Genetic mapping shows intraspecific variation and transgressive segregation for caterpillar‐induced aphid resistance in maize

Plants in nature have inducible defences that sometimes lead to targeted resistance against particular herbivores, but susceptibility to others. The metabolic diversity and genetic resources available for maize (Zea mays) make this a suitable system for a mechanistic study of within‐species variation in such plant‐mediated interactions between herbivores. Beet armyworms (Spodoptera exigua) and corn leaf aphids (Rhopalosiphum maidis) are two naturally occurring maize herbivores with different feeding habits. Whereas chewing herbivore‐induced methylation of 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one glucoside (DIMBOA‐Glc) to form 2‐hydroxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one glucoside (HDMBOA‐Glc) promotes caterpillar resistance, lower DIMBOA‐Glc levels favour aphid reproduction. Thus, caterpillar‐induced DIMBOA‐Glc methyltransferase activity in maize is predicted to promote aphid growth. To test this hypothesis, the impact of S. exigua feeding on R. maidis progeny production was assessed using seventeen genetically diverse maize inbred lines. Whereas aphid progeny production was increased by prior caterpillar feeding on lines B73, Ki11, Ki3 and Tx303, it decreased on lines Ky21, CML103, Mo18W and W22. Genetic mapping of this trait in a population of B73 × Ky21 recombinant inbred lines identified significant quantitative trait loci on maize chromosomes 1, 7 and 10. There is a transgressive segregation for aphid resistance, with the Ky21 alleles on chromosomes 1 and 7 and the B73 allele on chromosome 10 increasing aphid progeny production. The chromosome 1 QTL coincides with a cluster of three maize genes encoding benzoxazinoid O‐methyltransferases that convert DIMBOA‐Glc to HDMBOA‐Glc. Gene expression studies and benzoxazinoid measurements indicate that S. exigua ‐induced responses in this pathway differentially affect R. maidis resistance in B73 and Ky21.     

Within‐plant distribution of 1,4‐benzoxazin‐3‐ones contributes to herbivore niche differentiation in maize

Plant defences vary in space and time, which may translate into specific herbivore‐foraging patterns and feeding niche differentiation. To date, little is known about the effect of secondary metabolite patterning on within‐plant herbivore foraging. We investigated how variation in the major maize secondary metabolites, 1,4‐benzoxazin‐3‐one derivatives (BXDs), affects the foraging behaviour of two leaf‐chewing herbivores. BXD levels varied substantially within plants. Older leaves had higher levels of constitutive BXDs while younger leaves were consistently more inducible. These differences were observed independently of plant age, even though the concentrations of most BXDs declined markedly in older plants. Larvae of the well‐adapted maize pest Spodoptera frugiperda preferred and grew better on young inducible leaves irrespective of plant age, while larvae of the generalist Spodoptera littoralis preferred and tended to grow better on old leaves. In BXD‐free mutants, the differences in herbivore weight gain between old and young leaves were absent for both species, and leaf preferences of S. frugiperda were attenuated. In contrast, S. littoralis foraging patterns were not affected. In summary, our study shows that plant secondary metabolites differentially affect performance and foraging of adapted and non‐adapted herbivores and thereby likely contribute to feeding niche differentiation.     

Metabolomics reveals herbivore‐induced metabolites of resistance and susceptibility in maize leaves and roots

