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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.

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.

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.

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.

Insect parents that experienced an immune challenge are known to prepare (prime) the immune activity of their offspring for improved defence. This phenomenon has intensively been studied by analysing especially immunity-related proteins. However, it is unknown how transgenerational immune priming affects transcript levels of immune-relevant genes of the offspring upon an actual threat. Here, we investigated how an immune challenge of Manduca sexta parents affects the expression of immune-related genes in their eggs that are attacked by parasitoids. Furthermore, we addressed the question whether the transgenerational immune priming of expression of genes in the eggs is still traceable in adult offspring. Our study revealed that a parental immune challenge did not affect the expression of immune-related genes in unparasitised eggs. However, immune-related genes in parasitised eggs of immune-challenged parents were upregulated to a higher level than those in parasitised eggs of unchallenged parents. Hence, this transgenerational immune priming of the eggs was detected only "on demand", i.e. upon parasitoid attack. The priming effects were also traceable in adult female progeny of immune-challenged parents which showed higher transcript levels of several immune-related genes in their ovaries than non-primed progeny. Some of the primed genes showed enhanced expression even when the progeny was left unchallenged, whereas other genes were upregulated to a greater extent in primed female progeny than non-primed ones only when the progeny itself was immune-challenged. Thus, the detection of transgenerational immune priming strongly depends on the analysed genes and the presence or absence of an actual threat for the offspring. We suggest that M. sexta eggs laid by immune-challenged parents "afford" to upregulate the transcription of immunity-related genes only upon attack, because they have the chance to be endowed by parentally directly transferred protective proteins.

Anthropogenic land use may shape vegetation composition and affect trophic interactions by altering concentrations of host plant metabolites. Here, we investigated the hypotheses that: (1) plant N and defensive secondary metabolite contents of the herb Plantago lanceolata are affected by land use intensity (LUI) and the surrounding vegetation composition (=plant species richness and P. lanceolata density), and that (2) changes in plant chemistry affect abundances of the herbivorous weevils Mecinus pascuorum and Mecinus labilis, as well as their larval parasitoid Mesopolobus incultus, in the field. We determined plant species richness, P. lanceolata density, and abundances of the herbivores and the parasitoid in 77 grassland plots differing in LUI index in three regions across Germany. We also measured the N and secondary metabolite [the iridoid glycosides (IGs) aucubin and catalpol] contents of P. lanceolata leaves. Mixed-model analysis revealed that: (1) concentrations of leaf IGs were positively correlated with plant species richness; leaf N content was positively correlated with the LUI index. Furthermore: (2) herbivore abundance was not related to IG concentrations, but correlated negatively with leaf N content. Parasitoid abundance correlated positively only with host abundance over the three regions. Structural equation models revealed a positive impact of IG concentrations on parasitoid abundance in one region. We conclude that changes in plant chemistry due to land use and/or vegetation composition may affect higher trophic levels and that the manifestation of these effects may depend on local biotic or abiotic features of the landscape.

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.

The cuticular hydrocarbon (CHC) profile of the mustard leaf beetle Phaedon cochleariae is known to mediate mate recognition and is dependent on food plant species; beetles previously were shown to prefer mates that fed on the same plant species and which have a similar CHC pattern. In order to elucidate whether the pattern of ingested fatty acids affects the CHC pattern of P. cochleariae adults, we fed beetles: (a) with two different host plant species differing in fatty acid profile; and (b) artificial diets differing mainly in their composition of mono-, di-, and triunsaturated fatty acids. Analyses of the beetles' CHCs revealed that ingestion of different fatty acid blends results in quantitative effects on the beetle's straight-chain and methyl-branched CHCs. Interestingly, CHC patterns of males and females were affected differently by ingestion of fatty acids. In contrast to the effect on mating caused by feeding on different host plant species, beetles that were fed with different artificial diets, leading to different beetle CHC profiles, did not exhibit mating preference. We suggest that the occurrence of CHC-dependent assortative mating in P. cochleariae does not depend on the dietary fatty acids offered to the beetles in this study, but on other food constituents that affect CHC biosynthesis.

Knowledge of the plasticity of Phratora vulgatissima (Colepotera: Chrysomelidae) behavioural responses to cues of its willow host plants is essential for understanding host affiliations of this species and for developing management strategies. 2 We investigated how the experience obtained by adult P. vulgatissima with two different willow species shapes olfactory, feeding and oviposition preferences in the laboratory. The willow species differed in their leaf odours and phenolic glycoside contents. 3 Females that had experienced Salix viminalis (Salicaceae) neither discriminated between odours of S. viminalis and Salix dasyclados, nor between blends of green leaf volatiles (GLV) that mimic quantitatively those released by S. viminalis and S. dasyclados. However, when females had experienced S. dasyclados, they preferred the experienced odour of dasyclados and the respective GLV blend to the odour of S. viminalis or its GLV blend. 4 By contrast, regardless of their experience obtained in the adult stage, females preferred S. viminalis overS. dasyclados for feeding and oviposition. 5 Exposure of beetles to odour of stands with various willow species might affect the experience-induced olfactory preference for a single species and thus impair host location success.

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.

