Tomato Chemical Defenses Intensify Corn Earworm (Helicoverpa zea) Mortality from Opportunistic Bacterial Pathogens.

Insect herbivores face multiple challenges to their ability to grow and reproduce. Plants can produce a series of defenses that disrupt and damage the herbivore digestive system, which are heightened upon injury by insect feeding. Additionally, insects face threats from virulent microorganisms that can incur their own set of potential costs to hosts. Microorganisms that invade through the digestive system may function in concert with defenses generated by plants, creating combined assailments on host insects. In our study, we evaluated how tomato defenses interact with an enteric bacterial isolate, Serratia marcescens, in the corn earworm (Helicoverpa zea). We performed bioassays using different tomato cultivars that were induced by methyl jasmonate and larvae orally inoculated with a S. marcescens isolate. Untreated corn earworm larval mortality was low on constitutive tomato, while larvae inoculated with S. marcescens exhibited > 50% mortality within 5 days. Induction treatments elevated both control mortality (~ 45%) and in combination with S. marcescens (> 95%). Larvae also died faster when encountering induced defenses and Serratia. Using a tomato mutant, foliar polyphenol oxidase activity likely had stronger impacts on S. marcescens-mediated larval mortality. Induction treatments also elevated the number of bacterial colony-forming units in the hemolymph of larvae inoculated with Serratia. Larval mortality by S. marcescens was low (< 10%) on artificial diets. Our results demonstrate that plant chemical defenses enhance larval mortality from an opportunistic gut microbe. We propose that the combined damage from both the plant and microbial agent overwhelm the herbivore to increase mortality rates and expedite host death.

Spotted Lanternfly (Hemiptera: Fulgoridae) Can Complete Development and Reproduce Without Access to the Preferred Host, Ailanthus altissima.

Despite its broad host range, the spotted lanternfly Lycorma delicatula (White), is known to have a marked preference for Ailanthus altissima. However, whether this polyphagous phloem feeder can complete its life cycle in the absence of A. altissima is unknown. We examined the performance of L. delicatula with and without access to A. altissima by tracking development, survival, host tree species association, and oviposition in large enclosures planted with Salix babylonica and Acer saccharinum along with either A. altissima or Betula nigra. We monitored enclosures from late May 2019 through June 2020. Lycorma delicatula survival was slightly higher in enclosures with A. altissima and 50% of individuals in A. altissima enclosures reached the adult stage ~6.5 d earlier than in enclosures without A. altissima. In the presence of A. altissima, nymphs were most frequently observed on this host while adults were found at similar frequencies on A. altissima and A. saccharinum. In the absence of A. altissima, nymphs were most frequently associated with S. babylonica and A. saccharinum, while adults were most often found on A. saccharinum. Females laid a total of 46 and 6 egg masses in enclosures with and without A. altissima, respectively, before freezing temperatures killed the remaining adults. The proportion of eggs that hatched per egg mass did not differ between treatments. Although L. delicatula can complete development and reproduce on other host species without access to A. altissima, fitness was reduced. These findings have implications for management that relies exclusively on treatment of A. altissima.

Parasitic Wasp Mediates Plant Perception of Insect Herbivores.

Microplitis croceipes is a solitary parasitoid that specializes on noctuid larvae of Helicoverpa zea and Heliothis virescens. Both the parasitoid and its hosts are naturally distributed across a large part of North America. When parasitoids deposit their eggs into hosts, venom and polydnaviruses (PDVs) are also injected into the caterpillars, which can suppress host immune responses, thus allowing parasitoid larvae to develop. In addition, PDVs can regulate host oral cues, such as glucose oxidase (GOX). The purpose of this study was to determine if parasitized caterpillars differentially induce plant defenses compared to non-parasitized caterpillars using two different caterpillar host/plant systems. Heliothis virescens caterpillars parasitized by M. croceipes had significantly lower salivary GOX activity than non-parasitized caterpillars, resulting in lower levels of tomato defense responses, which benefited parasitoid performance by increasing the growth rate of parasitized caterpillars. In tobacco plants, parasitized Helicoverpa zea caterpillars had lower GOX activity but induced higher plant defense responses. The higher tobacco defense responses negatively affected parasitoid performance by reducing the growth rate of parasitized caterpillars, causing longer developmental periods, and reduced cocoon mass and survival of parasitoids. These studies demonstrate a species-specific effect in different plant-insect systems. Based on these results, plant perception of insect herbivores can be affected by parasitoids and lead to positive or negative consequences to higher trophic levels depending upon the particular host-plant system.

