Hyperparasitoids use herbivore-induced plant volatiles to locate their parasitoid host.

Plants respond to herbivory with the emission of induced plant volatiles. These volatiles may attract parasitic wasps (parasitoids) that attack the herbivores. Although in this sense the emission of volatiles has been hypothesized to be beneficial to the plant, it is still debated whether this is also the case under natural conditions because other organisms such as herbivores also respond to the emitted volatiles. One important group of organisms, the enemies of parasitoids, hyperparasitoids, has not been included in this debate because little is known about their foraging behaviour. Here, we address whether hyperparasitoids use herbivore-induced plant volatiles to locate their host. We show that hyperparasitoids find their victims through herbivore-induced plant volatiles emitted in response to attack by caterpillars that in turn had been parasitized by primary parasitoids. Moreover, only one of two species of parasitoids affected herbivore-induced plant volatiles resulting in the attraction of more hyperparasitoids than volatiles from plants damaged by healthy caterpillars. This resulted in higher levels of hyperparasitism of the parasitoid that indirectly gave away its presence through its effect on plant odours induced by its caterpillar host. Here, we provide evidence for a role of compounds in the oral secretion of parasitized caterpillars that induce these changes in plant volatile emission. Our results demonstrate that the effects of herbivore-induced plant volatiles should be placed in a community-wide perspective that includes species in the fourth trophic level to improve our understanding of the ecological functions of volatile release by plants. Furthermore, these findings suggest that the impact of species in the fourth trophic level should also be considered when developing Integrated Pest Management strategies aimed at optimizing the control of insect pests using parasitoids.

Folivory affects composition of nectar, floral odor and modifies pollinator behavior.

Herbivory induces changes in plants that influence the associated insect community. The present study addresses the potential trade-off between plant phytochemical responses to insect herbivory and interactions with pollinators. We used a multidisciplinary approach and have combined field and greenhouse experiments to investigate effects of herbivory in plant volatile emission, nectar production, and pollinator behavior, when Pieris brassicae caterpillars were allowed to feed only on the leaves of Brassica nigra plants. Interestingly, volatile emission by flowers changed upon feeding by herbivores on the leaves, whereas, remarkably, volatile emission by leaves did not significantly differ between infested and non-infested flowering plants. The frequency of flower visits by pollinators was generally not influenced by herbivory, but the duration of visits by honeybees and butterflies was negatively affected by herbivore damage to leaves. Shorter duration of pollinator visits could be beneficial for a plant, because it sustains pollen transfer between flowers while reducing nectar consumption per visit. Thus, no trade-off between herbivore-induced plant responses and pollination was evident. The effects of herbivore-induced plant responses on pollinator behavior underpin the importance of including ecological factors, such as herbivore infestation, in studies of the ecology of plant pollination.

Inhibition of lipoxygenase affects induction of both direct and indirect plant defences against herbivorous insects.

Herbivore-induced plant defences influence the behaviour of insects associated with the plant. For biting-chewing herbivores the octadecanoid signal-transduction pathway has been suggested to play a key role in induced plant defence. To test this hypothesis in our plant-herbivore-parasitoid tritrophic system, we used phenidone, an inhibitor of the enzyme lipoxygenase (LOX), that catalyses the initial step in the octadecanoid pathway. Phenidone treatment of Brussels sprouts plants reduced the accumulation of internal signalling compounds in the octadecanoid pathway downstream of the step catalysed by LOX, i.e. 12-oxo-phytodienoic acid (OPDA) and jasmonic acid. The attraction of Cotesia glomerata parasitoids to host-infested plants was significantly reduced by phenidone treatment. The three herbivores investigated, i.e. the specialists Plutella xylostella, Pieris brassicae and Pieris rapae, showed different oviposition preferences for intact and infested plants, and for two species their preference for either intact or infested plants was shown to be LOX dependent. Our results show that phenidone inhibits the LOX-dependent defence response of the plant and that this inhibition can influence the behaviour of members of the associated insect community.

Real-time tracking of Tomato brown rugose fruit virus (ToBRFV) outbreaks in the Netherlands using Nextstrain.

