performance. Here, we tested the PPH by studying responses of seven Brassicaceae plant species to oviposition by two cabbage white species. When including the egg phase, our study supports the ‘mother-knows-best hypothesis’: larvae of Pieris rapae (solitary) or P. brassicae (gregarious) gained most weight on those plant species which had received most eggs (B. nigra or B. montana, respectively). However, our experiments did not reveal any relationship between oviposition preference and egg survival. Brassicaceous species are known to respond to these butterfly eggs with a hypersensitive response (HR)-like necrosis, which can lower egg survival. Pieris eggs frequently induced necrosis in five of the tested plant species. Survival of clustered P. brassicae eggs was unaffected by HR-like in four of the five species. Therefore, our experiments did not reveal any relationship between P. brassicae egg survival and oviposition preference. Females of P. rapae preferred oviposition on plant species which most frequently showed HR-like necrosis. Remarkably, although egg survival was lower on HR-like plants, larval biomass was higher compared to plants without a necrosis. We conclude that egg survival does not seem to be a deciding factor for oviposition choices. However, egg-mediated plant responses might be important to explain the PPH of the two Pieris species.Lay summaryEgg-laying preferences of herbivorous insects can often be linked to offspring performance. Commonly, the fate of insect eggs and the plant responses to the eggs have been ignored when studying the link between preference and performance. By including the egg phase, our study supports the ‘mother-knows-best hypothesis’, explained by butterfly oviposition and associated egg and larval performances on different plant species. We especially found that egg-mediated responses seem a deciding factor for butterfly oviposition choices.

Main conclusion Elms, which have received insect eggs as a ‘warning’ of larval herbivory, enhance their anti-herbivore defences by accumulating salicylic acid and amplifying phenylpropanoid-related transcriptional and metabolic responses to hatching larvae. Abstract Plant responses to insect eggs can result in intensified defences against hatching larvae. In annual plants, this egg-mediated effect is known to be associated with changes in leaf phenylpropanoid levels. However, little is known about how trees—long-living, perennial plants—improve their egg-mediated, anti-herbivore defences. The role of phytohormones and the phenylpropanoid pathway in egg-primed anti-herbivore defences of a tree species has until now been left unexplored. Using targeted and untargeted metabolome analyses we studied how the phenylpropanoid pathway of Ulmus minor responds to egg-laying by the elm leaf beetle and subsequent larval feeding. We found that when compared to untreated leaves, kaempferol and quercetin concentrations increased in feeding-damaged leaves with prior egg deposition, but not in feeding-damaged leaves without eggs. PCR analyses revealed that prior insect egg deposition intensified feeding-induced expression of phenylalanine ammonia lyase (PAL), encoding the gateway enzyme of the phenylpropanoid pathway. Salicylic acid (SA) concentrations were higher in egg-treated, feeding-damaged leaves than in egg-free, feeding-damaged leaves, but SA levels did not increase in response to egg deposition alone—in contrast to observations made of Arabidopsis thaliana. Our results indicate that prior egg deposition induces a SA-mediated response in elms to feeding damage. Furthermore, egg deposition boosts phenylpropanoid biosynthesis in subsequently feeding-damaged leaves by enhanced PAL expression, which results in the accumulation of phenylpropanoid derivatives. As such, the elm tree shows similar, yet distinct, responses to insect eggs and larval feeding as the annual model plant A. thaliana.

Known elicitors of plant defenses against eggs of herbivorous insects are low-molecular-weight organic compounds associated with the eggs. However, previous studies provided evidence that also proteinaceous compounds present in secretion associated with eggs of the herbivorous sawfly Diprion pini can elicit defensive responses in Pinus sylvestris. Pine responses induced by the proteinaceous secretion are known to result in enhanced emission of (E)-β-farnesene, which attracts egg parasitoids killing the eggs. Here, we aimed to identify the defense-eliciting protein and elucidate its function. After isolating the defense-eliciting protein from D. pini egg secretion by ultrafiltration and gel electrophoresis, we identified it by MALDI-ToF mass spectrometry as an annexin-like protein, which we named “diprionin”. Further GC-MS analyses showed that pine needles treated with heterologously expressed diprionin released enhanced quantities of (E)-β-farnesene. Our bioassays confirmed attractiveness of diprionin-treated pine to egg parasitoids. Expression of several pine candidate genes involved in terpene biosynthesis and regulation of ROS homeostasis was similarly affected by diprionin and natural sawfly egg deposition. However, the two treatments had different effects on expression of pathogenesis related genes (PR1, PR5). Diprionin is the first egg-associated proteinaceous elicitor of indirect plant defense against insect eggs described so far.

