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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.

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.

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.

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.

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.

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.

The insect integument is covered by cuticular hydrocarbons (CHCs) which provide protection against environmental stresses, but are also used for communication. Here we review current knowledge on environmental and insect-internal factors which shape phenotypic plasticity of solitary living insects, especially herbivorous ones. We address the dynamics of changes which may occur within minutes, but may also last weeks, depending on the species and conditions. Two different modes of changes are suggested, i.e. stepwise and gradual. A switch between two distinct environments (e.g. host plant switch by phytophagous insects) results in stepwise formation of two distinct adaptive phenotypes, while a gradual environmental change (e.g. temperature gradients) induces a gradual change of numerous adaptive CHC phenotypes. We further discuss the ecological and evolutionary consequences of phenotypic plasticity of insect CHC profiles by addressing the question at which conditions is CHC phenotypic plasticity beneficial. The high plasticity of CHC profiles might be a trade-off for insects using CHCs for communication. We discuss how insects cope with the challenge to produce and "understand" a highly plastic, environmentally dependent CHC pattern that conveys reliable and comprehensible information. Finally, we outline how phenotypic plasticity of CHC profiles may promote speciation in insects that rely on CHCs for mate recognition.

Parasitic wasps which attack insects infesting processed stored food need to locate their hosts hidden inside these products. Their host search is well-known to be guided by host kairomones, perceived via olfaction or contact. Among contact kairomones, host cuticular hydrocarbons (CHCs) may provide reliable information for a parasitoid. However, the chemistry of CHC profiles of hosts living in processed stored food products is largely unknown. Here we showed that the ectoparasitoid Holepyris sylvanidis uses CHCs of its host Tribolium confusum, a worldwide stored product pest, as kairomones for host location and recognition at short range. Chemical analysis of T. confusum larval extracts by gas chromatography coupled with mass spectrometry revealed a rich blend of long-chain (C25-C30) hydrocarbons, including n-alkanes, mono-, and dimethylalkanes. We further studied whether host larvae leave sufficient CHCs on a substrate where they walk along, thus allowing parasitoids to perceive a CHC trail and follow it to their host larvae. We detected 18 CHCs on a substrate that had been exposed to host larvae. These compounds were also found in crude extracts of host larvae and made up about a fifth of the CHC amount extracted. Behavioral assays showed that trails of host CHCs were followed by the parasitoids and reduced their searching time until successful host recognition. Host CHC trails deposited on different substrates were persistent for about a day. Hence, the parasitoid H. sylvanidis exploits CHCs of T. confusum larvae for host finding by following host CHC trails and for host recognition by direct contact with host larvae.

Pinus sylvestris responds to insect egg deposition by ROS accumulation linked with reduced activity of the ROS scavenger catalase. Egg mortality in needles with hypersensitive response (HR)-like symptoms is enhanced. Aggressive reactive oxygen species (ROS) play an important role in plant defence against biotic stressors, including herbivorous insects. Plants may even generate ROS in response to insect eggs, thus effectively fighting against future larval herbivory. However, so far nothing is known on how ROS-mediated plant defence against insect eggs is enzymatically regulated. Neither do we know how insects cope with egg-induced plant ROS. We addressed these gaps of knowledge by studying the activities of ROS-related enzymes in Pinus sylvestris deposited with eggs of the herbivorous sawfly Diprion pini. This species cuts a slit into pine needles and inserts its eggs into the needle tissue. About a quarter of egg-deposited needles show chlorotic tissue at the oviposition sites, indicating hypersensitive response-like direct defence responses resulting in reduced larval hatching from eggs. Hydrogen peroxide and peroxidase sensitive staining of sections of egg-deposited pine needles revealed the presence of hydrogen peroxide and peroxidase activity in needle tissue close to the eggs. Activity of ROS-producing NADPH-oxidase did not increase after egg deposition. However, the activity of the ROS-detoxifying enzyme catalase decreased after egg deposition and ovipositional wounding of needles. These results show that local ROS accumulation at the oviposition site is not caused by increased NADPH-oxidase activity, but reduced activity of pine needle catalase may contribute to it. However, our data suggest that pine sawflies can counteract the egg deposition-induced hydrogen peroxide accumulation in pine needles by high catalase activity in their oviduct secretion which is released with the eggs into pine tissue.

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.

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.

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

Insectivorous birds are attracted to insect-damaged deciduous trees by plant cues. However, it is unknown whether birds can locate insects on coniferous trees by plant cues induced by insect feeding activity. Feeding damage may induce plant chemical changes even in parts of a tree where no actual infestation is present. In a laboratory set-up, we studied the behavioural responses of Great Tits (Parus major) and Blue Tits (Cyanistes caeruleus) to Scots pine (Pinus sylvestris) that had been systemically herbivore-induced by pine sawfly larvae (Diprion pini). When birds were simultaneously offered an untreated control Scots pine branch and a systemically herbivore-induced test branch (i.e. a branch without actually feeding larvae) in a study booth, they visited the systemically herbivore-induced test branch first significantly more often than the control branch. In order to elucidate whether this discrimination was based on visual and/or olfactory plant cues, we compared the light reflectance and odour of test and control branches in a separate experiment. The control branches tended to show higher light reflectance throughout the avian vision range of 300–700 nm than the test branches, but the discrimination threshold model suggests that birds are not able to detect this difference. The systemically herbivore-induced branches emitted the same 29 volatile compounds as control branches, but 21 of them were released in greater quantities by the test branches. Our study shows that herbivore-induced Scots pine branches emit olfactory cues that may allow the birds to discriminate between uninfested and insect-infested trees, while the role of visual, systemically induced plant cues is less obvious and needs further investigation.