Divergent host plant utilization by adults and offspring is related to intra-plant variation in chemical defences.

Adult and juvenile herbivores of the same species can use divergent feeding strategies, and thus may inhabit and consume different parts of the plant. Because the expression of chemical defences often differs between host plant tissues, this variation may result in disparate performance outcomes for adult and juvenile conspecifics that feed on distinct dietary substrates. The goal of this study was to evaluate how host range may differ between adults and juveniles in a generalist herbivore. We addressed the impacts of among- and within-plant defence variation using the wood-feeding Asian longhorned beetle (Anoplophora glabripennis) and three host plants having a range of putative resistance. Impacts of host plants on adult and offspring performance were assessed using a series of controlled bioassays. We evaluated adult-feeding and egg-laying behaviours in choice and no-choice experiments using the different hosts, and subsequent offspring establishment. We then evaluated host plant chemical composition related to nutrition and defence. Different plants had strong impacts on adult performance, but these patterns did not extend to effects on offspring. Females were capable of developing eggs when provided Acer rubrum, but not Populus deltoides or Populus tomentosa. Females that produced eggs by feeding on A. rubrum, however, deposited eggs into all three plant species. Larvae hatched and consumed tissues in all three hosts. The differences between adult and juvenile utilization of Populus spp. were reflected in markedly higher salicinoid phenolic concentrations in bark (>2% dw), while wood had trace quantities. Our results demonstrate that plant resistance mechanisms can differentially act upon adult and juvenile life stages of a polyphagous herbivore when there is differential expression of chemical defences among plant tissue types. Anoplophora glabripennis has been a globally successful invader due in part to its broad host range, and our results suggest a mechanism that permits the beetle to exploit marginally resistant plants. This study has implications for how host range differs between insect feeding stages, which is particularly important for invasive, polyphagous species encountering novel food sources.

Pathogen-Mediated Tritrophic Interactions: Baculovirus-Challenged Caterpillars Induce Higher Plant Defenses than Healthy Caterpillars.

Although the tritrophic interactions of plants, insect herbivores and their natural enemies have been intensely studied for several decades, the roles of entomopathogens in their indirect modulation of plant-insect relationships is still unclear. Here, we employed a sublethal dose of a baculovirus with a relatively broad host range (AcMNPV) to explore if feeding by baculovirus-challenged Helicoverpa zea caterpillars induces direct defenses in the tomato plant. We examined induction of plant defenses following feeding by H. zea, including tomato plants fed on by healthy caterpillars, AcMNPV-challenged caterpillars, or undamaged controls, and subsequently compared the transcript levels of defense related proteins (i.e., trypsin proteinase inhibitors, peroxidase and polyphenol oxidase) and other defense genes (i.e., proteinase inhibitor II and cysteine proteinase inhibitor) from these plants, in addition to comparing caterpillar relative growth rates. As a result, AcMNPV-challenged caterpillars induced the highest plant anti-herbivore defenses. We examined several elicitors and effectors in the secretions of these caterpillars (i.e., glucose oxidase, phospholipase C, and ATPase hydrolysis), which surprisingly did not differ between treatments. Hence, we suggest that the greater induction of plant defenses by the virus-challenged caterpillars may be due to differences in the amount of these secretions deposited during feeding or to some other unknown factor(s).

Host plant and population source drive diversity of microbial gut communities in two polyphagous insects.