The transition from pollinator-mediated outbreeding to selfing has occurred many times in angiosperms. This is generally accompanied by a reduction in traits attracting pollinators, including reduced emission of floral scent. In Capsella, emission of benzaldehyde as a main component of floral scent has been lost in selfing C. rubella by mutation of cinnamate-CoA ligase CNL1. However, the biochemical basis and evolutionary history of this loss remain unknown, as does the reason for the absence of benzaldehyde emission in the independently derived selfer Capsella orientalis. We used plant transformation, in vitro enzyme assays, population genetics and quantitative genetics to address these questions. CNL1 has been inactivated twice independently by point mutations in C. rubella, causing a loss of enzymatic activity. Both inactive haplotypes are found within and outside of Greece, the centre of origin of C. rubella, indicating that they arose before its geographical spread. By contrast, the loss of benzaldehyde emission in C. orientalis is not due to an inactivating mutation in CNL1. CNL1 represents a hotspot for mutations that eliminate benzaldehyde emission, potentially reflecting the limited pleiotropy and large effect of its inactivation. Nevertheless, even closely related species have followed different evolutionary routes in reducing floral scent.

Plants need to cope with changing environmental conditions, be it variable light or temperature, different availability of water or nutrients, or attack by pathogens or insects. Some of these changing conditions can become stressful and require strong countermeasures to ensure plant survival. Plants have evolved numerous distinct sensing and signalling mechanisms to perceive and respond appropriately to a variety of stresses. Because of the unpredictable nature of numerous stresses, resource-saving stress response mechanisms are inducible and become activated only upon a stress experience. Furthermore, plants have evolved mechanisms by which they can remember past stress events and prime their responses in order to react more rapidly or more strongly to recurrent stress. Research over the last decade has revealed mechanisms of this information storage and retrieval, which include epigenetic regulation, transcriptional priming, primed conformation of proteins, or specific hormonal or metabolic signatures. There is also increasing understanding of the ecological constraints and relevance of stress priming and memory. This special issue presents research articles and reviews addressing various aspects of this exciting and growing field of research. Here, we introduce the topic by referring to the articles published in this issue, and we outline open questions and future directions of research.

Successful host search by parasitic wasps is often mediated by host-associated chemical cues. The ectoparasitoid Holepyris sylvanidis is known to follow chemical trails released by host larvae of the confused flour beetle, Tribolium confusum, for short-range host location. Although the hexane-extractable trails consist of stable, long-chain cuticular hydrocarbons (CHCs) with low volatility, the kairomonal activity of a trail is lost two days after release. Here, we studied whether this loss of kairomonal activity is due to changes in the chemical trail composition induced by microbial activity. We chemically analyzed trails consisting of hexane extracts of T. confusum larvae after different time intervals past deposition under sterile and non-sterile conditions. GC-MS analyses revealed that the qualitative and quantitative pattern of the long-chain CHCs of larval trails did not significantly change over time, neither under non-sterile nor sterile conditions. Hence, our results show that the loss of kairomonal activity of host trails is not due to microbially induced changes of the CHC pattern of a trail. Interestingly, the kairomonal activity of trails consisting of host larval CHC extracts was recoverable after two days by applying hexane to them. After hexane evaporation, the parasitoids followed the reactivated host trails as they followed freshly laid ones. Cryo-scanning electron microscopy showed that the trails gradually formed filament-shaped microstructures within two days. This self-assemblage of CHCs was reversible by hexane application. Our study suggests that the long-chain CHCs of a host trail slowly undergo solidification by a self-assembling process, which reduces the accessibility of CHCs to the parasitoid’s receptors as such that the trail is no longer eliciting trail-following behavior.

