Multidisciplinary Approach to Unravelling the Relative Contribution of Different Oxylipins in Indirect Defense of Arabidopsis thaliana

The oxylipin pathway is commonly involved in induced plant defenses, and is the main signal-transduction pathway induced by insect folivory. Herbivory induces the production of several oxylipins, and consequently alters the so-called ‘oxylipin signature’ in the plant. Jasmonic acid (JA), as well as pathway intermediates are known to induce plant defenses. Indirect defense against herbivorous insects comprises the production of herbivore-induced plant volatiles (HIPVs). To unravel the precise oxylipin signal-transduction underlying the production of HIPVs in Arabidopsis thaliana and the resulting attraction of parasitoid wasps, we used a multidisciplinary approach that includes molecular genetics, metabolite analysis, and behavioral analysis. Mutant plants affected in the jasmonate pathway (18:0 and/or 16:0 -oxylipin routes; mutants dde2-2, fad5, opr3) were studied to assess the effects of JA and its oxylipin intermediates 12-oxo-phytodienoate (OPDA) and dinor-OPDA (dnOPDA) on HIPV emission and parasitoid (Diadegma semiclausum) attraction. Interference with the production of the oxylipins JA and OPDA altered the emission of HIPVs, in particular terpenoids and the phenylpropanoid methyl salicylate, which affected parasitoid attraction. Our data show that the herbivore-induced attraction of parasitoid wasps to Arabidopsis plants depends on HIPVs that are induced through the 18:0 oxylipin-derivative JA. Furthermore, our study shows that the 16:0-oxylipin route towards dnOPDA does not play a role in HIPV induction, and that the role of 18:0 derived oxylipin-intermediates, such as OPDA, is either absent or limited

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

Identification of Biologically Relevant Compounds in Aboveground and Belowground Induced Volatile Blends

Plants under attack by aboveground herbivores emit complex blends of volatile organic compounds (VOCs). Specific compounds in these blends are used by parasitic wasps to find their hosts. Belowground induction causes shifts in the composition of aboveground induced VOC blends, which affect the preference of parasitic wasps. To identify which of the many volatiles in the complex VOC blends may explain parasitoid preference poses a challenge to ecologists. Here, we present a case study in which we use a novel bioinformatics approach to identify biologically relevant differences between VOC blends of feral cabbage (Brassica oleracea L.). The plants were induced aboveground or belowground with jasmonic acid (JA) and shoot feeding caterpillars (Pieris brassicae or P. rapae). We used Partial Least Squares—Discriminant Analysis (PLSDA) to integrate and visualize the relation between plant-emitted VOCs and the preference of female Cotesia glomerata. Overall, female wasps preferred JA-induced plants over controls, but they strongly preferred aboveground JA-induced plants over belowground JA-induced plants. PLSDA revealed that the emission of several monoterpenes was enhanced similarly in all JA-treated plants, whereas homoterpenes and sesquiterpenes increased exclusively in aboveground JA-induced plants. Wasps may use the ratio between these two classes of terpenes to discriminate between aboveground and belowground induced plants. Additionally, it shows that aboveground applied JA induces different VOC biosynthetic pathways than JA applied to the root. Our bioinformatic approach, thus, successfully identified which VOCs matched the preferences of the wasps in the various choice tests. Additionally, the analysis generated novel hypotheses about the role of JA as a signaling compound in aboveground and belowground induced responses in plants

Prey and Non-prey Arthropods Sharing a Host Plant: Effects on Induced Volatile Emission and Predator Attraction

It is well established that plants infested with a single herbivore species can attract specific natural enemies through the emission of herbivore-induced volatiles. However, it is less clear what happens when plants are simultaneously attacked by more than one species. We analyzed volatile emissions of lima bean and cucumber plants upon multi-species herbivory by spider mites (Tetranychus urticae) and caterpillars (Spodoptera exigua) in comparison to single-species herbivory. Upon herbivory by single or multiple species, lima bean and cucumber plants emitted volatile blends that comprised mostly the same compounds. To detect additive, synergistic, or antagonistic effects, we compared the multi-species herbivory volatile blend with the sum of the volatile blends induced by each of the herbivore species feeding alone. In lima bean, the majority of compounds were more strongly induced by multi-species herbivory than expected based on the sum of volatile emissions by each of the herbivores separately, potentially caused by synergistic effects. In contrast, in cucumber, two compounds were suppressed by multi-species herbivory, suggesting the potential for antagonistic effects. We also studied the behavioral responses of the predatory mite Phytoseiulus persimilis, a specialized natural enemy of spider mites. Olfactometer experiments showed that P. persimilis preferred volatiles induced by multi-species herbivory to volatiles induced by S. exigua alone or by prey mites alone. We conclude that both lima bean and cucumber plants effectively attract predatory mites upon multi-species herbivory, but the underlying mechanisms appear different between these species