Tomato brown rugose fruit virus (ToBRFV) is a Tobamovirus that was first observed in 2014 and 2015 on tomato plants in Israel and Jordan respectively. Since the first description, the virus has been reported from all continents except Oceania and Antarctica, and has been found infecting both tomato and pepper crops. In October 2019, the Dutch National Plant Protection Organization received a ToBRFV infected tomato sample as part of a generic survey targeting tomato pests. Presence of the virus was verified using Illumina sequencing. A follow-up survey was initiated to determine the extent of ToBRFV presence in the Dutch tomato horticulture and identify possible linkages between ToBRFV genotypes, companies and epidemiological traits. Nextstrain was used to visualize these potential connections. By November 2019, 68 companies had been visited of which 17 companies were found to be infected. The 50 ToBRFV genomes from these outbreak locations group in three main clusters, which are hypothesized to represent three original sources. No correlation was found between genotypes, companies and epidemiological traits, and the source(s) of the Dutch ToBRFV outbreak remain unknown. This paper describes a Nextstrain build containing ToBRFV genomes up to and including November 2019. Sharing data with this interactive online tool will enable the plant virology field to better understand and communicate the diversity and spread of this new virus. Organizations are invited to share data or materials for inclusion in the Nextstrain build, which can be accessed at https://nextstrain.nrcnvwa.nl/ToBRFV/20191231.

Comparing induction at an early and late step in signal transduction mediating indirect defence in Brassica oleracea.

The induction of plant defences involves a sequence of steps along a signal transduction pathway, varying in time course. In this study, the effects of induction of an early and a later step in plant defence signal transduction on plant volatile emission and parasitoid attraction are compared. Ion channel-forming peptides represent a class of inducers that induce an early step in signal transduction. Alamethicin (ALA) is an ion channel-forming peptide mixture from the fungus Trichoderma viride that can induce volatile emission and increase endogenous levels of jasmonic acid (JA) and salicylic acid in plants. ALA was used to induce an early step in the defence response in Brussels sprouts plants, Brassica oleracea var. gemmifera, and to study the effect on volatile emission and on the behavioural response of parasitoids to volatile emission. The parasitoid Cotesia glomerata was attracted to ALA-treated plants in a dose-dependent manner. JA, produced through the octadecanoid pathway, activates a later step in induced plant defence signal transduction, and JA also induces volatiles that are attractive to parasitoids. Treatment with ALA and JA resulted in distinct volatile blends, and both blends differed from the volatile blends emitted by control plants. Even though JA treatment of Brussels sprouts plants resulted in higher levels of volatile emission, ALA-treated plants were as attractive to C. glomerata as JA-treated plants. This demonstrates that on a molar basis, ALA is a 20 times more potent inducer of indirect plant defence than JA, although this hormone has more commonly been used as a chemical inducer of plant defence.

Jasmonic acid-induced volatiles of Brassica oleracea attract parasitoids: effects of time and dose, and comparison with induction by herbivores.

Caterpillar feeding induces direct and indirect defences in brassicaceous plants. This study focused on the role of the octadecanoid pathway in induced indirect defence in Brassica oleracea. The effect of induction by exogenous application of jasmonic acid (JA) on the responses of Brussels sprouts plants and on host-location behaviour of associated parasitoid wasps was studied. Feeding by the biting-chewing herbivores Pieris rapae and Plutella xylostella resulted in significantly increased endogenous levels of JA, a central component in the octadecanoid signalling pathway that mediates induced plant defence. The levels of the intermediate 12-oxophyto-dienoic acid (OPDA) were significantly induced only after P. rapae feeding. Three species of parasitoid wasps, Cotesia glomerata, C. rubecula, and Diadegma semiclausum, differing in host range and host specificity, were tested for their behavioural responses to volatiles from herbivore-induced, JA-induced, and non-induced plants. All three species were attracted to volatiles from JA-induced plants compared with control plants; however, they preferred volatiles from herbivore-induced plants over volatiles from JA-induced plants. Attraction of C. glomerata depended on both timing and dose of JA application. JA-induced plants produced larger quantities of volatiles than herbivore-induced and control plants, indicating that not only quantity, but also quality of the volatile blend is important in the host-location behaviour of the wasps.

Sensitivity and speed of induced defense of cabbage (Brassica oleracea L.): dynamics of BoLOX expression patterns during insect and pathogen attack.

The lipoxygenase pathway is involved in the early steps of plant responses to herbivorous insects and phytopathogens. Induced defenses in the crucifer Brassica oleracea have been well documented. Here, we have cloned a LIPOXYGENASE (LOX) from B. oleracea (BoLOX). The sequence reveals that the BoLOX protein has a transit peptide for chloroplast targeting, which is characteristic for class 2 LOXs involved in jasmonic acid (JA) biosynthesis which takes place in the chloroplast. Phylogenetic analysis shows that BoLOX is closely related to B. napus BnLOX2fl and Arabidopsis thaliana AtLOX2, which mediates JA biosynthesis. BoLOX also shares functional characteristics with AtLOX2; BoLOX is inducible by wounding, JA treatment, and herbivores such as caterpillars (Pieris rapae, P. brassicae, and Mamestra brassicae), spider mites (Tetranychus urticae), locusts (Schistocerca gregaria), and a bacterial pathogen (Pseudomonas syringae pv. tomato). Of these, Pieris spp. caterpillars also induce AtLOX2 and JA biosynthesis in Arabidopsis. However, the aphid Myzus persicae did not induce BoLOX, which agrees with previous reports that this aphid induces neither AtLOX2 nor JA biosynthesis in Arabidopsis. Quantitative expression analysis of temporal, spatial, and density-dependent BoLOX transcript levels through real-time quantitative polymerase chain reaction demonstrated that BoLOX is maximally expressed after feeding by only two first-instar caterpillars for 24 h. Systemic expression was approximately 10-fold lower than local expression for herbivore-induced responses. The good correlation of BoLOX transcript levels with reports in the literature on induced defenses of B. oleracea is discussed.