Eggs of the elm leaf beetle Xanthogaleruca luteola are often heavily attacked by the chalcidoid wasp Oomyzus gallerucae. We studied the chemical signals mediating interactions between the egg parasitoid, its host, and the plant Ulmus campestris. Olfactometer bioassays with O. gallerucae showed that volatiles of the host-plant complex attract the parasitoid. In order to determine the source of attractive volatiles within this host-plant-complex, we tested separately the effect of odours of eggs, gravid elm leaf beetle females, faeces of the beetles and elm twigs (with undamaged leaves and leaves damaged either mechanically or by feeding of the beetles). Odours of faeces of the elm leaf beetle were attractive, whereas neither volatiles from eggs nor from gravid females acted as attractants. Volatiles from undamaged or damaged plants did not elicit a positive reaction in O. gallerucae, whereas volatiles from feeding-damaged plants onto which host eggs had been deposited were attractive. This latter result suggests that it is not feeding but deposition of host eggs onto elm leaves that induces the production of plant volatiles attractive to the egg parasitoid. Investigations of the search patterns of O. gallerucae within the habitat by laboratory bioassays revealed that the egg parasitoid encounters host eggs by chance. Contact kairomones from faeces were demonstrated to be important in microhabitat acceptance, while contact kairomones isolated from the host eggs are relevant for host recognition.

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.

Chemical defense of leaf beetle larvae (Chrysomelidae) against enemies is provided by secretions containing a wide range of deterrent compounds or by unpalatable hemolymph constituents. Here we report a new, very strong feeding deterrent against ants released by larvae of the alder leaf beetle Agelastica alni when attacked. The larvae release a defensive fluid from openings of pairwise, dorsolaterally located tubercles on the first to the eighth abdominal segments. The fluid, consisting of hemolymph and probably a glandular cell secretion, has previously been shown to contain a very stable, non-volatile feeding deterrent. The major deterrent component was isolated by repeated HPLC separation and analyzed by NMR and MS. The compound proved to be gamma-L-glutamyl-L-2-furylalanine (1), a novel dipeptide containing the unusual amino acid L-2-furylalanine. This amino acid, although synthetically well known, has not previously been reported from natural sources. The absolute configuration of the natural compound was elucidated by enantioselective gas chromatography after derivatization. The structure of the dipeptide was verified by the synthesis of several isomeric dipeptides. In bioassays a concentration of 1 microg microL(-1) was sufficient to deter polyphagous Myrmica rubra ants from feeding.

Pinus sylvestris (Scots pine) is known to respond to eggs laid by the sawfly Diprion pini on its needles by releasing a blend of terpenoids, including the sesquiterpene ( E )- β -farnesene. These compounds attract a wasp, Closterocerus ruforum , which parasitizes sawfly eggs. D . pini oviposition also enhances the transcription of two sesquiterpene synthases, an ( E )- β -caryophyllene/ α -humulene synthase ( PsTPS 1) and a 1(10),5-germacradiene-4-ol synthase ( PsTPS 2). To gain a better understanding of the function of these sesquiterpenes in promoting insect egg parasitism, we compared the outcome of D . pini oviposition on P . sylvestris with interactions between other pine and sawfly species: Neodiprion sertifer eggs on P . sylvestris , Gilpinia pallida eggs on P . sylvestris , D . pini eggs on Pinus nigra . The first of these attracts the parasitoid C . ruforum , while the latter two do not. As determined by quantitative real-time PCR, both PsTPS 1 and PsTPS 2 transcripts increased significantly only for those species combinations where the odor of egg–laden pine needles was attractive to C . ruforum . Moreover, enhanced transcription of these genes was found only at those time periods when odor was attractive, i.e. 3 days after oviposition. Thus, the PsTPS 1 and PsTPS 2 genes are good markers for parasitoid attraction. We also characterized a sesquiterpene synthase from P . sylvestris ( PsTPS 5) which produces ( E )- β -farnesene, the compound previously determined to be responsible for C . ruforum attraction. However, transcript levels of PsTPS 5 were not enhanced by oviposition of sawfly species that cause C . ruforum attraction. More research on this experimental system is required to determine the role of oviposition-induced sesquiterpenes in attracting egg parasitoids and the role of sesquiterpene synthases in regulating sesquiterpene formation.

Olfactory orientation by insects may be guided by specific volatile blends released from sites where resources are present. Such blends need to be recognized against the odorous background of the habitat. The egg parasitoid Closterocerus ruforum is known to be attracted to plant volatiles induced by egg deposition of its herbivorous host, the pine sawfly Diprion pini. The parasitoid has to detect this volatile signal against a background of other pine volatiles (i.e. odour from pine without host eggs). Previous studies have shown that attractive resource-indicating odour (pine with host eggs) and nonattractive habitat odour (pine without eggs) differ only by enhanced quantities of (E)-β-farnesene in the resource-indicating odour. However, (E)-β-farnesene per se was not attractive. We studied the relevance of quantitative ratios of (E)-β-farnesene and other pine volatiles present in the habitat for parasitoid orientation by electrophysiological methods (EAG) and behavioural (olfactometer) assays. Seven of 12 terpenoid pine volatiles elicited an EAG response. In tests of the parasitoid's behavioural response to EAG-active volatiles, a blend of five components (including (E)-β-farnesene) was sufficient to attract the parasitoid but only when the ratio of (E)-β-farnesene and the other compounds matched that in odour from egg-laden pine. Since the quantities of volatiles from egg-laden and egg-free pine are not significantly different except for (E)-β-farnesene, our results show that the quantity of a resource-indicating key compound (here (E)-β-farnesene) is validated by its ratio to quantities of background volatiles emitted by both the resource and the habitat.