Plants respond to herbivory by reprogramming their metabolism. Most research in this context has focused on locally induced compounds that function as toxins or feeding deterrents. We developed an ultra‐high‐pressure liquid chromatography time‐of‐flight mass spectrometry (UHPLC‐TOF‐MS)‐based metabolomics approach to evaluate local and systemic herbivore‐induced changes in maize leaves, sap, roots and root exudates without any prior assumptions about their function. Thirty‐two differentially regulated compounds were identified from Spodoptera littoralis‐infested maize seedlings and isolated for structure assignment by microflow nuclear magnetic resonance (CapNMR). Nine compounds were quantified by a high throughput direct nano‐infusion tandem mass spectrometry/mass spectrometry (MS/MS) method. Leaf infestation led to a marked local increase of 1,3‐benzoxazin‐4‐ones, phospholipids, N‐hydroxycinnamoyltyramines, azealic acid and tryptophan. Only few changes were found in the root metabolome, but 1,3‐benzoxazin‐4‐ones increased in the vascular sap and root exudates. The role of N‐hydroxycinnamoyltyramines in plant–herbivore interactions is unknown, and we therefore tested the effect of the dominating p‐coumaroyltyramine on S. littoralis. Unexpectedly, p‐coumaroyltyramine was metabolized by the larvae and increased larval growth, possibly by providing additional nitrogen to the insect. Taken together, this study illustrates that herbivore attack leads to the induction of metabolites that can have contrasting effects on herbivore resistance in the leaves and roots.     

Degradation of cytokinins by maize cytokinin dehydrogenase is mediated by free radicals generated by enzymatic oxidation of natural benzoxazinones

Hydroxamic acid 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐one (DIMBOA) was isolated from maize phloem sap as a compound enhancing the degradation of isopentenyl adenine by maize cytokinin dehydrogenase (CKX), after oxidative conversion by either laccase or peroxidase. Laccase and peroxidase catalyze oxidative cleavage of DIMBOA to 4‐nitrosoresorcinol‐1‐monomethyl ether (coniferron), which serves as a weak electron acceptor of CKX. The oxidation of DIMBOA and coniferron generates transitional free radicals that are used by CKX as effective electron acceptors. The function of free radicals in the CKX‐catalyzed reaction was also verified with a stable free radical of 2,2′‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid. Application of exogenous cytokinin to maize seedlings resulted in an enhanced benzoxazinoid content in maize phloem sap. The results indicate a new function for DIMBOA in the metabolism of the cytokinin group of plant hormones.     

Insect eggs suppress plant defence against chewing herbivores

Plants activate direct and indirect defences in response to insect egg deposition. However, whether eggs can manipulate plant defence is unknown. In Arabidopsis thaliana, oviposition by the butterfly Pieris brassicae triggers cellular and molecular changes that are similar to the changes caused by biotrophic pathogens. In the present study, we found that the plant defence signal salicylic acid (SA) accumulates at the site of oviposition. This is unexpected, as the SA pathway controls defence against fungal and bacterial pathogens and negatively interacts with the jasmonic acid (JA) pathway, which is crucial for the defence against herbivores. Application of P. brassicae or Spodoptera littoralis egg extract onto leaves reduced the induction of insect‐responsive genes after challenge with caterpillars, suggesting that egg‐derived elicitors suppress plant defence. Consequently, larval growth of the generalist herbivore S. littoralis, but not of the specialist P. brassicae, was significantly higher on plants treated with egg extract than on control plants. In contrast, suppression of gene induction and enhanced S. littoralis performance were not seen in the SA‐deficient mutant sid2‐1, indicating that it is SA that mediates this phenomenon. These data reveal an intriguing facet of the cross‐talk between SA and JA signalling pathways, and suggest that insects have evolved a way to suppress the induction of defence genes by laying eggs that release elicitors. We show here that egg‐induced SA accumulation negatively interferes with the JA pathway, and provides an advantage for generalist herbivores.     

Drought changes plant chemistry and causes contrasting responses in lepidopteran herbivores