Metabolomic plasticity in GM and non-GM potato leaves in response to aphid herbivory and virus infection.

An important aspect of ecological safety of genetically modified (GM) plants is the evaluation of unintended effects on plant-insect interactions. These interactions are to a large extent influenced by the chemical composition of plants. This study uses NMR-based metabolomics to establish a baseline of chemical variation to which differences between a GM potato line and its parent cultivar are compared. The effects of leaf age, virus infection, and aphid herbivory on plant metabolomes were studied. The metabolome of the GM line differed from its parent only in young leaves of noninfected plants. This effect was small when compared to the baseline. Consistently, aphid performance on excised leaves was influenced by leaf age, while no difference in performance was found between GM and non-GM plants. The metabolomic baseline approach is concluded to be a useful tool in ecological safety assessment.

Increasing insight into induced plant defense mechanisms using elicitors and inhibitors.

One of the strategies that plants employ to defend themselves against herbivore attack is the induced production of carnivore-attracting volatiles. Using elicitors and inhibitors of different steps of the signal-transduction pathways can improve our understanding of the mechanisms underlying induced plant defenses. For instance, we recently showed that application of jasmonic acid, a key hormone in the octadecanoid pathway involved in herbivore-induced defense, to Brassica oleracea affects gene expression, hormone levels, and volatile emission, as well as oviposition by herbivores and host location behavior by parasitoids. Such defense responses vary with the dose of the elicitor and with time since application. This addendum describes how the use of inhibitors, in addition to the use of elicitors like jasmonic acid, can be applied in bio-assays to investigate the role of signal-transduction pathways involved in induced plant defense. We show how inhibition of different steps of the octadecanoid pathway affects host location behavior by parasitoids.

Jasmonic acid-induced changes in Brassica oleracea affect oviposition preference of two specialist herbivores.

Jasmonic acid (JA) is a key hormone involved in plant defense responses. The effect of JA treatment of cabbage plants on their acceptability for oviposition by two species of cabbage white butterflies, Pieris rapae and P. brassicae, was investigated. Both butterfly species laid fewer eggs on leaves of JA-treated plants compared to control plants. We show that this is due to processes in the plant after JA treatment rather than an effect of JA itself. The oviposition preference for control plants is adaptive, as development time from larval hatch until pupation of P. rapae caterpillars was longer on JA-treated plants. Total glucosinolate content in leaf surface extracts was similar for control and treated plants; however, two of the five glucosinolates were present in lower amounts in leaf surface extracts of JA-treated plants. When the butterflies were offered a choice between the purified glucosinolate fraction isolated from leaf surface extracts of JA-treated plants and that from control plants, they did not discriminate. Changes in leaf surface glucosinolate profile, therefore, do not seem to explain the change in oviposition preference of the butterflies after JA treatment, suggesting that as yet unknown infochemicals are involved.

Differences in effects of pyrrolizidine alkaloids on five generalist insect herbivore species.

The evolution of the diversity in plant secondary compounds is often thought to be driven by insect herbivores, although there is little empirical evidence for this assumption. To investigate whether generalist insect herbivores could play a role in the evolution of the diversity of related compounds, we examined if (1) related compounds differ in their effects on generalists, (2) there is a synergistic effect among compounds, and (3) effects of related compounds differed among insect species. The effects of pyrrolizidine alkaloids (PAs) were tested on five generalist insect herbivore species of several genera using artificial diets or neutral substrates to which PAs were added. We found evidence that structurally related PAs differed in their effects to the thrips Frankliniella occidentalis, the aphid Myzus persicae, and the locust Locusta migratoria. The individual PAs had no effect on Spodoptera exigua and Mamestra brassicae caterpillars. For S. exigua, we found indications for synergistic deterrent effects of PAs in PA mixtures. The relative effects of PAs differed between insect species. The PA senkirkine had the strongest effect on the thrips, but had no effect at all on the aphids. Our results show that generalist herbivores could potentially play a role in the evolution and maintenance of the diversity of PAs.