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Neonicotinoid insecticides are used to manage spotted lanternfly (Lycorma delicatula (White); hereafter SLF), a recently introduced pest in the United States. Neonicotinoids can harm nontargets, such as pollinators potentially exposed via floral resources of treated plants. We quantified neonicotinoid residues in whole flowers of two SLF host plant species, red maple (Acer rubrum L. [Sapindales: Sapindaceae]) and tree-of-heaven (Ailanthus altissima (Mill.) [Sapindales: Simaroubaceae]), treated with post-bloom imidacloprid or dinotefuran applications that differed in timing and method of application. In red maple flowers, dinotefuran residues from fall applications were significantly higher than summer applications, while imidacloprid residues from fall applications were significantly lower than summer applications. Residues did not differ between application methods or sites. In tree-of-heaven flowers, dinotefuran residues were only detected in one of 28 samples at a very low concentration. To assess acute mortality risk to bees from oral exposure to residues in these flowers, we calculated risk quotients (RQ) using mean and 95% prediction interval residue concentrations from treatments in this study and lethal concentrations obtained from acute oral bioassays for Apis mellifera (L. (Hymenoptera: Apidae)) and Osmia cornifrons (Radoszkowski (Hymenoptera: Megachilidae)), then compared these RQs to a level of concern. For A. mellifera, only one treatment group, applied at 2X maximum label rate, had an RQ that exceeded this level. However, several RQs for O. cornifrons exceeded the level of concern, suggesting potential acute risk to solitary bees. Further studies are recommended for more comprehensive risk assessments to nontargets from neonicotinoid use for SLF management.

The International Standard of Phytosanitary Measures No. 15 (ISPM-15) requires signatory countries of the International Plant Protection Convention to use approved treatment methods on wood packaging materials used in international trade to significantly reduce the risk of spreading alien species. One of the approved methods utilizes dielectric heating (DH) to raise the temperature in the wood to 60°C through the profile of the workload for 1 minute to eradicate pests. To improve the uniform distribution of heat within a wood workload using radio frequency, a form of DH, we tested the use of a wool blanket as a thermal insulation material for wood pallet components. Three species commonly used in the pallet industry—yellow poplar (Liriodendron tulipifera L.), northern red oak (Quercus rubra L.), and eastern white pine (Pinus strobus L.)—of low moisture content were used to generate matched sets of stringer samples. Internal wood temperatures were monitored continuously during treatment to comply with the ISPM-15 schedule. Applying a wool (keratin) insulation blanket increased the rate of heating, improved heating uniformity throughout the workload, and reduced moisture loss compared to uninsulated controls for some wood species, but not all. These findings were most significant for pallet materials with higher moisture content and more permeability (white pine and yellow poplar). By reducing treatment time, power consumption costs can be reduced using wool insulation when implementing the ISPM-15 DH treatment schedule.

Since 2000, wild black cherry (Prunus serotina Ehrh.) has produced erratic seed crops, especially in the Allegheny National Forest where poor seed production has been implicated in difficulties with black cherry regeneration in forest stands. Given that black cherry is insect pollinated and unable to produce viable seed from self-pollination, a reduction in seed crops could be due to a pollination deficit; however, its key pollinators are unknown. Identifying the pollinators and factors that influence pollinator abundance and fidelity is critical for supporting and enhancing seed production for this valuable timber species. Over a two-year period in developed, semideveloped, and forested areas in Pennsylvania, we identified the potential pollinators of black cherry and examined how their abundance, along with several other abiotic and biotic factors, influenced viable seed production. We found that andrenid (Andrenidae: Hymenoptera) bees are likely the most important pollinators. The proportion of viable seeds increased as the number of andrenids increased, and these ground nesting bees were most abundant on forest edges, highlighting this habitat’s potential to support pollination services. Andrenids carried an average of 347-fold more black cherry pollen than flies and 18-fold more than halictid (Halictidae: Hymenoptera) bees. We did not find a significant relationship between the abundance of any other taxa besides andrenids and viable seed production. Black cherry flowers also provide resources for natural enemies such as the economically important parasitoid of Popillia japonica Newman (Scarabaeidae: Coleoptera), Tiphia vernalis Rohwer (Tiphiidae: Hymenoptera), which was observed feeding on black cherry nectar in this study.