Sequence-Based Genotyping for Marker Discovery and Co-Dominant Scoring in Germplasm and Populations

Conventional marker-based genotyping platforms are widely available, but not without their limitations. In this context, we developed Sequence-Based Genotyping (SBG), a technology for simultaneous marker discovery and co-dominant scoring, using next-generation sequencing. SBG offers users several advantages including a generic sample preparation method, a highly robust genome complexity reduction strategy to facilitate de novo marker discovery across entire genomes, and a uniform bioinformatics workflow strategy to achieve genotyping goals tailored to individual species, regardless of the availability of a reference sequence. The most distinguishing features of this technology are the ability to genotype any population structure, regardless whether parental data is included, and the ability to co-dominantly score SNP markers segregating in populations. To demonstrate the capabilities of SBG, we performed marker discovery and genotyping in Arabidopsis thaliana and lettuce, two plant species of diverse genetic complexity and backgrounds. Initially we obtained 1,409 SNPs for arabidopsis, and 5,583 SNPs for lettuce. Further filtering of the SNP dataset produced over 1,000 high quality SNP markers for each species. We obtained a genotyping rate of 201.2 genotypes/SNP and 58.3 genotypes/SNP for arabidopsis (n = 222 samples) and lettuce (n = 87 samples), respectively. Linkage mapping using these SNPs resulted in stable map configurations. We have therefore shown that the SBG approach presented provides users with the utmost flexibility in garnering high quality markers that can be directly used for genotyping and downstream applications. Until advances and costs will allow for routine whole-genome sequencing of populations, we expect that sequence-based genotyping technologies such as SBG will be essential for genotyping of model and non-model genomes alike

Increased Terpenoid Accumulation in Cotton (Gossypium hirsutum) Foliage is a General Wound Response

The subepidermal pigment glands of cotton accumulate a variety of terpenoid products, including monoterpenes, sesquiterpenes, and terpenoid aldehydes that can act as feeding deterrents against a number of insect herbivore species. We compared the effect of herbivory by Spodoptera littoralis caterpillars, mechanical damage by a fabric pattern wheel, and the application of jasmonic acid on levels of the major representatives of the three structural classes of terpenoids in the leaf foliage of 4-week-old Gossypium hirsutum plants. Terpenoid levels increased successively from control to mechanical damage, herbivory, and jasmonic acid treatments, with E-β-ocimene and heliocide H1 and H4 showing the highest increases, up to 15-fold. Herbivory or mechanical damage to older leaves led to terpenoid increases in younger leaves. Leaf-by-leaf analysis of terpenes and gland density revealed that higher levels of terpenoids were achieved by two mechanisms: (1) increased filling of existing glands with terpenoids and (2) the production of additional glands, which were found to be dependent on damage intensity. As the relative response of individual terpenoids did not differ substantially among herbivore, mechanical damage, and jasmonic acid treatments, the induction of terpenoids in cotton foliage appears to represent a non-specific wound response mediated by jasmonic acid

Do Leaf Cutting Ants Cut Undetected? Testing the Effect of Ant-Induced Plant Defences on Foraging Decisions in Atta colombica

Leaf-cutting ants (LCAs) are polyphagous, yet highly selective herbivores. The factors that govern their selection of food plants, however, remain poorly understood. We hypothesized that the induction of anti-herbivore defences by attacked food plants, which are toxic to either ants or their mutualistic fungus, should significantly affect the ants' foraging behaviour. To test this “induced defence hypothesis,” we used lima bean (Phaseolus lunatus), a plant that emits many volatile organic compounds (VOCs) upon herbivore attack with known anti-fungal or ant-repellent effects. Our results provide three important insights into the foraging ecology of LCAs. First, leaf-cutting by Atta ants can induce plant defences: Lima bean plants that were repeatedly exposed to foraging workers of Atta colombica over a period of three days emitted significantly more VOCs than undamaged control plants. Second, the level to which a plant has induced its anti-herbivore defences can affect the LCAs' foraging behaviour: In dual choice bioassays, foragers discriminated control plants from plants that have been damaged mechanically or by LCAs 24 h ago. In contrast, strong induction levels of plants after treatment with the plant hormone jasmonic acid or three days of LCA feeding strongly repelled LCA foragers relative to undamaged control plants. Third, the LCA-specific mode of damaging leaves allows them to remove larger quantities of leaf material before being recognized by the plant: While leaf loss of approximately 15% due to a chewing herbivore (coccinelid beetle) was sufficient to significantly increase VOC emission levels after 24 h, the removal of even 20% of a plant's leaf area within 20 min by LCAs did not affect its VOC emission rate after 24 h. Taken together, our results support the “induced defence hypothesis” and provide first empirical evidence that the foraging behaviour of LCAs is affected by the induction of plant defence responses