Drought events are predicted to increase due to climate change, yet consequences for plant–insect interactions are only partially understood. Drought‐mediated interactions between herbivores and their host plants are affected by a combination of factors, including characteristics of the affected plant, its associated herbivore and of the prevailing drought. Studying the effect of these factors in combination may provide important insight into plant and herbivore responses to drought. We studied drought effects on plant resistance to two leaf‐chewing herbivores by considering differing growth conditions, plant chemistry and insect responses in concert. We exposed Alliaria petiolata plants from several wild populations to different intensities of intermittent drought stress and quantified drought‐mediated changes in plant chemistry. Simultaneously, we assessed behavior (feeding preference) and performance of two lepidopteran herbivores: Pieris brassicae, a specialist, and Spodoptera littoralis, a generalist. Drought led to lowest concentrations of secondary defense compounds in severely stressed plants, without affecting total nitrogen content. Additionally, drought evoked opposite patterns in feeding preferences (plant palatability) between the herbivore species. Pieris brassicae consumed most of well‐watered plants, while S. littoralis preferred severely drought‐stressed plants. Hence, feeding preferences of S. littoralis reflected changes in plant secondary chemistry. Contrary to their feeding preference, P. brassicae performed better on drought‐stressed than on well‐watered plants, with faster development and higher attained pupal mass (plant suitability). Spodoptera littoralis showed retarded development in all treatments. In conclusion, drought caused plant secondary defense compounds to decrease consistently across all studied plant populations, which evoked contrasting feeding preferences of two herbivore species of the same feeding guild. These results suggest herbivore specificity as a possible explanation for herbivore responses to drought and emphasize the importance of herbivore characteristics such as feeding specialization in understanding and predicting consequences of future drought events.     

Volatiles produced by soil‐borne endophytic bacteria increase plant pathogen resistance and affect tritrophic interactions

Volatile organic compounds (VOCs) released by soil microorganisms influence plant growth and pathogen resistance. Yet, very little is known about their influence on herbivores and higher trophic levels. We studied the origin and role of a major bacterial VOC, 2,3‐butanediol (2,3‐BD), on plant growth, pathogen and herbivore resistance, and the attraction of natural enemies in maize. One of the major contributors to 2,3‐BD in the headspace of soil‐grown maize seedlings was identified as Enterobacter aerogenes, an endophytic bacterium that colonizes the plants. The production of 2,3‐BD by E. aerogenes rendered maize plants more resistant against the Northern corn leaf blight fungus Setosphaeria turcica. On the contrary, E. aerogenes‐inoculated plants were less resistant against the caterpillar Spodoptera littoralis. The effect of 2,3‐BD on the attraction of the parasitoid Cotesia marginiventris was more variable: 2,3‐BD application to the headspace of the plants had no effect on the parasitoids, but application to the soil increased parasitoid attraction. Furthermore, inoculation of seeds with E. aerogenes decreased plant attractiveness, whereas inoculation of soil with a total extract of soil microbes increased parasitoid attraction, suggesting that the effect of 2,3‐BD on the parasitoid is indirect and depends on the composition of the microbial community.     

Effects of Bt maize expressing Cry1Ab and Bt spray on Spodoptera littoralis

Various studies have been conducted to assess the damage caused by secondary lepidopteran pests to transgenic Bt maize expressing Cry1Ab. However, to date little is known on the effects of transgenic maize on Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), a polyphagous herbivore which is considered a pest in Mediterranean maize growing areas. Here we present results on the effects of Bt maize (Bt‐11) and Bt spray (Dipel) on the various life stage parameters of this herbivore. We further assess the expression of Cry1Ab in different leaves and leaf parts in maize at a given plant growth stage, and determine whether the feeding damage of 3rd instar S. littoralis is influenced by Bt toxin expression. Contrary to previous literature reporting that S. littoralis is not sensitive to Bt Cry1Ab toxin, our results show that insects fed on either transgenic or Bt sprayed plants were negatively affected. Young S. littoralis larvae (1st and 2nd instars) were found to be the most sensitive to the Bt toxin. This was represented by a higher mortality and a slower developmental time of larvae maintained on transgenic or sprayed plants when compared to insects maintained on control plants. Moreover, Bt maize had a stronger and prolonged detrimental effect on insects when compared to Bt spray in maize. This was revealed by the fact that insects maintained on transgenic plants from 3rd instar to pupation took longer to reach adult emergence compared to insects that were maintained on sprayed plants. This was likely due to the continuous exposure of insects to the toxin when kept on transgenic maize. ELISA results showed a variation in the amount of Bt toxin among different leaf sections in transgenic maize at a given plant growth stage. These differences in Bt toxin were primarily found in the youngest leaf of growing plants. Although the lowest amounts of Bt toxin were detected in the growing leaf section of young leaves, this difference did not appear to influence the feeding behavior of 3rd instar S. littoralis.     