We investigated the role that cuticular hydrocarbons (CHC) play in sexual communication by the mustard leaf beetle, Phaedon cochleariae (Coleoptera: Chrysomelidae). In laboratory bioassays, male P. cochleariae attempted to copulate with living or freeze-killed females as often as with males. However, the duration of copulation with females was longer than with males. To elucidate the impact of CHC on this behavior, cuticular compounds of adults of both sexes were extracted with dichloromethane. Male mating attempts with glass beads treated with the dichloromethane extract were nearly as frequent as with living beetles. The dichloromethane extract was fractionated by silica gel chromatography, and the biological activity of the fractions was tested by applying them to glass beads. A non-polar hexane fraction significantly elicited mating behavior, whereas the polar methanol fraction did not, likely because it contained defensive compounds from exocrine glands located in the elytra and pronota. Interestingly, a mixture of both the non-polar and polar fraction tended to elicit more mating attempts than did the non-polar hexane fraction alone. Further fractionation of the significantly active hexane fraction by silver nitrate column chromatography revealed that saturated CHC elicited mating behavior, but the olefins did not. GC-MS analyses of dichloromethane cuticular extracts showed that the male and female CHC profiles were qualitatively identical, but differed in their relative composition. Canonical discriminant analysis showed that CHC profiles of males and females formed separate clusters. Nevertheless, the results of our bioassays demonstrated that male and female CHC did not elicit sex discriminative male behavior, but induced mating by males regardless of the sex of the partner.

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.

Parasitoids of herbivorous larvae are well known to be able to associatively learn a response to feeding-induced plant volatiles. However, little is known of the abilities of egg parasitoids to learn a response to herbivore oviposition-induced plant volatiles (OVIV). The eulophid wasp Chrysonotomyia ruforum is known to be attracted by OVIV from Scots pine released 3 days after host egg deposition. This study aimed to elucidate (1) the impact of the nutritional state of the parasitoid on its response to OVIV and (2) the effect of learning and response to the temporal change of OVIV. Supply of food as a reward during associative learning trials inevitably influences the nutritional state and thus leads to an experimental constraint. Nevertheless, the results indicate that the parasitoids have to associate OVIV with host presence and that they need to be supplied with food to learn and to respond to OVIV. Furthermore, the parasitoids' positive response to OVIV from pine twigs laden with eggs for 3 days was independent of the induction time of pine twigs experienced. However, a positive response was given only to OVIV from pine twigs with 3-day-old eggs, while OVIV from twigs with other induction times (1, 2, 4 days) were not attractive. Thus, our findings indicate an unusual learning process in this specialized egg parasitoid. We suggest that the parasitoid is using a learning strategy by which common information is filtered out of similar odour blends (here: 1- to 4-day-long induced pine twigs), but a behavioural response to this learned information becomes evident only in a specific odour context (here: 3-day-long induced twigs).

Plants can recruit parasitic wasps in response to egg deposition by herbivorous insects–a sophisticated indirect plant defense mechanism. Oviposition by the Large Cabbage White butterfly Pieris brassicae on Brussels sprout plants induces phytochemical changes that arrest the egg parasitoid Trichogramma brassicae . Here, we report the identification of an elicitor of such an oviposition-induced plant response. Eliciting activity was present in accessory gland secretions released by mated female butterflies during egg deposition. In contrast, gland secretions from virgin female butterflies were inactive. In the male ejaculate, P. brassicae females receive the anti-aphrodisiac benzyl cyanide (BC) that reduces the females' attractiveness for subsequent mating. We detected this pheromone in the accessory gland secretion released by mated female butterflies. When applied onto leaves, BC alone induced phytochemical changes that arrested females of the egg parasitoid. Microarray analyses revealed a similarity in induced plant responses that may explain the arrest of T. brassicae to egg-laden and BC-treated plants. Thus, a male-derived compound endangers the offspring of the butterfly by inducing plant defense. Recently, BC was shown to play a role in foraging behavior of T. brassicae , by acting as a cue to facilitate phoretic transport by mated female butterflies to oviposition sites. Our results suggest that the anti-aphrodisiac pheromone incurs fitness costs for the butterfly by both mediating phoretic behavior and inducing plant defense.

Plants can respond to attack by herbivorous insects very soon after herbivores start producing a new generation by depositing eggs onto their leaves. Egg-induced plant responses may result in killing the attacker in its egg stage. However, if the eggs do survive, they can also prime feeding-induced plant defenses against the larvae hatching from eggs. In this paper we focus first on egg-induced plant responses that resemble hypersensitive responses (HR) to phytopathogens and lead to egg desiccation or detachment from plants. We then summarize the current knowledge about egg-mediated effects on feeding-induced plant defenses against larvae. Finally, we discuss the insect species specificity of plant responses to eggs and the variability of insect susceptibility to these responses.

The role of soil characteristics for cue-directed behaviour of rhizophagous insects remains largely unexplored to date. Here, we studied behavioural responses of polyphagous cockchafer larvae (Melolontha melolontha; Scarabaeidae) to roots of one of its preferred host plants, dandelion (Taraxacum sectio ruderalia; Asteraceae). Plants were grown in substrates with different adsorptive capacities, i.e. in vermiculite or sand. A behavioural assay was developed which allowed both monitoring the release of attractive CO2 from roots and observation of larval behaviour at the same time. In sand, larvae got closer to the roots than in vermiculite where they eventually stopped orienting towards the roots: However, their total locomotory activity and the number of turning events/stationary points were higher in vermiculite. Larval behaviour was not correlated with CO2 concentrations in both vermiculite and sand. On average, larvae kept a greater distance to vermiculite-grown roots. However, a feeding bioassay revealed that the larvae consumed more of vermiculite-grown roots than of sand-grown ones. This result showed that the weaker orientation of larvae towards vermiculite-grown roots was not due to the lower palatability of these roots. Vermiculite might adsorb foraging-relevant cues of dandelion roots, alter composition of root exudates, and/or physically impair oriented movements of soil-dwelling insects. Our study shows that the type of substrate affects responses of rhizophagous larvae to host roots at multiple levels independently of root-derived CO2 gradients.