Different Transcript Patterns in Response to Specialist and Generalist Herbivores in the Wild Arabidopsis Relative Boechera divaricarpa

BACKGROUND: Plants defend themselves against herbivorous insects, utilizing both constitutive and inducible defenses. Induced defenses are controlled by several phytohormone-mediated signaling pathways. Here, we analyze transcriptional changes in the North American Arabidopsis relative Boechera divaricarpa in response to larval herbivory by the crucifer specialist lepidopteran Plutella xylostella (diamondback moth) and by the generalist lepidopteran Trichoplusia ni (cabbage semilooper), and compare them to wounding and exogenous phytohormone application. METHODOLOGY/PRINCIPAL FINDINGS: We use a custom macroarray constructed from B. divaricarpa herbivory-regulated cDNAs identified by suppression subtractive hybridization and from known stress-responsive A. thaliana genes for transcript profiling after insect herbivory, wounding and in response to jasmonate, salicylate and ethylene. In addition, we introduce path analysis as a novel approach to analyze transcript profiles. Path analyses reveal that transcriptional responses to the crucifer specialist P. xylostella are primarily determined by direct effects of the ethylene and salicylate pathways, whereas responses to the generalist T. ni are influenced by the ethylene and jasmonate pathways. Wound-induced transcriptional changes are influenced by all three pathways, with jasmonate having the strongest effect. CONCLUSIONS/SIGNIFICANCE: Our results show that insect herbivory is distinct from simple mechanical plant damage, and that different lepidopteran herbivores elicit different transcriptional responses

Olfactory receptors on the maxillary palps of small ermine moth larvae: evolutionary history of benzaldehyde sensitivity

In lepidopterous larvae the maxillary palps contain a large portion of the sensory equipment of the insect. Yet, knowledge about the sensitivity of these cells is limited. In this paper a morphological, behavioral, and electrophysiological investigation of the maxillary palps of Yponomeuta cagnagellus (Lepidoptera: Yponomeutidae) is presented. In addition to thermoreceptors, CO2 receptors, and gustatory receptors, evidence is reported for the existence of two groups of receptor cells sensitive to plant volatiles. Cells that are mainly sensitive to (E)-2-hexenal and hexanal or to (Z)-3-hexen-1-ol and 1-hexanol were found. Interestingly, a high sensitivity for benzaldehyde was also found. This compound is not known to be present in Euonymus europaeus, the host plant of the monophagous Yponomeuta cagnagellus, but it is a prominent compound in Rosaceae, the presumed hosts of the ancestors of Y. cagnagellus. To elucidate the evolutionary history of this sensitivity, and its possible role in host shifts, feeding responses of three Yponomeuta species to benzaldehyde were investigated. The results confirm the hypothesis that the sensitivity to benzaldehyde evolved during the ancestral shift from Celastraceae to Rosaceae and can be considered an evolutionary relict, retained in the recently backshifted Celastraceae-specialist Y. cagnagellus

Chrysolina herbacea Modulates Terpenoid Biosynthesis of Mentha aquatica L.

Interactions between herbivorous insects and plants storing terpenoids are poorly understood. This study describes the ability of Chrysolina herbacea to use volatiles emitted by undamaged Mentha aquatica plants as attractants and the plant's response to herbivory, which involves the production of deterrent molecules. Emitted plant volatiles were analyzed by GC-MS. The insect's response to plant volatiles was tested by Y-tube olfactometer bioassays. Total RNA was extracted from control plants, mechanically damaged leaves, and leaves damaged by herbivores. The terpenoid quantitative gene expressions (qPCR) were then assayed. Upon herbivory, M. aquatica synthesizes and emits (+)-menthofuran, which acts as a deterrent to C. herbacea. Herbivory was found to up-regulate the expression of genes involved in terpenoid biosynthesis. The increased emission of (+)-menthofuran was correlated with the upregulation of (+)-menthofuran synthase