Plants can respond to eggs laid by herbivorous insects on their leaves by preparing (priming) their defense against the hatching larvae. Egg-mediated priming of defense is known for several plant species, including Brassicaceae. However, it is unknown yet for how long the eggs need to remain on a plant until a primed defense state is reached, which is ecologically manifested by reduced performance of the hatching larvae. To address this question, we used Arabidopsis thaliana, which carried eggs of the butterfly Pieris brassicae for 1–6 days prior to exposure to larval feeding. Our results show that larvae gained less biomass the longer the eggs had previously been on the plant. The strongest priming effect was obtained when eggs had been on the plant for 5 or 6 days, i.e., for (almost) the entire development time of the Pieris embryo inside the egg until larval hatching. Transcript levels of priming-responsive genes, levels of jasmonic acid-isoleucine (JA-Ile), and of the egg-inducible phytoalexin camalexin increased with the egg exposure time. Larval performance studies on mutant plants revealed that camalexin is dispensable for anti-herbivore defense against P. brassicae larvae, whereas JA-Ile – in concert with egg-induced salicylic acid (SA) – seems to be important for signaling egg-mediated primed defense. Thus, A. thaliana adjusts the kinetics of its egg-primed response to the time point of larval hatching. Hence, the plant is optimally prepared just in time prior to larval hatching.

Cuticular hydrocarbons (CHCs) of host insects are used by many parasitic wasps as contact kairomones for host location and recognition. As the chemical composition of CHCs varies from species to species, the CHC pattern represents a reliable indicator for parasitoids to discriminate host from non-host species. Holepyris sylvanidis is an ectoparasitoid of beetle larvae infesting stored products. Previous studies demonstrated that the larval CHC profile of the confused flour beetle, Tribolium confusum, comprises long chain linear and methyl-branched alkanes (methyl alkanes), which elicit trail following and host recognition in H. sylvanidis. Here we addressed the question, whether different behavioral responses of this parasitoid species to larvae of other beetle species are due to differences in the larval CHC pattern. Our study revealed that H. sylvanidis recognizes and accepts larvae of T. confusum, T. castaneum and T. destructor as hosts, whereas larvae of Oryzaephilus surinamensis were rejected. However, the latter species became attractive after applying a sample of T. confusum larval CHCs to solvent extracted larvae. Chemical analyses of the larval extracts revealed that CHC profiles of the Tribolium species were similar in their composition, while that of O. surinamensis differed qualitatively and quantitatively, i.e. methyl alkanes were present as minor components on the cuticle of all Tribolium larvae, but were absent in the O. surinamensis CHC profile. Furthermore, the parasitoid successfully recognized solvent extracted T. confusum larvae as hosts after they had been treated with a fraction of methyl alkanes. Our results show that methyl alkanes are needed for host recognition by H. sylvanidis.

Plants can respond to eggs laid by herbivorous insects on their leaves by preparing (priming) their defense against the hatching larvae. Egg-mediated priming of defense is known for several plant species, including Brassicaceae. However, it is unknown yet for how long the eggs need to remain on a plant until a primed defense state is reached, which is ecologically manifested by reduced performance of the hatching larvae. To address this question, we usedArabidopsis thaliana, which carried eggs of the butterflyPieris brassicaefor 1–6 days prior to exposure to larval feeding. Our results show that larvae gained less biomass the longer the eggs had previously been on the plant. The strongest priming effect was obtained when eggs had been on the plant for 5 or 6 days, i.e., for (almost) the entire development time of thePierisembryo inside the egg until larval hatching. Transcript levels of priming-responsive genes, levels of jasmonic acid-isoleucine (JA-Ile), and of the egg-inducible phytoalexin camalexin increased with the egg exposure time. Larval performance studies on mutant plants revealed that camalexin is dispensable for anti-herbivore defense againstP. brassicaelarvae, whereas JA-Ile – in concert with egg-induced salicylic acid (SA) – seems to be important for signaling egg-mediated primed defense. Thus,A. thalianaadjusts the kinetics of its egg-primed response to the time point of larval hatching. Hence, the plant is optimally prepared just in time prior to larval hatching.

