Butterfly eggs prime anti-herbivore defense in an annual but not perennial Arabidopsis species.

MAIN CONCLUSION: Unlike Arabidopsis thaliana, defenses of Arabidopsis lyrata against Pieris brassicae larval feeding are not primable by P. brassicae eggs. Thus, egg primability of plant anti-herbivore defenses is not phylogenetically conserved in the genus Arabidopsis. While plant anti-herbivore defenses of the annual species Arabidopsis thaliana were shown to be primable by Pieris brassicae eggs, the primability of the phylogenetically closely related perennial Arabidopsis lyrata has not yet been investigated. Previous studies revealed that closely related wild Brassicaceae plant species, the annual Brassica nigra and the perennial B. oleracea, exhibit an egg-primable defense trait, even though they have different life spans. Here, we tested whether P. brassicae eggs prime anti-herbivore defenses of the perennial A. lyrata. We exposed A. lyrata to P. brassicae eggs and larval feeding and assessed their primability by (i) determining the biomass of P. brassicae larvae after feeding on plants with and without prior P. brassicae egg deposition and (ii) investigating the plant transcriptomic response after egg deposition and/or larval feeding. For comparison, these studies were also conducted with A. thaliana. Consistent with previous findings, A. thaliana's response to prior P. brassicae egg deposition negatively affected conspecific larvae feeding upon A. thaliana. However, this was not observed in A. lyrata. Arabidopsis thaliana responded to P. brassicae eggs with strong transcriptional reprogramming, whereas A. lyrata responses to eggs were negligible. In response to larval feeding, A. lyrata exhibited a greater transcriptome change compared to A. thaliana. Among the strongly feeding-induced A. lyrata genes were those that are egg-primed in feeding-induced A. thaliana, i.e., CAX3, PR1, PR5, and PDF1.4. These results suggest that A. lyrata has evolved a robust feeding response that is independent from prior egg exposure.

Sawfly Sex Pheromones: Analysis of Their Impact on Pine Odor Attractive to Egg Parasitoids.

Pinus sylvestris trees are known to efficiently defend themselves against eggs of the herbivorous sawfly Diprion pini. Their direct defense against eggs is primable by prior exposure to the sex pheromones of this species and their indirect defense involves attraction of egg parasitoids by egg-induced pine needle odor. But it is unknown whether exposure of pine to D. pini sex pheromones also affects pine indirect defense against sawfly eggs. In this study, we investigated the influence of exposure of P. sylvestris trees to the sex pheromones of D. pini on indirect defense mediated by egg parasitoids. Behavioral assays with Closterocerus ruforum, a key parasitoid of sawfly eggs, revealed no significant attraction to odor from egg-free pines pre-exposed to pheromones. Chemical analyses of odor from egg-free pines showed no pheromone-induced change in the emission rates of the known key terpenoids promoting parasitoid attraction. Further comparative analyses of odor from egg-laden pines pre-exposed to the sex pheromones and of odor from egg-laden pines unexposed to pheromones neither revealed significant differences in the emission rates of terpenoids relevant for parasitoid attraction. The results suggest that a pheromone-induced or pheromone-primed, egg-induced pine indirect defense seems to be redundant in addition to the known pheromone-primable pine direct defense against the eggs and the known egg-inducible indirect defense.

Pine Response to Sawfly Pheromones: Effects on Sawfly's Oviposition and Larval Growth.

Insect pheromones have been intensively studied with respect to their role in insect communication. However, scarce knowledge is available on the impact of pheromones on plant responses, and how these in turn affect herbivorous insects. A previous study showed that exposure of pine (Pinus sylvestris) to the sex pheromones of the pine sawfly Diprion pini results in enhanced defenses against the eggs of this sawfly; the egg survival rate on pheromone-exposed pine needles was lower than that on unexposed pine. The long-lasting common evolutionary history of D. pini and P. sylvestris suggests that D. pini has developed counter-adaptations to these pine responses. Here, we investigated by behavioral assays how D. pini copes with the defenses of pheromone-exposed pine. The sawfly females did not discriminate between the odor of pheromone-exposed and unexposed pine. However, when they had the chance to contact the trees, more unexposed than pheromone-exposed trees received eggs. The exposure of pine to the pheromones did not affect the performance of larvae and their pupation success. Our findings indicate that the effects that responses of pine to D. pini sex pheromones exert on the sawfly eggs and sawfly oviposition behavior do not extend to effects on the larvae.