Among numerous other factors, host-plant volatiles may affect selection of food plants by herbivorous insects. The blue willow leaf beetle, Phratora vulgatissima (L.) (Coleoptera: Chrysomelidae), is known to differentiate between willow species and genotypes. However, so far no knowledge is available on the physiological abilities of this leaf beetle to respond to willow volatiles. In this study, we recorded electroantennograms of male and female P. vulgatissima to volatiles from two Salix viminalis L. (Salicaceae) genotypes: Jorr and 78021. The headspace of these genotypes were analysed by coupled gas chromatography-mass spectrometry. In addition to known green leaf volatiles (GLV), several terpenoid components were found. Both males and females of P. vulgatissima showed strong responses to the GLVs (Z)-3-hexenol and (Z)-3-hexenyl acetate, and moderate responses to (E)-ocimene and β-caryophyllene. Females, but not males, also responded to R-(+)-limonene. This work represents a further step to identify substances relevant for the orientation of P. vulgatissima to host plants.

Wolbachia are obligatory, cytoplasmatically inherited alpha-proteobacteria, which are common endosymbionts in arthropods where they may cause reproductive abnormalities. Many insects are well known to protect themselves from deleterious microorganisms by antibiotic components. In this study, we addressed the question whether Wolbachia are able to infect insects containing antimicrobial anthraquinones and anthrones, and if so, whether these genotypes of Wolbachia comprise a monophyletic cluster within one of the known supergroups. Leaf beetles of the taxon Galerucini (Galerucinae) are known to contain 1,8-dihydroxylated anthraquinones and anthrones. Also, the scale insect Dactylopius contains an anthraquinone glycoside, carminic acid. Our analyses revealed that a representative of the Galerucini, Galeruca tanaceti and Dactylopius, are indeed infected by endosymbiotic Wolbachia bacteria. Phylogenetic analysis of the wsp and ftsZ genes of these bacteria revealed that strains in G. tanaceti cluster in supergroup A, whereas those present in Dactylopius are distinctive from each other and from those of G. tanaceti. They are clustering in supergroups A and B. Wolbachia strains present in close, but anthraquinone-free relatives of G. tanaceti were shown to belong also to supergroup A. From these results, we can conclude (1) a double infection in Dactylopius, (2) that the presence of antimicrobial compounds such as anthraquinones does not necessarily protect insects from infection by Wolbachia, and (3) that genotypes of Wolbachia-infecting anthraquinone-containing insects most likely do not comprise a unique genotype. These results show that Wolbachia bacteria might be adapted to cope even with conditions usually detrimental to other bacteria and that these adaptations are widespread among Wolbachia supergroups.

An unusual and complex tropane alkaloid pattern has been detected in the root bark of Astripomoea malvacea and in the whole plant of Falkia repens (Convolvulaceae) by GC-MS analysis. The specific profile of both species is characterized by the presence of aliphatic 3- acyloxytropanes/-nortropanes (exclusively in A. malvacea; predominantly in F. repens in cooccurrence with a few aromatic as well as arylalkyl acyl congeners). The principal alkaloid of A. malvacea, astrimalvine A N-oxide [3β -(3-tigloyloxy-2-methylbutyryloxy)tropane N-oxide], isolated and structurally elucidated by detailed spectroscopic analysis, represents the first N-oxide of a 3β -tropanol derivative in the Convolvulaceae. Its minor tertiary congener astrimalvine B [3β -(3-hydroxy-2-methylbutyryloxy)tropane] turned out to be a metabolite of both convolvulaceaous species. This is the first phytochemical report on the African genera Astripomoea and Falkia.

Contains fulltext : 36396.pdf (Publisher’s version ) (Closed access) fence in response to attack by an herbivorous sawfly. Egg deposition by the sawfly Diprion pini L. induces pine to release, three days after egg laying, locally and systemically terpenoid volatiles that attract parasitoids to kill the eggs. The elicitor of the pine's response is located in the sawfly's oviduct secretion enveloping the eggs after deposition. Application of this secretion on twigs with artificially conducted ovipositional woundings mimics the effects of egg deposition. Furthermore, jasmonic acid (JA) induces a volatile pattern similar, but not identical, to the one induced by egg deposition. To gain deeper insight into the transcluction of plant signals induced by herbivore egg deposition, it was investigated whether ethylene emission from pine is affected by sawfly egg deposition. Systemically induced ethylene emission from differently treated pine twigs was monitored for a period of 3 d after treatment. Ethylene emissions from untreated control twigs were compared with those from twigs treated as follows: (i) sawfly egg secretion [= oviduct secretion (OVI)] was transferred on artificially wounded pine needles (attractive volatiles), (ii) needles were artificially wounded (non-attractive volatiles), and (iii) the twig was supplied with JA (attractive volatiles). Ethylene emission from systemically OVI-induced twigs was significantly lower than from untreated controls, whereas artificial wounding had no detectable effect. JA-treated twigs released much more ethylene and showed higher variability of ethylene emission than artificially wounded twigs and OVI-treated ones. Ethylene emissions from pine after the various treatments studied here are discussed with respect to known effects of insect feeding on ethylene release from plants.