Plants respond to insect eggs with transcriptional changes, resulting in enhanced defence against hatching larvae. However, it is unknown whether phylogenetically distant plant species show conserved transcriptomic responses to insect eggs and subsequent larval feeding. We used Generally Applicable Gene set Enrichment (GAGE) on gene ontology terms to answer this question and analysed transcriptome data from Arabidopsis thaliana, wild tobacco (Nicotiana attenuata), bittersweet nightshade (Solanum dulcamara) and elm trees (Ulmus minor) infested by different insect species. The different plant–insect species combinations showed considerable overlap in their transcriptomic responses to both eggs and larval feeding. Within these conformable responses across the plant–insect combinations, the responses to eggs and feeding were largely analogous, and about one-fifth of these analogous responses were further enhanced when egg deposition preceded larval feeding. This conserved transcriptomic response to eggs and larval feeding comprised gene sets related to several phytohormones and to the phenylpropanoid biosynthesis pathway, of which specific branches were activated in different plant–insect combinations. Since insect eggs and larval feeding activate conserved sets of biological processes in different plant species, we conclude that plants with different lifestyles share common transcriptomic alarm responses to insect eggs, which likely enhance their defence against hatching larvae.

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 F(1) larval traits. We tested the effects depending on whether F(1) 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 F(1) larvae feeding on the parental plant species. However, the parental treatment had a detrimental effect on F(1) larval biomass when F(1) 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 F(1) 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.

Significance Plant defenses against herbivorous insects can target the feeding stages and the egg stage. Feeding-induced plant defenses are known to be primed by cues indicating imminent infestation, including sex pheromones. However, priming of egg-induced plant defenses has been unknown so far. Therefore, we studied whether a plant’s response to insect sex pheromones, which might indicate imminent egg depositions, primes defenses against the eggs. Indeed, exposure of pine to the sex pheromones of an herbivorous sawfly primes the tree’s defense against sawfly eggs. The priming effect results in enhanced egg mortality, enhanced accumulation of hydrogen peroxide in egg-laden needles, and differential expression of several defense-related pine genes. These findings open up exciting research perspectives in plant protection from insect infestation., Plants respond to insect infestation with defenses targeting insect eggs on their leaves and the feeding insects. Upon perceiving cues indicating imminent herbivory, such as damage-induced leaf odors emitted by neighboring plants, they are able to prime their defenses against feeding insects. Yet it remains unknown whether plants can amplify their defenses against insect eggs by responding to cues indicating imminent egg deposition. Here, we tested the hypothesis that a plant strengthens its defenses against insect eggs by responding to insect sex pheromones. Our study shows that preexposure of Pinus sylvestris to pine sawfly sex pheromones reduces the survival rate of subsequently laid sawfly eggs. Exposure to pheromones does not significantly affect the pine needle water content, but results in increased needle hydrogen peroxide concentrations and increased expression of defense-related pine genes such as SOD (superoxide dismutase), LOX (lipoxygenase), PAL (phenylalanine ammonia lyase), and PR-1 (pathogenesis related protein 1) after egg deposition. These results support our hypothesis that plant responses to sex pheromones emitted by an herbivorous insect can boost plant defensive responses to insect egg deposition, thus highlighting the ability of a plant to mobilize its defenses very early against an initial phase of insect attack, the egg deposition.

Background In native environments plants frequently experience simultaneous or sequential unfavourable abiotic and biotic stresses. The plant’s response to combined stresses is usually not the sum of the individual responses. Here we investigated the impact of cold on plant defense against subsequent herbivory by a generalist and specialist insect. Results We determined transcriptional responses of Arabidopsis thaliana to low temperature stress (4 °C) and subsequent larval feeding damage by the lepidopteran herbivores Mamestra brassicae (generalist), Pieris brassicae (specialist) or artificial wounding. Furthermore, we compared the performance of larvae feeding upon cold-experienced or untreated plants. Prior experience of cold strongly affected the plant’s transcriptional anti-herbivore and wounding response. Feeding by P. brassicae, M. brassicae and artificial wounding induced transcriptional changes of 1975, 1695, and 2239 genes, respectively. Of these, 125, 360, and 681 genes were differentially regulated when cold preceded the tissue damage. Overall, prior experience of cold mostly reduced the transcriptional response of genes to damage. The percentage of damage-responsive genes, which showed attenuated transcriptional regulation when cold preceded the tissue damage, was highest in M. brassicae damaged plants (98%), intermediate in artificially damaged plants (89%), and lowest in P. brassicae damaged plants (69%). Consistently, the generalist M. brassicae performed better on cold-treated than on untreated plants, whereas the performance of the specialist P. brassicae did not differ. Conclusions The transcriptional defense response of Arabidopsis leaves to feeding by herbivorous insects and artificial wounding is attenuated by a prior exposure of the plant to cold. This attenuation correlates with improved performance of the generalist herbivore M. brassicae, but not the specialist P. brassicae, a herbivore of the same feeding guild. Electronic supplementary material The online version of this article (10.1186/s12870-019-1943-3) contains supplementary material, which is available to authorized users.