Symbioses between insects and microbes are ubiquitous, but vary greatly in terms of function, transmission mechanism, and location in the insect. Lepidoptera (butterflies and moths) are one of the largest and most economically important insect orders; yet, in many cases, the ecology and functions of their gut microbiomes are unresolved. We used high-throughput sequencing to determine factors that influence gut microbiomes of field-collected fall armyworm (Spodoptera frugiperda) and corn earworm (Helicoverpa zea). Fall armyworm midgut bacterial communities differed from those of corn earworm collected from the same host plant species at the same site. However, corn earworm bacterial communities differed between collection sites. Subsequent experiments using fall armyworm evaluating the influence of egg source and diet indicated that that host plant had a greater impact on gut communities. We also observed differences between regurgitant (foregut) and midgut bacterial communities of the same insect host, suggesting differential colonization. Our findings indicate that host plant is a major driver shaping gut microbiota, but differences in insect physiology, gut region, and local factors can also contribute to variation in microbiomes. Additional studies are needed to assess the mechanisms that affect variation in insect microbiomes, as well as the ecological implications of this variability in caterpillars.

Herbivore-Induced Defenses in Tomato Plants Enhance the Lethality of the Entomopathogenic Bacterium, Bacillus thuringiensis var. kurstaki.

Plants can influence the effectiveness of microbial insecticides through numerous mechanisms. One of these mechanisms is the oxidation of plant phenolics by plant enzymes, such as polyphenol oxidases (PPO) and peroxidases (POD). These reactions generate a variety of products and intermediates that play important roles in resistance against herbivores. Oxidation of the catecholic phenolic compound chlorogenic acid by PPO enhances the lethality of the insect-killing bacterial pathogen, Bacillus thuringiensis var. kurstaki (Bt) to the polyphagous caterpillar, Helicoverpa zea. Since herbivore feeding damage often triggers the induction of higher activities of oxidative enzymes in plant tissues, here we hypothesized that the induction of plant defenses would enhance the lethality of Bt on those plants. We found that the lethality of a commercial formulation of Bt (Dipel® PRO DF) on tomato plants was higher if it was applied to plants that were induced by H. zea feeding or induced by the phytohormone jasmonic acid. Higher proportions of H. zea larvae killed by Bt were strongly correlated with higher levels of PPO activity in the leaflet tissue. Higher POD activity was only weakly associated with higher levels of Bt-induced mortality. While plant-mediated variation in entomopathogen lethality is well known, our findings demonstrate that plants can induce defensive responses that work in concert with a microbial insecticide/entomopathogen to protect against insect herbivores.

Host-plant induced changes in microbial community structure and midgut gene expression in an invasive polyphage (Anoplophora glabripennis).

Polyphagous insect herbivores possess diverse mechanisms to overcome challenges of feeding in multiple plant species including, but not limited to, transcriptional plasticity and associations with obligate or facultative symbionts. The Asian longhorned beetle (Anoplophora glabripennis) is a polyphagous wood-feeder capable of developing on over 100 tree species and, like other polyphages, its genome contains amplifications of digestive and detoxification genes. This insect also possesses a diverse gut microbial community, which has the metabolic potential to augment digestive physiology. While the genomic repertoires of A. glabripennis and its microbial community have been studied previously, comparatively less is known about how the gut transcriptome and community change in response to feeding in different hosts. In this study, we show that feeding in two suitable hosts (Acer spp. and Populus nigra) altered the expression levels of multicopy genes linked to digestion and detoxification. However, feeding in a host with documented resistance (Populus tomentosa) induced changes in the transcriptome and community beyond what was observed in insects reared in P. nigra, including the downregulation of numerous ß-glucosidases, odorant binding proteins, and juvenile hormone binding proteins, the upregulation of several cuticular genes, and the loss of one major bacterial family from the gut community.

Gut-Associated Bacteria of Helicoverpa zea Indirectly Trigger Plant Defenses in Maize.