Identity and combinations of arbuscular mycorrhizal fungal isolates influence plant resistance and insect preference

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The interaction between host and host plant influences the oviposition and performance of a generalist ectoparasitoid

The successful development of parasitoids of herbivores depends on the quality of their host, which is often affected by the host plant. Therefore, a parasitoid’s oviposition decisions will directly depend on the host, but also on plant quality. Here, we investigated the direct effects of host species and the indirect effects of the host’s food plant on the oviposition decisions and performance of the gregarious ectoparasitoid Euplectrus platyhypenae Howard (Hymenoptera: Eulophidae). With a series of no‐choice experiments, we determined the oviposition and performance of the parasitoid on: (1) two caterpillar species, fall armyworm, Spodoptera frugiperda JE Smith (Lepidoptera: Noctuidae), and velvet armyworm, Spodoptera latifascia Walker, reared on maize (Zea mays L., Poaceae), (2) the same caterpillars reared on maize, bean (Phaseolus vulgaris L., Fabaceae), or squash (Cucurbita pepo L., Cucurbitaceae) leaves, and (3) S. latifascia caterpillars reared on leaves of wild and cultivated lima bean, Phaseolus lunatus L. All these insects and plants originate from Mesoamerica where they have coexisted for thousands of years in the traditional agricultural system known as Milpa in which maize, beans, and squash are planted together. We found that the preferred and best combination of host and host plant for parasitoid performance was S. frugiperda on maize. Parasitoids laid larger clutches, had higher survival, and more females and larger adults emerged from S. frugiperda reared on maize. However, when both caterpillar species were reared on squash, S. latifascia was the better host. Contrary to the literature, S. frugiperda was not able to develop on bean plants. Results from the lima bean experiment showed that parasitoid performance was best when S. latifascia was reared on leaves of cultivated compared to wild lima bean. These findings are discussed in the context of mixed cropping in which the ability of generalist parasitoids to switch among hosts and host plant species could be advantageous for pest management.     

Stress magnitude matters: different intensities of pulsed water stress produce non‐monotonic resistance responses of host plants to insect herbivores

Abstract 1. Water stress may increase or reduce the suitability of plants for herbivores. The recently proposed ‘pulsed stress hypothesis’ suggests consideration of stress phenology (pulsed vs. continuous stress) to explain these conflicting effects of plant water stress on herbivore performance. 2. This hypothesis was tested for the effect of differing stress intensity on performance and preference of insect herbivores belonging to different feeding guilds, namely leaf‐chewing insects (Spodoptera littoralis caterpillars) and phloem‐feeding insects (Aphis pomi aphids), on apple plants (Malus domestica). The plants were non‐stressed or exposed to a low or high intensity of pulsed water stress. 3. Plant responses to the different stress levels were generally monotonic. Growth, stomatal conductance (g_s), leaf water, and old‐leaf nitrogen concentration decreased, whereas young‐leaf nitrogen concentration and leaf mass per area (LMA) increased with increasing stress intensity. The stable isotope composition of foliar carbon (δ¹³C) responded non‐monotonically to the drought treatments. The δ¹³C values were highest in low‐stress plants, intermediate in high‐stress plants, and lowest in non‐stressed plants. 4. The preference and performance responses of the caterpillars were also non‐monotonic. Non‐stressed plants were intermediately, low‐stress plants least, and high‐stress plants most attractive or suitable. Aphid population growth was highest on non‐stressed plants and lowest on low‐stress plants. 5. The results highlight the importance of water stress intensity for the outcome of interactions between herbivores and drought‐affected plants. They show that pulsed water stress may enhance or reduce insect herbivore performance and plant resistance, depending on stress intensity.     