Plants respond to insect infestation with defenses targeting insect eggs on their leaves and the feeding insects. Upon perceiving cues indicating imminent herbivory, such as damage-induced leaf odors emitted by neighboring plants, they are able to prime their defenses against feeding insects. Yet it remains unknown whether plants can amplify their defenses against insect eggs by responding to cues indicating imminent egg deposition. Here, we tested the hypothesis that a plant strengthens its defenses against insect eggs by responding to insect sex pheromones. Our study shows that preexposure of Pinus sylvestris to pine sawfly sex pheromones reduces the survival rate of subsequently laid sawfly eggs. Exposure to pheromones does not significantly affect the pine needle water content, but results in increased needle hydrogen peroxide concentrations and increased expression of defense-related pine genes such as SOD (superoxide dismutase), LOX (lipoxygenase), PAL (phenylalanine ammonia lyase), and PR-1 (pathogenesis related protein 1) after egg deposition. These results support our hypothesis that plant responses to sex pheromones emitted by an herbivorous insect can boost plant defensive responses to insect egg deposition, thus highlighting the ability of a plant to mobilize its defenses very early against an initial phase of insect attack, the egg deposition.

Global change has complex eco-evolutionary consequences for organisms and ecosystems, but related concepts (e.g., novel ecosystems) do not cover their full range. Here we propose an umbrella concept of “ecological novelty” comprising (1) a site-specific and (2) an organism-centered, eco-evolutionary perspective. Under this umbrella, complementary options for studying and communicating effects of global change on organisms, ecosystems, and landscapes can be included in a toolbox. This allows researchers to address ecological novelty from different perspectives, e.g., by defining it based on (a) categorical or continuous measures, (b) reference conditions related to sites or organisms, and (c) types of human activities. We suggest striving for a descriptive, non-normative usage of the term “ecological novelty” in science. Normative evaluations and decisions about conservation policies or management are important, but require additional societal processes and engagement with multiple stakeholders., BioScience, 69 (11)

The enormous species richness of flowering plants is at least partly due to floral diversification driven by interactions between plants and their animal pollinators [1, 2]. Specific pollinator attraction relies on visual and olfactory floral cues [3-5]; floral scent can not only attract pollinators but also attract or repel herbivorous insects [6-8]. However, despite its central role for plant-animal interactions, the genetic control of floral scent production and its evolutionary modification remain incompletely understood [9-13]. Benzenoids are an important class of floral scent compounds that are generated from phenylalanine via several enzymatic pathways [14-17]. Here we address the genetic basis of the loss of floral scent associated with the transition from outbreeding to selfing in the genus Capsella. While the outbreeding C. grandiflora emits benzaldehyde as a major constituent of its floral scent, this has been lost in the selfing C. rubella. We identify the Capsella CNL1 gene encoding cinnamate: CoA ligase as responsible for this variation. Population genetic analysis indicates that CNL1 has been inactivated twice independently in C. rubella via different novel mutations to its coding sequence. Together with a recent study in Petunia [18], this identifies cinnamate: CoA ligase as an evolutionary hotspot for mutations causing the loss of benzenoid scent compounds in association with a shift in the reproductive strategy of Capsella from pollination by insects to self-fertilization., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe, 904