Parasitoids are confronted with many different infochemicals of their hosts and food plants during host selection. Here, we investigated the effect of kairomones from the adult host Pieris brassicae and of cues present on Brussels sprout plants infested by P. brassicae eggs on the behavioral response of the egg parasitoid Trichogramma evanescens. Additionally, we tested whether the parasitoid¿s acceptance of P. brassicae eggs changes with different host ages. The wasps did not discriminate between olfactory cues from mated and virgin females or between mated females and males of P. brassicae. T. evanescens randomly climbed on the butterflies, showing a phoretic behavior without any preference for a certain sex. The parasitoid was arrested on leaf parts next to 1-day-old host egg masses. This arrestment might be due to cues deposited during oviposition. The wasps parasitized host eggs up to 3 days old equally well. Our results were compared with former studies on responses by T .brassicae showing that T. evanescens makes less use of infochemicals from P. brassicae than T. brassicae.

Eggs of several insect species are protected against natural enemies by noxious components. However, almost nothing is known about the fate of these defensive substances during egg development nor their site of biosynthesis. The eggs of several leaf beetle species of the taxon Galerucini contain components that are unusual in insects: 1,8-dihydroxylated anthraquinones and anthrones that deter predators such as ants and birds. These components, i.e., the anthrones dithranol and chrysarobin, and the anthraquinones chrysazin and chrysophanol, are not sequestered from host plants. We asked whether the amounts of these components in the overwintering eggs of Galeruca tanaceti change from deposition to larval hatching. Gas chromatography-mass spectroscopy (GC-MS) analyses of eggs revealed a significant decrease in total amounts of dithranol and chrysophanol from egg deposition in autumn to the next spring 5 months later. Thus, these results do not provide any hint of active anthraquinone biosynthesis within eggs. Instead, the anthrones and anthraquinones that must be incorporated by the female into the eggs seem to be degraded to some extent either by the embryo or endosymbionts. GC-MS analyses showed that parasitization of eggs had some effects on the quantities of anthrones and anthraquinones.

Melolontha cockchafer males search for mates using green leaf volatiles (GLV), released by host plants after female feeding. Thus, the feeding-induced plant volatiles act as sexual kairomones. Males of both Melontha hippocastani and Melontha melolontha are strongly attracted by the GLV (Z)-3-hexen-1-ol (Z-3-ol). Sex pheromones enhance the attractiveness of Z-3-ol and have been identified as toluquinone (TQ) in M. melolontha, and 1,4-benzoquinone (BQ) in M. hippocastani. Additionally, phenol acts as a male attractant in both species. From the perspective of potential application, we investigated by field experiments with volatile-baited traps the ways of enhancing the number of captured males by the use of specific binary or ternary blends of Z-3-ol with phenol, and TQ or BQ respectively. The data show that in both species binary lures containing Z-3-ol combined with TQ or BQ at a ratio of 10 : 1 are the most potent male attractants.

Allopatric populations of the leaf beetle Chrysomela lapponica are known to feed upon either willow (Salicaceae) or birch (Betulaceae). This study aimed to elucidate the differentiation process of these allopatric populations. We investigated whether these allopatric populations specialized on different host plants are still able to produce fertile offspring when interbreeding. Individuals from a population in Finland (willow specialists) and one in the Czech Republic (birch specialists) were crossed in laboratory. Hybrid formation succeeded only between females from the Czech, birch specialized population and males from the Finnish, willow specialized population, while no eggs were produced by females of the willow specialists having mated males of the birch specialists. Behavioral, morphological, physiological, and chemical features of the F1 hybrids were studied. The chemical composition of larval defensive secretion and feeding preferences of the resulting F1 hybrids mainly showed similarities with the paternal phenotype, while the area of black coloring on the offspring¿s elytra was intermediate between those of the parental elytra. F1 hybrids did not accept the host plant (birch) of their mothers for feeding and only survived on willow. Thus, since mothers only lay eggs on birch, we found evidence for a postzygotic isolation mechanism between the individuals of the two investigated populations: when having been mated with willow specialized Finnish males, the birch specialized Czech mothers place the hybrid eggs on a plant species (birch), on which the hatching larvae cannot survive.

SUMMARYEgg deposition by the phytophagous sawfly Diprion pini L.(Hymenoptera, Diprionidae) is known to induce locally and systemically the emission of volatiles in Scots pine (Pinus sylvestris L.) that attract the egg parasitoid Chrysonotomyia ruforum Krausse(Hymenoptera, Eulophidae). The egg parasitoids kill the eggs and thus prevent damage to the plant from feeding sawfly larvae. The elicitor inducing the pine's response is known to be located in the oviduct secretion which the female sawfly applies to the eggs when inserting them into a slit in the pine needle using the sclerotized ovipositor valves. In this study we have characterized the elicitor. The elicitor was still active when isolated from the oviduct and applied directly to slits made in the pine needles. However,as soon as the oviduct secretion was dissolved in Aqua dest. and stored for 3 h at room temperature or kept frozen at -80°C, its activity was lost. In contrast, oviduct secretion kept its eliciting activity, when dissolved in Ringer solution (pH 7.2) both after storage at room temperature and after freezing. The activity of the elicitor vanished after treatment of the oviduct secretion with proteinase K, which destroyed all proteins. This suggests that the elicitor in the oviduct secretion is a peptide or protein, or a component bound to these. SDS-PAGE revealed a similar, but not identical protein pattern from hemolymph and oviduct secretion. Hemolymph itself has no eliciting effect. The elicitor in the oviduct secretion is only active when transferred to slit pine needles, since its application on undamaged needles did not induce the emission of attractive volatiles.