The invasive planthopper, spotted lanternfly (SLF), Lycorma delicatula (White) (Hemiptera: Fulgoridae), feeds on a broad range of plants including species of economic importance such as grape. Although SLF feeds on wild and cultivated grape, the effect of grapevines on the insect’s life history traits is unknown. This study examined the effect of cultivated Concord grapevines (Vitis labrusca) and the insect’s preferred host tree of heaven (TOH), Ailanthus altissima, on SLF development, survival, reproduction, and body mass. Newly emerged nymphs were allowed to feed on either TOH, Concord grapevines or a mixed diet of Concord grapevines plus TOH through adulthood until death. Development, mortality, and oviposition of paired adults were tracked daily to calculate the SLF rate of development, survival, and reproduction among treatments. When feeding exclusively on Concord grapevines, SLF was able to develop and reproduce but had higher mortality, slower development, and produced fewer eggs. SLF fed on the mixed diet of grapevines plus TOH exhibited faster nymphal development, laid more eggs, and had higher body mass compared with those fed only on grape or TOH. SLF had greater survival when fed on either the mixed diet or on TOH alone. We conclude that Concord grapevines are a poor-quality host for SLF, but when combined with TOH, SLF fitness increases above that of feeding on TOH alone. This study supports the elimination of TOH as a part of SLF vineyard management practices.

Insects frequently confront different microbial assemblages. Bacteria inhabiting an insect gut are often commensal, but some can become pathogenic when the insect is compromised from different stressors. Herbivores are often confronted by various forms of plant resistance, but how defenses generate opportunistic microbial infections from residents in the gut are not well understood. In this study, we evaluated the pathogenic tendencies of Serratia isolated from the digestive system of healthy fall armyworm larvae (Spodoptera frugiperda) and how it interfaces with plant defenses. We initially selected Serratia strains that varied in their direct expression of virulence factors. Inoculation of the different isolates into the fall armyworm body cavity indicated differing levels of pathogenicity, with some strains exhibiting no effects while others causing mortality 24 h after injection. Oral inoculations of pathogens on larvae provided artificial diets caused marginal (

The spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae) (White, 1845), is an invasive pest in the Mid-Atlantic region of the United States. Understanding this pest’s dispersion patterns is fundamental for development of management and surveillance programs. To address this knowledge gap, we quantified spotted lanternfly nymph dispersion patterns by instar for rural and urban/suburban habitats, and we compared the number of sample units required for sticky traps and in situ visual counts to estimate population densities at several precisions. In addition, we assessed the ability of two experimental designs (completely random and randomized complete block) to detect management practices’ impacts in the field. All instars typically followed an aggregated dispersion pattern. Sample size and time requirements for checking and replacing sticky traps and for conducting in situ counts were similar, but in situ counts do not require purchasing traps, installation time, or delays before treatment, and do not remove insects. Although the cost for using in situ counts is likely less than for sticky traps, early instar spotted lanternfly nymph populations are harder to visually detect than later instars because of their small size, which may negate any cost advantage when treatments are applied early. In general, using a randomized complete block design resulted in higher statistical power than a completely random design, allowing detection of proportional population reductions of 10–20% less with equal replication. Studies aiming to evaluate treatments that reduce spotted lanternfly numbers by less than 60% will require researchers to evaluate the feasibility of using the required large sample sizes.

Lycorma delicatula (spotted lanternfly) has a broad host range with a strong preference for the invasive host plant from its native range, tree of heaven (Ailanthus altissima); it had long been speculated that L. delicatula could not develop or reproduce without access to tree of heaven. In 2019, we found that this assumption was incorrect, but fitness was reduced in the absence of A. altissima in that the number of egg masses laid was dramatically fewer for insects reared on suitable non-A. altissima host plants that had recently been established. We hypothesized that longer established, larger trees (of the same species) would improve the fitness of L. delicatula in the absence of tree of heaven. In spring 2020, we examined insect performance with and without access to A. altissima by tracking development, survival, host tree association and oviposition in large enclosures with trees planted two years prior to the study. Each enclosure included one each of Juglans nigra, Salix babylonica and Acer saccharinum along with either one A. altissima or one Betula nigra; these trees had twice the diameter of the same trees the previous year. We reared nymphs with and without access to A. altissima, released them into the corresponding large enclosures as third instars, and monitored them from early July 2020 through November 2020. We also determined whether lack of access to A. altissima by parents of L. delicatula have any fitness effects on offspring performance. To ensure adequate adult populations for comparing fecundity between treatments, third instars were released into the multi-tree enclosures due to high mortality in earlier instars that occurred in a similar study in 2019. Insect survival was higher and development faster with access to A. altissima. Third and fourth instar nymphs were most frequently observed on A. altissima when it was present, while adults were equally associated with A. saccharinum and A. altissima. In the absence of A. altissima, nymphs were most frequently found on S. babylonica, while adults were most often on A. saccharinum. Females with access to A. altissima deposited nearly 7-fold more egg masses than those without access to A. altissima, which is consistent with the difference in egg mass numbers between the two treatments the previous year; thus, our hypothesis was rejected. The offspring of parents that had been reared without access to A. altissima showed similar survival and development time from egg to adult as offspring from parents that never had access to A. altissima. These findings suggest that managers need to be aware that even in the absence of A. altissima in the landscape, several hardwood host trees can be utilized by L. delicatula to develop and reproduce, but fitness without A. altissima is likely to still be reduced.