Maladaptive sexual interactions among heterospecific individuals (sexual interference) can prevent the coexistence of animal species. Thus, the avoidance of sexual interference by divergence of mate recognition systems is crucial for a stable coexistence in sympatry. Mate recognition systems are thought to be under tight genetic control. However, we demonstrate that mate recognition systems of two closely related sympatric leaf beetle species show a high level of host-induced phenotypic plasticity. Mate choice in the mustard leaf beetles, Phaedon cochleariae and P. armoraciae, is mediated by cuticular hydrocarbons (CHCs). Divergent host plant use causes a divergence of CHC phenotypes, whereas similar host use leads to their convergence. Consequently, both species exhibit significant behavioral isolation when they feed on alternative host species, but mate randomly when using a common host. Thus, sexual interference between these syntopic leaf beetles is prevented by host-induced phenotypic plasticity rather than by genotypic divergence of mate recognition systems.

Oviposition by Spodoptera exigua on Nicotiana attenuata primes plant defence against its larvae that consequently suffer reduced performance. To reveal whether this is a general response of tobacco to insect oviposition or species-specific, we investigated whether also Manduca sexta oviposition primes N. attenuata's anti-herbivore defence. The plant response to M. sexta and S. exigua oviposition overlapped in the egg-primed feeding-induced production of the phenylpropanoid caffeoylputrescine. While M. sexta larvae were unaffected in their performance, they showed a novel response to the oviposition-mediated plant changes: a reduced antimicrobial activity in their haemolymph. In a cross-resistance experiment, S. exigua larvae suffered reduced performance on M. sexta-oviposited plants like they did on S. exigua-oviposited plants. The M. sexta oviposition-mediated plant effects on the S. exigua larval performance and on M. sexta larval immunity required expression of the NaMyb8 transcription factor that is governing biosynthesis of phenylpropanoids such as caffeoylputrescine. Thus, NaMyb8-dependent defence traits mediate the effects that oviposition by both lepidopteran species exerts on the plant's anti-herbivore defence. These results suggest that oviposition by lepidopteran species on N. attenuata leaves may generally prime the feeding-induced production of certain plant defence compounds but that different herbivore species show different susceptibility to egg-primed plant effects.

The insect's microbiota is well acknowledged as a “hidden” player influencing essential insect traits. The gut microbiome of butterflies and moths (Lepidoptera) has been shown to be highly variable between and within species, resulting in a controversy on the functional relevance of gut microbes in this insect order. Here, we aim to (i) review current knowledge on the composition of gut microbial communities across Lepidoptera and (ii) elucidate the drivers of the variability in the lepidopteran gut microbiome and provide an overview on (iii) routes of transfer and (iv) the putative functions of microbes in Lepidoptera. To find out whether Lepidopterans possess a core gut microbiome, we compared studies of the microbiome from 30 lepidopteran species. Gut bacteria of the Enterobacteriaceae, Bacillaceae, and Pseudomonadaceae families were the most widespread across species, with Pseudomonas, Bacillus, Staphylococcus, Enterobacter, and Enterococcus being the most common genera. Several studies indicate that habitat, food plant, and age of the host insect can greatly impact the gut microbiome, which contributes to digestion, detoxification, or defense against natural enemies. We mainly focus on the gut microbiome, but we also include some examples of intracellular endosymbionts. These symbionts are present across a broad range of insect taxa and are known to exert different effects on their host, mostly including nutrition and reproductive manipulation. Only two intracellular bacteria genera (Wolbachia and Spiroplasma) have been reported to colonize reproductive tissues of Lepidoptera, affecting their host's reproduction. We explore routes of transmission of both gut microbiota and intracellular symbionts and have found that these microbes may be horizontally transmitted through the host plant, but also vertically via the egg stage. More detailed knowledge about the functions and plasticity of the microbiome in Lepidoptera may provide novel leads for the control of lepidopteran pest species.

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.