Dissemination of the biocontrol agent Vairimorpha necatrix by the spined soldier bug, Podisus maculiventris

The ability of the spined soldier bug, Podisus maculiventris (Say) (Heteroptera: Pentatomidae), to disseminate infective forms of two lepidopteran pathogens, Vairimorpha necatrix (Kramer) (Microspora: Microsporidia) and Lacanobia oleracea granulovirus (LoGV) was investigated. Individual female P. maculiventris that had fed on Lacanobia oleracea L. (Lepidoptera: Noctuidae) larvae, infected with V. necatrix, excreted approximately 6 × 10⁸V. necatrix spores during the subsequent 7 days. Excreted spores were fed to L. oleracea larvae, causing 100% mortality, indicating that the spores remained viable after passing through the gut of the predator. Podisus maculiventris that had fed on V. necatrix or LoGV‐infected larvae were allowed to defecate on the foliage of tomato plants, prior to the infestation of the plants with L. oleracea or Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) larvae. This proved to be an effective way of infecting the pest larvae with the pathogens, particularly when five predatory bugs were used per plant. After 20 days, the number of S. littoralis and L. oleracea surviving on the plants was reduced by 75% and 61%, respectively. Female P. maculiventris maintained on V. necatrix‐infected prey showed reduced egg production and longevity, whilst those fed on LoGV‐infected prey showed only reduced egg production. The potential for P. maculiventris to disseminate insect pathogens is discussed in the context of improved biological control of lepidopteran pests.     

Genetically engineered maize plants reveal distinct costs and benefits of constitutive volatile emissions in the field

Genetic manipulation of plant volatile emissions is a promising tool to enhance plant defences against herbivores. However, the potential costs associated with the manipulation of specific volatile synthase genes are unknown. Therefore, we investigated the physiological and ecological effects of transforming a maize line with a terpene synthase gene in field and laboratory assays, both above‐ and below ground. The transformation, which resulted in the constitutive emission of (E)‐β‐caryophyllene and α‐humulene, was found to compromise seed germination, plant growth and yield. These physiological costs provide a possible explanation for the inducibility of an (E)‐β‐caryophyllene‐synthase gene in wild and cultivated maize. The overexpression of the terpene synthase gene did not impair plant resistance nor volatile emission. However, constitutive terpenoid emission increased plant apparency to herbivores, including adults and larvae of the above ground pest Spodoptera frugiperda, resulting in an increase in leaf damage. Although terpenoid overproducing lines were also attractive to the specialist root herbivore Diabrotica virgifera virgifera below ground, they did not suffer more root damage in the field, possibly because of the enhanced attraction of entomopathogenic nematodes. Furthermore, fewer adults of the root herbivore Diabrotica undecimpunctata howardii were found to emerge near plants that emitted (E)‐β‐caryophyllene and α‐humulene. Yet, overall, under the given field conditions, the costs of constitutive volatile production overshadowed its benefits. This study highlights the need for a thorough assessment of the physiological and ecological consequences of genetically engineering plant signals in the field to determine the potential of this approach for sustainable pest management strategies.     

Inga laurina trypsin inhibitor (ILTI) obstructs Spodoptera frugiperda trypsins expressed during adaptive mechanisms against plant protease inhibitors

Plant protease inhibitors (PIs) are elements of a common plant defense mechanism induced in response to herbivores. The fall armyworm, Spodoptera frugiperda, a highly polyphagous lepidopteran pest, responds to various PIs in its diet by expressing genes encoding trypsins. This raises the question of whether the PI‐induced trypsins are also inhibited by other PIs, which we posed as the hypothesis that Inga laurina trypsin inhibitor (ILTI) inhibits PI‐induced trypsins in S. frugiperda. In the process of testing our hypothesis, we compared its properties with those of selected PIs, soybean Kunitz trypsin inhibitor (SKTI), Inga vera trypsin inhibitor (IVTI), Adenanthera pavonina trypsin inhibitor (ApTI), and Entada acaciifolia trypsin inhibitor (EATI). We report that ILTI is more effective in inhibiting the induced S. frugiperda trypsins than SKTI and the other PIs, which supports our hypothesis. ILTI may be more appropriate than SKTI for studies regarding adaptive mechanisms to dietary PIs.     