Insect-associated microbes can contribute to the physiological and ecological functions of insects. Despite a few examples in beetles and piercing-sucking insects, the varied mechanisms of how insect-associated bacteria mediate plant-insect interactions are still not fully understood. The polyphagous herbivore Helicoverpa zea is a major agricultural pest that harbors certain microbes in their digestive systems. Enterobacter ludwigii is one of the gut-associated bacteria identified from field-collected caterpillars, and it has been shown to indirectly induce defenses in the dicot plant tomato by triggering the biosynthesis of salivary elicitors, but there are no clear mechanisms to show how gut microbes alter these salivary cues and how a different host plant responds to these inducible elicitors. Here, we conducted a series of assays to determine whether infection with E. ludwigii affects H. zea larval growth, immunity, and salivary responses and thus influences induced defenses of maize to herbivory. Inoculating lab-reared caterpillars with E. ludwigii, did not significantly affect the growth of caterpillars, but two immunity-related genes glucose oxidase (GOX) and lysozyme (LYZ) were more highly expressed in both salivary glands and midguts compared with MgCl2 solution-treated caterpillars. Oral elicitors were evaluated for their role in triggering maize-specific defense responses. Our results show that saliva and its main component protein glucose oxidase (GOX) from E. ludwigii-inoculated caterpillars played a role in inducing maize anti-herbivore responses. These findings provide a novel concept that introducing bacteria to an herbivore may be an important approach to pest control through alteration of insect immune responses and thus indirect induction of plant resistance.

Helicoverpa zea gut-associated bacteria indirectly induce defenses in tomato by triggering a salivary elicitor(s).

Insect gut-associated microbes modulating plant defenses have been observed in beetles and piercing-sucking insects, but the role of caterpillar-associated bacteria in regulating plant induced defenses has not been adequately examined. We identified bacteria from the regurgitant of field-collected Helicoverpa zea larvae using 16S ribosomal RNA (rRNA) gene sequencing and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. A combination of biochemical, molecular, and confocal electron microscopy methods were used to determine the role of caterpillar-associated bacteria in mediating defenses in Solanum lycopersicum (tomato). Laboratory-reared H. zea inoculated with one of the bacteria identified in field-collected H. zea, Enterobacter ludwigii, induced expression of the tomato defense-related enzyme polyphenol oxidase and genes regulated by jasmonic acid (JA), whereas the salicylic acid (SA)-responsive pathogenesis-related gene was suppressed. Additionally, saliva and its main component glucose oxidase from inoculated caterpillars played an important role in elevating tomato anti-herbivore defenses. However, there were only low detectable amounts of regurgitant or bacteria on H. zea-damaged tomato leaves. Our results suggest that H. zea gut-associated bacteria indirectly mediate plant-insect interactions by triggering salivary elicitors. These findings provide a proof of concept that introducing gut bacteria to a herbivore may provide a novel approach to pest management through indirect induction of plant resistance.

Fall Armyworm-Associated Gut Bacteria Modulate Plant Defense Responses.

Mechanical damage caused by insect feeding along with components present in insect saliva and oral secretions are known to induce jasmonic acid-mediated defense responses in plants. This study investigated the effects of bacteria from oral secretions of the fall armyworm Spodoptera frugiperda on herbivore-induced defenses in tomato and maize plants. Using culture-dependent methods, we identified seven different bacterial isolates belonging to the family Enterobacteriacea from the oral secretions of field-collected caterpillars. Two isolates, Pantoea ananatis and Enterobacteriaceae-1, downregulated the activity of the plant defensive proteins polyphenol oxidase and trypsin proteinase inhibitors (trypsin PI) but upregulated peroxidase (POX) activity in tomato. A Raoultella sp. and a Klebsiella sp. downregulated POX but upregulated trypsin PI in this plant species. Conversely, all of these bacterial isolates upregulated the expression of the herbivore-induced maize proteinase inhibitor (mpi) gene in maize. Plant treatment with P. ananatis and Enterobacteriaceae-1 enhanced caterpillar growth on tomato but diminished their growth on maize plants. Our results highlight the importance of herbivore-associated microbes and their ability to mediate insect plant interactions differently in host plants fed on by the same herbivore.

Herbivore Oral Secreted Bacteria Trigger Distinct Defense Responses in Preferred and Non-Preferred Host Plants.