Drought induces moderate, diverse changes in the odour of grassland species.

Plants react to drought stress with numerous changes including altered emissions of volatile organic compounds (VOC) from leaves, which provide protection against oxidative tissue damage and mediate numerous biotic interactions. Despite the share of grasslands in the terrestrial biosphere, their importance as carbon sinks and their contribution to global biodiversity, little is known about the influence of drought on VOC profiles of grassland species. Using coupled gas chromatography-mass spectrometry, we analysed the odorants emitted by 22 European grassland species exposed to an eight-week-lasting drought treatment (DT; 30% water holding capacity, WHC). We focused on the odorants emitted during the light phase from whole plant shoots in their vegetative stage. Emission rates were standardised to the dry weight of each shoot. Well-watered (WW) plants (70% WHC) served as control. Drought-induced significant changes included an increase in total emission rates of plant VOC in six and a decrease in three species. Diverging effects on the number of emitted VOC (chemical richness) or on the Shannon diversity of the VOC profiles were detected in 13 species. Biosynthetic pathways-targeted analyses revealed 13 species showing drought-induced higher emission rates of VOC from one, two, three, or four major biosynthetic pathways (lipoxygenase, shikimate, mevalonate and methylerythritol phosphate pathway), while six species exhibited reduced emission rates from one or two of these pathways. Similarity trees of odorant profiles and their drought-induced changes based on a biosynthetically informed distance metric did not match species phylogeny. However, a phylogenetic signal was detected for the amount of terpenoids released by the studied species under WW and DT conditions. A comparative analysis of emission rates of single compounds released by WW and DT plants revealed significant VOC profile dissimilarities in four species only. The moderate drought-induced changes in the odorant emissions of grassland species are discussed with respect to their impact on trophic interactions across the food web. (294 words).

Microbial associates of the elm leaf beetle: uncovering the absence of resident bacteria and the influence of fungi on insect performance.

Microbial symbionts play crucial roles in the biology of many insects. While bacteria have been the primary focus of research on insect-microbe symbiosis, recent studies suggest that fungal symbionts may be just as important. The elm leaf beetle (ELB, Xanthogaleruca luteola) is a serious pest species of field elm (Ulmus minor). Using culture-dependent and independent methods, we investigated the abundance and species richness of bacteria and fungi throughout various ELB life stages and generations, while concurrently analyzing microbial communities on elm leaves. No persistent bacterial community was found to be associated with the ELB or elm leaves. By contrast, fungi were persistently present in the beetle's feeding life stages and on elm leaves. Fungal community sequencing revealed a predominance of the genera Penicillium and Aspergillus in insects and on leaves. Culture-dependent surveys showed a high prevalence of two fungal colony morphotypes closely related to Penicillium lanosocoeruleum and Aspergillus flavus. Among these, the Penicillium morphotype was significantly more abundant on feeding-damaged compared with intact leaves, suggesting that the fungus thrives in the presence of the ELB. We assessed whether the detected prevalent fungal morphotypes influenced ELB's performance by rearing insects on (i) surface-sterilized leaves, (ii) leaves inoculated with Penicillium spores, and (iii) leaves inoculated with Aspergillus spores. Insects feeding on Penicillium-inoculated leaves gained more biomass and tended to lay larger egg clutches than those consuming surface-sterilized leaves or Aspergillus-inoculated leaves. Our results demonstrate that the ELB does not harbor resident bacteria and that it might benefit from associating with Penicillium fungi.IMPORTANCEOur study provides insights into the still understudied role of microbial symbionts in the biology of the elm leaf beetle (ELB), a major pest of elms. Contrary to expectations, we found no persistent bacterial symbionts associated with the ELB or elm leaves. Our research thus contributes to the growing body of knowledge that not all insects rely on bacterial symbionts. While no persistent bacterial symbionts were detectable in the ELB and elm leaf samples, our analyses revealed the persistent presence of fungi, particularly Penicillium and Aspergillus on both elm leaves and in the feeding ELB stages. Moreover, when ELB were fed with fungus-treated elm leaves, we detected a potentially beneficial effect of Penicillium on the ELB's development and fecundity. Our results highlight the significance of fungal symbionts in the biology of this insect.