Male Melolontha cockchafers are known to use green leaf volatiles induced by female feeding on host plants for their mate location. Earlier studies of the response of the European cockchafer, Melolontha melolontha L. (Coleoptera: Scarabaeidae), to different green leaf aldehydes, alcohols, and acetates revealed that only green leaf alcohols were attractive to males in the field. Females were not attracted at all by these volatiles. Here, we present a study that aimed to elucidate the structure-activity relationships of aliphatic alcohols. Both behavioural and physiological responses were studied in male and female M. melolontha by field tests and electroantennography. The compounds tested were saturated aliphatic alcohols with chain lengths between five and eight carbon atoms. Furthermore, the cockchafer's response to six-carbon alcohols with (E)-2-, (E)-3-, (Z)-2-, (Z)-3-, and (Z)-4-configurated double bonds was tested. All compounds elicited dose-dependent responses on the antennae of both sexes. In general, males showed a stronger normalized EAG response to the stimuli than females. However, only the naturally occurring six-carbon alcohols, i.e., 1-hexanol (E )-2-, (Z)-3, and (E)-3-hexen-1-ol were attractive to M. melolontha males in the field. Females were not attracted to any of the tested compounds, confirming previous results on the olfactory orientation of Melolontha cockchafers.

We investigated by olfactometry and feeding- and oviposition-choice-tests how the highly specialised elm leaf beetle, Xanthogaleruca luteola Muller (Coleoptera: Chrysomelidae), responds to conspecifically induced defences in the field elm Ulmus minor Miller (Ulmaceae). While egg deposition of the beetle induced elms to release volatiles attractive to the egg parasitoid Oomyzus gallerucae Fonscolombe (Hymenoptera: Eulophidae), feeding alone did not. In the present study, females of the elm leaf beetle showed preferences for the odours of twigs induced by low egg deposition and feeding over odours from uninfested twigs. In contrast, heavy infestation rendered elm odours less attractive to the beetles. Feeding and oviposition bioassays revealed an oviposition preference for leaves from uninfested twigs when compared to locally infested leaves. However, beetles preferred to feed upon systemically induced leaves compared to uninfested ones. The different preferences of the elm leaf beetle during host plant approach might be explained by a strategy that accounts for both gaining access to high quality nutrition and avoiding competition or parasitism.

Willows are often attacked by both herbivorous insects and rust fungi. Little is known about interactions between these two willow enemies. We studied whether feeding and oviposition behavior of the willow leaf beetle Plagiodera versicolora upon the willow hybrid Salix x cuspidata is affected when the rust fungus Melampsora allii-fragilis has attacked the plant. Laboratory bioassays revealed that adult willow leaf beetles significantly avoided feeding and oviposition on rust-infected leaves when compared to healthy leaves. Further bioassays aimed to elucidate the temporal and spatial scale of effects of rust infection on feeding behavior of adults. While infected parts of leaves were avoided at all times past infection tested (8, 12, and 16 days), symptom-free parts of infected leaves were only avoided 16 days past infection. Systemic effects extended only one leaf position up and two leaf positions down from the infection site.

The application aspects of chemicals which attract the forest cockchafer, Melolontha hippocastani F., were investigated in field and laboratory experiments. Previous studies have shown that males of M. hippocastani are attracted by a synthetic mixture of green leaf volatiles (GLV) and the sex pheromone 1,4-benzoquinone (BQ), that synergistically enhances the male response to GLV. In the present study, we demonstrated that BQ also synergised the male response to one single component of the GLV mixture, the leaf alcohol (Z)-3-hexen-1-ol (Z-3-ol). BQ enhanced the attractiveness of Z-3-ol at doses between 0.05 and 5 mg per trap, reaching a maximum at 5 × 10−1 mg day−1. The addition of an insecticide (cyhalothrin) to traps baited with BQ and Z-3-ol did not affect the lures’ attractiveness. However, when a conidiospore formulation of the entomopathogenic fungus Beauveria brongniartii (Saccardo) Petch was added, the attractiveness of baited traps was significantly reduced. Furthermore, two types of dispensers baited with a solution of BQ in Z-3-ol at 20 mg ml−1 were tested over the entire 4-week flight season. Both a membrane dispenser and a dispenser based on a porous polyethylene (PPE) absorbent disk attracted more males than controls over the entire 4 weeks. The membrane dispenser attracted as many males each week as a reference formulation that was renewed daily. Furthermore, the membrane dispenser attracted more males than the PPE dispenser in weeks 2–4, although laboratory experiments showed that the latter released even higher or at least equal amounts of the joined lure over the entire 4 weeks. However, estimation of the BQ/Z-3-ol ratios of the released material by solid phase microextraction (SPME) and coupled gas chromatography–mass spectrometry (GC-MS) revealed that the membrane dispenser released a higher proportion of BQ than the PPE dispenser in weeks 2–4. Therefore, a higher BQ/Z-3-ol ratio might be responsible for the advantage of the membrane dispenser in the field.

Slow growth in herbivores may lead to higher mortality, due to prolonged exposure to natural enemies. A number of studies has tested the 'slow-growth, high-mortality' hypothesis using predators or parasitoids as natural enemies of the herbivore, but the possible role of pathogens is poorly documented. We provide evidence that phytopathogenic infection of a plant enhances the susceptibility of herbivore larvae to a generalist entomopathogen. Larvae of the mustard leaf beetle Phaedon cochleariae grow more slowly when feeding on Chinese cabbage leaves infected by the phytopathogenic fungus Alternaria brassicae than larvae feeding on healthy leaves. Treatment of such larvae feeding on diseased plants with an LD(50) of the entomopathogenic fungus Metarhizium anisopliae resulted in 100% mortality, compared with a mortality rate of 54% in the control larvae feeding on uninfected leaves. This is the first demonstration of an interaction between a phyto- and an entomopathogenic fungus.