Insect symbiotic bacteria affect host physiology and mediate plant-insect interactions, yet there are few clear examples of symbiotic bacteria regulating defense responses in different host plants. We hypothesized that plants would induce distinct defense responses to herbivore- associated bacteria. We evaluated whether preferred hosts (horsenettle) or non-preferred hosts (tomato) respond similarly to oral secretions (OS) from the false potato beetle (FPB, Leptinotarsa juncta), and whether the induced defense triggered by OS was due to the presence of symbiotic bacteria in OS. Both horsenettle and tomato damaged by antibiotic (AB) treated larvae showed higher polyphenol oxidase (PPO) activity than those damaged by non-AB treated larvae. In addition, application of OS from AB treated larvae induced higher PPO activity compared with OS from non-AB treated larvae or water treatment. False potato beetles harbor bacteria that may provide abundant cues that can be recognized by plants and thus mediate corresponding defense responses. Among all tested bacterial isolates, the genera Pantoea, Acinetobacter, Enterobacter, and Serratia were found to suppress PPO activity in tomato, while only Pantoea sp. among these four isolates was observed to suppress PPO activity in horsenettle. The distinct PPO suppression caused by symbiotic bacteria in different plants was similar to the pattern of induced defense-related gene expression. Pantoea inoculated FPB suppressed JA-responsive genes and triggered a SA-responsive gene in both tomato and horsenettle. However, Enterobacter inoculated FPB eliminated JA-regulated gene expression and elevated SA-regulated gene expression in tomato, but did not show evident effects on the expression levels of horsenettle defense-related genes. These results indicate that suppression of plant defenses by the bacteria found in the oral secretions of herbivores may be a more widespread phenomenon than previously indicated.

Sex-specific trail pheromone mediates complex mate finding behavior in Anoplophora glabripennis.

Anoplophora glabripennis (Motsch.) is a polyphagous member of the Cerambycidae, and is considered, worldwide, to be one of the most serious quarantine pests of deciduous trees. We isolated four chemicals from the trail of A. glabripennis virgin and mated females that were not present in trails of mature males. These compounds were identified as 2-methyldocosane and (Z)-9-tricosene (major components), as well as (Z)-9-pentacosene and (Z)-7-pentacosene (minor components); every trail wash sample contained all four chemical components, although the amounts and ratios changed with age of the female. Males responded to the full pheromone blend, regardless of mating status, but virgin females chose the control over the pheromone, suggesting that they may use it as a spacing pheromone to avoid intraspecific competition and maximize resources. Virgin, but not mated, males also chose the major pheromone components in the absence of the minor components, over the control. Taken together, these results indicate that all four chemicals are components of the trail pheromone. The timing of production of the ratios of the pheromone blend components that produced positive responses from males coincided with the timing of sexual maturation of the female.

Herbivore exploits orally secreted bacteria to suppress plant defenses.

Induced plant defenses in response to herbivore attack are modulated by cross-talk between jasmonic acid (JA)- and salicylic acid (SA)-signaling pathways. Oral secretions from some insect herbivores contain effectors that overcome these antiherbivore defenses. Herbivores possess diverse microbes in their digestive systems and these microbial symbionts can modify plant-insect interactions; however, the specific role of herbivore-associated microbes in manipulating plant defenses remains unclear. Here, we demonstrate that Colorado potato beetle (Leptinotarsa decemlineata) larvae exploit bacteria in their oral secretions to suppress antiherbivore defenses in tomato (Solanum lycopersicum). We found that antibiotic-untreated larvae decreased production of JA and JA-responsive antiherbivore defenses, but increased SA accumulation and SA-responsive gene expression. Beetles benefit from down-regulating plant defenses by exhibiting enhanced larval growth. In SA-deficient plants, suppression was not observed, indicating that suppression of JA-regulated defenses depends on the SA-signaling pathway. Applying bacteria isolated from larval oral secretions to wounded plants confirmed that three microbial symbionts belonging to the genera Stenotrophomonas, Pseudomonas, and Enterobacter are responsible for defense suppression. Additionally, reinoculation of these bacteria to antibiotic-treated larvae restored their ability to suppress defenses. Flagellin isolated from Pseudomonas sp. was associated with defense suppression. Our findings show that the herbivore exploits symbiotic bacteria as a decoy to deceive plants into incorrectly perceiving the threat as microbial. By interfering with the normal perception of herbivory, beetles can evade antiherbivore defenses of its host.