Plants employ various defences killing the insect attacker in an early stage. Oviposition by cabbage white butterflies (Pieris spp.) on brassicaceous plants, including Brassica nigra, induces a hypersensitive response (HR) - like leaf necrosis promoting desiccation of eggs. To gain a deeper insight into the arms race between butterflies and plants, we conducted field and greenhouse experiments using different B. nigra genotypes. We investigated variation in HR and consequent survival of P. brassicae egg clusters. Impact of egg density, distribution type and humidity on HR formation and egg survival was tested. HR differed among plant genotypes as well as plant individuals. Egg density per plant did not affect HR formation. Remarkably, egg survival did not depend on the formation of HR, unless butterflies were forced to lay single eggs. Larval hatching success from single eggs was lower on plants expressing HR. This may be due to increased vulnerability of single eggs to low humidity conditions at necrotic leaf sites. We conclude that effectiveness of HR-like necrosis in B. nigra varies with plant genotype, plant individual and the type of egg laying behaviour (singly or clustered). By clustering eggs, cabbage white butterflies can escape the egg-killing, direct plant defence trait.

Summary Allelopathy is a phenomenon where plants have deleterious effects on growth of surrounding plants through the production of chemical substances. Soil hyphae of arbuscular mycorrhizal fungi may enhance transport processes of allelochemicals by providing ‘highways’ connecting plants below-ground. In three studies ranging from high ecological realism to experimental control, we showed that the presence of mycorrhizal hyphae may strongly contribute to the transport of allelochemicals. We analysed the accumulation of naturally released juglone in the field in intact or disrupted hyphal connections and determined its growth reducing effects on sensitive target plants in a bioassay. Secondly, we tested the effects of Juglans regia leaf litter addition in the presence or absence of the mycorrhizal fungus Rhizophagus irregularis on target plants, and finally, we added pure juglone to Lycopersicon lycopersicum plants in the presence or absence of Rhizophagus. Throughout, we found increased juglone transfer if mycorrhizal hyphae were present, resulting in reduced growth of target plants. Our results point to mycorrhizal hyphae playing an important role in extending the bioactive zone of allelochemicals. We suggest that hyphal networks increase the effectiveness of allelochemicals in natural systems and play a crucial role in chemical interaction processes and hence influence plant community structure.

Convolvulaceae provide a rich source of tropane alkaloids, however, 2-substi-tuted tropanes have been described for only few species of this taxon. In this note, 2,7-diesters such as ipvelutine [7β-acetoxy-2α-(tigloyloxy)tropane] isolated from the vegetative parts of the Australian Ipomoea velutina R. BR. are described as a new group of tropane diesters.

Plants can retain information about environmental stress and thus, prepare themselves for impending stress. In nature, it happens that environmental stimuli like ‘cold’ and ‘insect egg deposition’ precede insect herbivory. Both these stimuli are known to elicit transcriptomic changes in Arabidposis thaliana. It is unknown, however, whether they affect the plant’s anti- herbivore defence and feeding-induced transcriptome when they end prior to herbivory. Here we investigated the transcriptomic response of Arabidopsis to feeding by Pieris brassicae larvae after prior exposure to cold or oviposition. The transcriptome of plants that experienced a five-day-chilling period (4 °C) was not fully reset to the pre-chilling state after deacclimation (20 °C) for one day and responded differently to herbivory than that of chilling-inexperienced plants. In contrast, when after a five-day- lasting oviposition period the eggs were removed, one day later the transcriptome and, consistently, also its response to herbivory resembled that of egg-free plants. Larval performance was unaffected by previous exposure of plants to cold and to eggs, thus indicating P. brassicae tolerance to cold- mediated plant transcriptomic changes. Our results show strong differences in the persistence of the plant’s transcriptomic state after removal of different environmental cues, and consequently differential effects on the transcriptomic response to later herbivory.

The role of phenotypical plasticity in ecological speciation and the evolution of sexual isolation remains largely unknown. We investigated whether or not divergent host plant use in an herbivorous insect causes assortative mating by phenotypically altering traits involved in mate recognition. We found that males of the mustard leaf beetle Phaedon cochleariae preferred to mate with females that were reared on the same plant species to females provided with a different plant species, based on divergent cuticular hydrocarbon profiles that serve as contact pheromones. The cuticular hydrocarbon phenotypes of the beetles were host plant specific and changed within 2 weeks after a shift to a novel host plant species. We suggest that plant-induced phenotypic divergence in mate recognition cues may act as an early barrier to gene flow between herbivorous insect populations on alternative host species, preceding genetic divergence and thus, promoting ecological speciation.