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.

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)

Insectivorous birds feed upon all developmental stages of herbivorous insects, including insect eggs if larvae and adults are unavailable. Insect egg deposition on plants can induce plant traits that are subsequently exploited by egg parasitoids searching for hosts. However, it is unknown whether avian predators can also use egg-induced plant changes for prey localization. Here, we studied whether great tits (Parus major) and blue tits (Cyanistes caeruleus) are attracted by traits of the Scots pine (Pinus sylvestris) induced by pine sawfly (Diprion pini) egg deposition. We chose this plant – insect system because sawfly egg deposition on pine needles is known to locally and systemically induce a change in pine volatile organic compounds (VOCs), and tits are known to prey upon sawfly eggs. In dual choice laboratory experiments, we simultaneously offered the birds an egg-free control branch and a systemically egg-induced branch. Significantly more birds visited the egg-induced branch first. We confirmed by GC-MS analyses that systemically egg-induced branches released more (E)-β-farnesene compared to control branches. Spectrophotometric analyses showed that control branches reflected more light than egg-induced branches throughout the avian visual range. Although a discrimination threshold model for blue tits suggests that the birds are poor at discriminating this visual difference, the role of visual stimuli in attracting the birds to egg-induced pines cannot be discounted. Our study shows, for the first time, that egg-induced odorous and/or visual plant traits can help birds to locate insect eggs without smelling or seeing those eggs.

Plants can improve their antiherbivore defence by taking insect egg deposition as cue of impending feeding damage. Previous studies showed that Pieris brassicae larvae feeding upon egg-deposited Brassicaceae perform worse and gain less weight than larvae on egg-free plants. We investigated how P. brassicae oviposition on Arabidopsis thaliana affects the plant's molecular and chemical responses to larvae. A transcriptome comparison of feeding-damaged leaves without and with prior oviposition revealed about 200 differently expressed genes, including enhanced expression of PR5, which is involved in salicylic acid (SA)-signalling. SA levels were induced by larval feeding to a slightly greater extent in egg-deposited than egg-free plants. The adverse effect of egg-deposited wild-type (WT) plants on larval weight was absent in an egg-deposited PR5-deficient mutant or other mutants impaired in SA-mediated signalling, that is, sid2/ics1, ald1, and pad4. In contrast, the adverse effect of egg-deposited WT plants on larvae was retained in egg-deposited npr1 and wrky70 mutants impaired further downstream in SA-signalling. Oviposition induced accumulation of flavonols in WT plants with and without feeding damage, but not in the PR5-deficient mutant. We demonstrated that egg-mediated improvement of A. thaliana's antiherbivore defence involves SA-signalling in an NPR1-independent manner and is associated with accumulation of flavonols.

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.

Plants may take insect eggs on their leaves as a warning of future herbivory and intensify their defence against feeding larvae. Responsible agents are, however, largely unknown, and little knowledge is available on this phenomenon in perennial plants. We investigated how egg deposition affects the anti-herbivore defence of elm against the multivoltine elm leaf beetle. Prior egg deposition caused changes in the quality of feeding-damaged leaves that resulted in increased larval mortality and reduced reproductive capacity of the herbivore by harming especially female larvae. Chemical analyses of primary and secondary leaf metabolites in feeding-damaged, egg-free (F) and feeding-damaged, egg-deposited (EF)-leaves revealed only small differences in concentrations when comparing metabolites singly. However, a pattern-focused analysis showed clearly separable patterns of (F) and (EF)-leaves because of concentration differences in especially nitrogen and phenolics, of which robinin was consumed in greater amounts by larvae on (EF) than on (F)-leaves. Our study shows that insect egg deposition mediates a shift in the quantitative nutritional pattern of feeding-damaged leaves, and thus might limit the herbivore's population growth by reducing the number of especially female herbivores. This may be a strategy that pays off in a long run particularly in perennial plants against multivoltine herbivores.

Elms (Ulmus spp.) have long been appreciated for their environmental tolerance, landscape and ornamental value, and the quality of their wood. Although elm trees are extremely hardy against abiotic stresses such as wind and pollution, they are susceptible to attacks of biotic stressors. Over 100 phytopathogens and invertebrate pests are associated with elms: fungi, bacteria and insects like beetles and moths, and to a lesser extent aphids, mites, viruses and nematodes. While the biology of the pathogen and insect vector of the Dutch elm disease has been intensively studied, less attention has been paid so far to the defence mechanisms of elms to other biotic stressors. This review highlights knowledge of direct and indirect elm defences against biotic stressors focusing on morphological, chemical and gene regulation aspects. First, we report how morphological defence mechanisms via barrier formation and vessel occlusion prevent colonisation and spread of wood- and bark-inhabiting fungi and bacteria. Second, we outline how secondary metabolites such as terpenoids (volatile terpenoids, mansonones and triterpenoids) and phenolics (lignans, coumarins, flavonoids) in leaves and bark are involved in constitutive and induced chemical defence mechanisms of elms. Third, we address knowledge on how the molecular regulation of elm defence is orchestrated through the interaction of a huge variety of stress- and defence-related genes. We conclude by pointing to the gaps of knowledge on the chemical and molecular mechanisms of elm defence against pest insects and diseases. An in-depth understanding of defence mechanisms of elms will support the development of sustainable integrated management of pests and diseases attacking elms.