Gas chromatography – mass spectrometry analyses of the headspace volatiles of Scots pine (Pinus sylvestris) induced by egg deposition of the sawfly Diprion pini were conducted. The odor blend of systemically oviposition-induced pine twigs, attractive for the eulophid egg parasitoid Chrysonotomyia ruforum, was compared to volatiles released by damaged pine twigs (control) that are not attractive for the parasitoid. The mechanical damage inflicted to the control twigs mimicked the damage by a sawfly female prior to egg deposition. The odor blend released by oviposition-induced pine twigs consisted of numerous mono- and sesquiterpenes, which all were also present in the headspace of the artificially damaged control twigs. A quantitative comparison of the volatiles from oviposition-induced twigs and controls revealed that only the amounts of (E)-β-farnesene were significantly higher in the volatile blend of the oviposition-induced twigs. Volatiles from pine twigs treated with jasmonic acid (JA) also attract the egg parasitoid. No qualitative differences were detected when comparing the composition of the headspace of JA-treated pine twigs with the volatile blend of untreated control twigs. JA-treated pine twigs released significantly higher amounts of (E)-β-farnesene. However, the JA treatment induced a significant increase of the amount of further terpenoid components. The release of terpenoids by pine after wounding, egg deposition, and JA treatment is discussed with special respect to (E)-β-farnesene.

Aggregation behavior of herbivorous insects is mediated by a wide range of biotic and abiotic factors. It has been suggested that aggregation behavior of the blue willow leaf beetle Phratora vulgatissima is mediated by both host plant odor and by odor released by the beetles. Previous studies show that the beetles respond to plant odors according to their prior host plant experiences. Here, we analyzed the effect of the host plant species on odor released and perceived by adult P. vulgatissima. The major difference between the odor of beetles feeding on salicin-rich and salicin-poor host plants was the presence of salicylaldehyde in the odor of the former, where both males and females released this compound. Electrophysiological studies showed that the intensity of responses to single components of odor released by beetles was sex specific and dependent on the host plant species with which the beetles were fed. Finally, behavioral studies revealed that males feeding on salicin-rich willows were attracted by salicylaldehyde, whereas females did not respond behaviorally to this compound, despite showing clear antennal responses to it. Finally, the ecological relevance of the influence of a host plant species on the plasticity of beetle odor chemistry, perception, and behavior is discussed.

Plants exhibit multifarious defence traits against herbivory that are constitutively expressed or induced upon attack. Insect egg deposition often precedes impending larval attack, and several plants can increase their resistance against larvae after experiencing the oviposition by an herbivore. The nature of such oviposition-mediated resistance remains unknown, and here we aim to determine plant traits that explain it. We test whether oviposition on a host plant can induce plant defence responses or enhance (prime) the induction of defence traits in response to larval herbivory. We exposed Nicotiana attenuata plants to oviposition by moths of a generalist herbivore, Spodoptera exigua. Its larvae suffered higher mortality, retarded development and inflicted less feeding damage on oviposition-experienced than on oviposition-unexperienced plants. While oviposition alone did not induce any of the examined defence traits, oviposited plants exhibited a stronger inducibility of known defence traits, i.e. caffeoylputrescine (CP) and trypsin protease inhibitors (TPIs). We found no effects of oviposition on phytohormone levels, but on the feeding-inducible accumulation of the transcription factor NaMyb8 that is governing biosynthesis of phenylpropanoid–polyamine conjugates, including CP. Comparison of larval performance on wild-type plants, CP- deficient plants (silenced NaMyb8 gene), and TPI-deficient plants (silenced NaPI gene) revealed that priming of plant resistance to larvae by prior oviposition required NaMyb8-mediated defence traits. Our results show that plants can use insect egg deposition as a warning signal to prime their feeding-induced defence.

The response of the two most abundant cockchafer species in central Europe, Melolontha hippocastani and M. melolontha, towards phenol, mixtures of phenol with the leaf alcohol (Z)-3-hexen-1-ol and the known cockchafer pheromones, 1,4-benzoquinone (M. hippocastani) and toluquinone (M. melolontha), was investigated in the field. During the swarming period at dusk, phenol attracted males of both species, and enhanced the known attraction of cockchafer males towards (Z)-3-hexen-1-ol. A mixture of phenol plus (Z)-3-hexen-1-ol was less attractive for M. hippocastani males than a mixture of (Z)-3-hexen-1-ol plus 1,4- benzoquinone, whereas phenol plus (Z)-3-hexen-1-ol attracted as many M. melolontha males as a mixture of (Z)-3-hexen-1-ol plus toluquinone. In both species three component mixtures containing phenol, (Z)-3-hexen-1-ol, and the respective benzoquinone did not capture more males than two component mixtures consisting of only (Z)-3-hexen-1-ol and the benzoquinone. A possible role of phenol as another cockchafer sex pheromone component is discussed.