Occurrence and direct control potential of parasitoids and predators of the fall armyworm (Lepidoptera: Noctuidae) on maize in the subtropical lowlands of Mexico

Abstract 1 Native natural enemies have the potential to control fall armyworm Spodoptera frugiperda (Smith) in tropical maize grown in Mexico, where this insect pest causes severe economic losses to farmers. It has been proposed that enhancing herbivore‐induced volatile emissions in maize plants may help to increase the effectiveness of natural enemies, which use these volatiles to locate their prey. This will only be of immediate benefit to farmers if the activity of the natural enemies results in a direct reduction in herbivory. Here we report on field surveys for the most common natural enemies in a tropical maize‐growing region in Mexico and the potential effects of these enemies on herbivory by fall armyworm. 2 Caterpillars were collected in maize fields near Poza Rica in the state of Veracruz during January and February 1999, 2000 and 2001. Plants were either naturally infested by S. frugiperda, or artificially infested with laboratory‐reared larvae. Ten species of parasitoids emerged from the collected larvae and eight species of predators that are known to feed on larvae and eggs were observed on the plants. Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae) was the dominant parasitoid species, in 1999 and 2001. 3 Of the nine larval parasitoids collected, six (all solitary) are known to reduce herbivory, whereas one causes the host to eat more (for two species this is not known). This implies that enhancing the effectiveness of solitary endoparasitoids may benefit subsistence farmers in developing countries by immediately reducing herbivory. The overall benefit for the plant resulting from parasitoid activity also has important implications for the evolutionary role of parasitoids in contributing to selection pressures that shape indirect defences in plants.     

Oviposition by a moth suppresses constitutive and herbivore-induced plant volatiles in maize

Plant volatiles function as important signals for herbivores, parasitoids, predators, and neighboring plants. Herbivore attack can dramatically increase plant volatile emissions in many species. However, plants do not only react to herbivore-inflicted damage, but also already start adjusting their metabolism upon egg deposition by insects. Several studies have found evidence that egg deposition itself can induce the release of volatiles, but little is known about the effects of oviposition on the volatiles released in response to subsequent herbivory. To study this we measured the effect of oviposition by Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) moths on constitutive and herbivore-induced volatiles in maize (Zea mays L.). Results demonstrate that egg deposition reduces the constitutive emission of volatiles and suppresses the typical burst of inducible volatiles following mechanical damage and application of caterpillar regurgitant, a treatment that mimics herbivory. We discuss the possible mechanisms responsible for reducing the plant’s signaling capacity triggered by S. frugiperda oviposition and how suppression of volatile organic compounds can influence the interaction between the plant, the herbivore, and other organisms in its environment. Future studies should consider oviposition as a potential modulator of plant responses to insect herbivores.     

Starvation‐induced autophagy in Spodoptera frugiperda Sf9 ovarian cells

Autophagy controls insect development and can be targeted for pest control in agriculture. In the present study, starvation‐induced autophagy is investigated in the insect species Spodoptera frugiperda. Bioinformatics analysis and a search of the EST database (http://bioweb.ensam.inra.fr/spodobase) identifies a putative ATG8 gene of S. frugiperda. To generate a biomarker of autophagosome, the DNA sequence encoding the open reading frame of this gene is amplified and cloned into a pIEX‐4‐mCherry‐EGFP‐SfATG8 recombinant vector. Sf9 cells are then transfected with this expression vector and starved in phosphate‐buffered saline solution for 4 h to induce autophagy, which is examined by LysoTracker staining (Life Technologies, Grand Island, New York), western blotting and fluorescence microscopy. The results obtained show that starvation stimulates lipidation of SfATG8‐PE and the formation of autophagosomes, providing a foundation for further research with respect to autophagy in insects.