The impact of insect egg deposition on Pinus sylvestris transcriptomic and phytohormonal responses to larval herbivory.

Plants can improve their resistance to feeding damage by insects if they have perceived insect egg deposition prior to larval feeding. Molecular analyses of these egg-mediated defence mechanisms have until now focused on angiosperm species. It is unknown how the transcriptome of a gymnosperm species responds to insect eggs and subsequent larval feeding. Scots pine (Pinus sylvestris L.) is known to improve its defences against larvae of the herbivorous sawfly Diprion pini L. if it has previously received sawfly eggs. Here, we analysed the transcriptomic and phytohormonal responses of Scots pine needles to D. pini eggs (E-pine), larval feeding (F-pine) and to both eggs and larval feeding (EF-pine). Pine showed strong transcriptomic responses to sawfly eggs and-as expected-to larval feeding. Many egg-responsive genes were also differentially expressed in response to feeding damage, and these genes play an important role in biological processes related to cell wall modification, cell death and jasmonic acid signalling. EF-pine showed fewer transcriptomic changes than F-pine, whereas EF-treated angiosperm species studied so far showed more transcriptional changes to the initial phase of larval feeding than only feeding-damaged F-angiosperms. However, as with responses of EF-angiosperms, EF-pine showed higher salicylic acid concentrations than F-pine. Based on the considerable overlap of the transcriptomes of E- and F-pine, we suggest that the weaker transcriptomic response of EF-pine than F-pine to larval feeding damage is compensated by the strong, egg-induced response, which might result in maintained pine defences against larval feeding.

Plant defensive responses to insect eggs are inducible by general egg-associated elicitors.

Egg deposition by herbivorous insects is well known to elicit defensive plant responses. Our study aimed to elucidate the insect and plant species specificity of these responses. To study the insect species specificity, we treated Arabidopsis thaliana with egg extracts and egg-associated secretions of a sawfly (Diprion pini), a beetle (Xanthogaleruca luteola) and a butterfly (Pieris brassicae). All egg extracts elicited salicylic acid (SA) accumulation in the plant, and all secretions induced expression of plant genes known to be responsive to the butterfly eggs, among them Pathogenesis-Related (PR) genes. All secretions contained phosphatidylcholine derivatives, known elicitors of SA accumulation and PR gene expression in Arabidopsis. The sawfly egg extract did not induce plant camalexin levels, while the other extracts did. Our studies on the plant species specificity revealed that Solanum dulcamara and Ulmus minor responded with SA accumulation and cell death to P. brassicae eggs, i.e. responses also known for A. thaliana. However, the butterfly eggs induced neoplasms only in S. dulcamara. Our results provide evidence for general, phosphatidylcholine-based, egg-associated elicitors of plant responses and for conserved plant core responses to eggs, but also point to plant and insect species-specific traits in plant-insect egg interactions.

Elm tree defences against a specialist herbivore are moderately primed by an infestation in the previous season.

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.

Adaptive Plasticity of Insect Eggs in Response to Environmental Challenges.

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

Priming of Arabidopsis resistance to herbivory by insect egg deposition depends on the plant's developmental stage.

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.

Pine defense against eggs of an herbivorous sawfly is elicited by an annexin-like protein present in egg-associated secretion.

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)-ß-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)-ß-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.

Responses to larval herbivory in the phenylpropanoid pathway of Ulmus minor are boosted by prior insect egg deposition.

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. 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.

Cuticular Hydrocarbon Trails Released by Host Larvae Lose their Kairomonal Activity for Parasitoids by Solidification.

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.

Priming by Timing: Arabidopsis thaliana Adjusts Its Priming Response to Lepidoptera Eggs to the Time of Larval Hatching.

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 Importance of Methyl-Branched Cuticular Hydrocarbons for Successful Host Recognition by the Larval Ectoparasitoid Holepyris sylvanidis.

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.

Arabidopsis, tobacco, nightshade and elm take insect eggs as herbivore alarm and show similar transcriptomic alarm responses.

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.

Insectivorous birds can see and smell systemically herbivore-induced pines.

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.

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

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

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

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

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

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