1 Cross-effects between a herbivorous insect and a phytopathogenic fungus on their common host plant were examined. Specifically, we addressed the questions whether (i) infection of Chinese cabbage leaves by the fungus Alternaria brassicae affects the development and host selection behaviour of the leaf beetle Phaedon cochleariae and (ii) whether herbivory influences host suitability of Chinese cabbage for A. brassicae. 2 Feeding on fungus-infected leaves prolonged larval development and reduced pupal weight of P. cochleariae. Adult beetles avoided feeding and egg deposition on fungus-infected leaves. In contrast to these local effects, no systemic effect of phytopathogenic infection on the herbivore was detected. 3 Herbivory did not influence fungal growth neither locally nor systemically. 4 Thus, our results demonstrate an asymmetric relationship between herbivore and fungus. Whereas herbivory had no visible impact on fungal growth, fungal infection of the plant induced local resistance against P. cochleariae.

Plants may respond both to feeding and oviposition by herbivorous insects. While responses of plants to feeding damage by herbivores have been studied intensively during the past decades, only a few, but growing number of studies consider the reactions of plants towards egg deposition by herbivorous insects. Plants showing defensive response to oviposition by herbivores do not ‘wait’ until being damaged by feeding, but may instead react towards one of the initial steps of herbivore attack, the egg deposition. Direct plant defensive responses to feeding act directly against the feeding stages of the herbivores. However, a plant may also show direct defensive responses to egg deposition by (a) formation of neoplasms, (b) formation of necrotic tissue (= hypersensitive response), and (c) production of oviposition deterrents. All these plant reactions have directly negative effects on the eggs, hatching larvae, or on the ovipositing females. Indirect plant defensive responses to feeding result in the emission of volatiles (= synomones) that attract predators or parasitoids of the feeding stages. A few recent studies have shown that plants are able to emit volatiles also in response to egg deposition and that these volatiles attract egg parasitoids. Studies on the mechanisms of induction of synomones by egg deposition show several parallels to the mechanisms of induction of plant responses by feeding damage. When considering induced plant defence against herbivores from an evolutionary point of view, the question arises whether herbivores evolved the ability to circumvent or even to exploit the plant’s defensive responses. The reactions of herbivores to oviposition induced plant responses are compared with their reactions to feeding induced plant responses.

Herbivorous insects may be informed about the presence of competitors on the same host plant by a variety of cues. These cues can derive from either the competitor itself or the damaged plant. In the mustard leaf beetle Phaedon cochleariae (Coleoptera, Chrysomelidae), adults are known to be deterred from feeding and oviposition by the exocrine glandular secretion of conspecific co-occurring larvae. We hypothesised that the exocrine larval secretion released by feeding larvae may adsorb to the surface of Chinese cabbage leaves, and thus, convey the information about their former or actual presence. Further experiments tested the influence of leaves damaged by conspecific larvae, mechanically damaged leaves, larval frass and regurgitant on the oviposition and feeding behaviour of P. cochleariae. Finally, the effect of previous conspecific herbivory on larval development and larval host selection was assessed. Our results show that (epi)chrysomelidial, the major component of the exocrine secretion from P. cochleariae larvae, was detectable by GC-MS in surface extracts from leaves upon which larvae had fed. However, leaves exposed to volatiles of the larval secretion were not avoided by female P. cochleariae for feeding or oviposition. Thus, we conclude that secretion volatiles did not adsorb in sufficient amounts on the leaf surface to display deterrent activity towards adults. By contrast, gravid females avoided to feed and lay their eggs on leaves damaged by second-instar larvae for three days when compared to undamaged leaves. Mechanical damage of leaves and treatment of artificially damaged leaves with larval frass or regurgitant did not affect oviposition and feeding of P. cochleariae. Since no adverse effects of previous herbivory on larval development were detected, we suggest that female P. cochleariae avoid Chinese cabbage leaves damaged by feeding larvae for other reasons than escape from competition or avoidance of direct negative effects that result from consuming induced plant material.

Mate finding in the European cockchafer, Melolontha melolontha, has been investigated in the field. Our data suggest that mainly males show flight activity at dusk. In a landing cage bioassay, male cockchafers preferred cages baited with females over cages baited with males. Gas chromatographic analysis of beetle extracts with electronantennographic detection revealed the presence of electrophysiologically active compounds, among them toluquinone. In funnel trap bioassays none of these compounds was attractive towards males per se. A mixture of green leaf volatiles (GLV) mimicking the bouquet of mechanically damaged leaves was highly attractive to M. melolontha males. The attractiveness of the same GLV mixture was synergistically enhanced when toluquinone was added to the lure. GLV attract males to damaged leaves. Toluquinone as a sex pheromone indicates the presence of conspecific females and synergistically enhances the attractiveness of GLV.

SUMMARY Plant volatiles released in response to feeding insects are known to attract enemies of the feeding herbivores. In this study, egg deposition by a herbivorous insect was shown to induce a gymnosperm plant to emit volatiles that attract egg parasitoids. Odour from twigs of Pinus sylvestris laden with egg masses of the pine sawfly Diprion pini attracts the eulophid egg parasitoid Chrysonotomyia ruforum. Volatiles released from pine twigs without diprionid eggs are not attractive. Oviposition by the sawfly onto pine needles induces not only a local response in pine needles laden with eggs but also a systemic reaction. Needles without eggs but adjacent to those bearing diprionid eggs also release the volatiles that attract the egg parasitoid. The elicitor of the attractive volatiles was shown to be present in the oviduct secretion coating the eggs of D. pini. When pine twigs are treated with jasmonic acid, a well-known plant wound signal, they emit volatiles that attract the egg parasitoid. These results show, for the first time, that a gymnosperm plant is able to attract parasitoids as soon as a herbivore has deposited its eggs on it. Thus, the plant appears to defend itself against herbivores prior to being damaged by feeding larvae.