Plants can defend themselves against insect attack prior to larval feeding damage by responding to eggs laid on their leaves. Insect egg deposition can induce leaves to release a complex blend of volatiles attracting egg parasitoids which kill the eggs. Only a few studies have addressed the question which of these egg-induced plant volatiles are relevant for parasitoid attraction. Egg deposition by the elm leaf beetle Xanthogaleruca luteola on leaves of the European field elm Ulmus minor is known to induce the emission of a blend consisting mainly of terpenoids and some green leaf volatiles, which attracts a specialised egg parasitoid of X. luteola, the eulophid wasp Oomyzus gallerucae. Here, we investigated the role of oviposition-induced terpenoids from elm leaves for parasitoid attraction. Quantitative GC–MS analyses showed that inhibition of terpene biosynthesis in leaves by treatment with cerivastatin® and fosmidomycin reduced emission of (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), (E)-β-caryophyllene and a yet unidentified oxygenated sesquiterpene, but unexpectedly also that of green leaf volatiles (GLVs). Laboratory olfactometer assays revealed that inhibitor treatment rendered oviposition-induced elm leaves unattractive for the parasitoids. Further bioassays showed that single terpenoids per se attracted the parasitoids. Although the only tested GLV 1-hexanol was not attractive in olfactometer tests, we cannot rule out that other GLVs might play a role in parasitoid attraction. When attractiveness of DMNT was tested in the field, parasitoids were attracted to DMNT-baited traps in the presence of background odour emitted by a natural elm stand. We conclude that elms alert their egg parasitoid “helpers” after elm leaf beetle oviposition by means of one or more terpenoid volatiles.

Vegetation complexity is characterized by two major traits, i.e., plant chemical and plant structural complexity. Plant species diversity strongly determines these traits. Furthermore, plant structures affect microclimatic conditions, which in turn influence the emission and dispersion of plant volatiles (e.g., chemical complexity). Plant volatile chemical complexity may significantly affect orientation of herbivorous and carnivorous arthropods. Therefore, the way in which plant chemical and plant structural complexity act “in concert” may influence foraging and mating success of arthropods, and thus, finally, community composition. This review emphasizes an integrative view on the relationship between plant species diversity, plant structural complexity, plant volatiles (chemical complexity) and their effects on arthropods. Three new hypotheses are raised, which predict possible relations between plant volatile complexity and plant species diversity: (1) saturation-, (2) step-by-step, (3) incoherence-hypothesis. We conclude that arthropod orientation in natural environments is strongly determined by the relationship between plant volatile diversity and plant species diversity. Furthermore, we emphasize that structural complexity of the vegetation affects plant volatile diversity and thus, arthropod orientation. We review available information on how insects actually respond to complexity during olfactory and visual search and ask for both laboratory and field studies to further unravel the mechanisms of interactions between vegetation traits and their impact on arthropod orientation.

Plants are endangered at their roots by soil-dwelling rhizophagous insects. These below-ground living herbivores may orient to the source of carbon dioxide (CO 2 ), an ubiquitous volatile released by respiring plant roots. Here, we studied the interaction of CO 2 and other plant root-derived chemical stimuli with regard to the chemical orientation of the polyphagous larvae of Melolontha melolontha L. (Scarabaeidae). A soil arena was developed that enabled both determination of the actual soil CO 2 concentration and the behavioural response of an insect to (a) CO 2 gradients per se , (b) chemical stimuli released from respiring, undamaged roots of plants potted into vermiculite in this arena and (c) combinations of CO 2 gradients and root-derived stimuli. In a root-free arena, larvae of M. melolontha oriented to the source of synthetic CO 2 . However, similar CO 2 gradients generated by host plant roots did not attract the larvae. Neither did a synthetic CO 2 gradient combined with aqueous extracts from rhizospheres with undamaged plant roots elicit an attractive effect. Our data suggest that orientation of cockchafer larvae within CO 2 gradients generated by respiring roots is ‘masked’ by an aqueous extract from a rhizosphere with undamaged roots. The results emphasise that effects of behaviour modifying plant-derived compounds need to be investigated against the background of naturally co-occurring chemicals. The significance of our results for orientation of soil living insects is discussed with respect to abiotic conditions in natural soil and the role of soil microorganisms for the attractiveness of plant roots.