Plant chemistry plays an important role in mediating resistance against insect herbivores. Depending on insect species and feeding strategies, plant defenses can prevent insect attacks, impact digestive physiology and influence reproductive output. The Asian longhorned beetle (Anoplophora glabripennis) is an invasive, tree-killing insect that exploits multiple host plant species. Anoplophora glabripennis is documented to attack Populus spp., but the ability for adults to consume Populus varies between beetle populations. In this study, we explored the influence of salicinoid phenolic glycosides, known to be potent chemical defenses in Populus against generalist insects, on A. glabripennis adult behavior and fecundity. We performed a series of choice- and no-choice experiments using extracts of salicinoids applied to plants that do not produce these compounds. Salicinoids affected A. glabripennis performance in a dose-dependent manner, with higher concentrations significantly reducing feeding. Egg development was reduced by > 90% in females fed salicinoids at ~ 1.3% dry weight. We then compared salicinoids present in feeding material vs. frass and found both a decrease in concentration and a shift in composition of these compounds. However, some of the more complex salicinoids were unchanged in the excrement, suggesting minimal detoxification had occurred. Our results show that salicinoids disrupt egg development in A. glabripennis, thereby reducing fitness. These results have implications for how A. glabripennis metabolizes plant defense chemicals and how tolerance may vary among populations.

The effect of temperature on insect-plant interactions in the face of changing climate is complex as the plant, its herbivores and their interactions are usually affected differentially leading to an asymmetry in response. Using experimental warming and a combination of biochemical and herbivory bioassays, the effects of elevated temperatures and herbivore damage (Helicoverpa zea) on resistance and tolerance traits of Solanum lycopersicum var. Better boy (tomato), as well as herbivory performance and salivary defense elicitors were examined. Insects and plants were differentially sensitive towards warming within the experimental temperature range. Herbivore growth rate increased with temperature, whereas plants growth as well as the ability to tolerate stress measured by photosynthesis recovery and regrowth ability were compromised at the highest temperature regime. In particular, temperature influenced the caterpillars’ capacity to induce plant defenses due to changes in the amount of a salivary defense elicitor, glucose oxidase (GOX). This was further complexed by the temperature effects on plant inducibility, which was significantly enhanced at an above-optimum temperature; this paralleled with an increased plants resistance to herbivory but significantly varied between previously damaged and undamaged leaves. Elevated temperatures produced asymmetry in species’ responses and changes in the relationship among species, indicating a more complicated response under a climate change scenario. Electronic supplementary material The online version of this article (10.1007/s10886-020-01201-6) contains supplementary material, which is available to authorized users.

Insects possess specific immune responses to protect themselves from different types of pathogens. Activation of immune cascades can inflict significant developmental costs on the surviving host. To characterize infection kinetics in a surviving host that experiences baculovirus inoculation, it is crucial to determine the timing of immune responses. Here, we investigated time-dependent immune responses and developmental costs elicited by inoculations from each of two wild-type baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Helicoverpa zea single nucleopolyhedrovirus (HzSNPV), in their common host H. zea. As H. zea is a semi-permissive host of AcMNPV and fully permissive to HzSNPV, we hypothesized there are differential immune responses and fitness costs associated with resisting infection by each virus species. Newly molted 4th-instar larvae that were inoculated with a low dose (LD15 ) of either virus showed significantly higher hemolymph FAD-glucose dehydrogenase (GLD) activities compared to the corresponding control larvae. Hemolymph phenoloxidase (PO) activity, protein concentration and total hemocyte numbers were not increased, but instead were lower than in control larvae at some time points post-inoculation. Larvae that survived either virus inoculation exhibited reduced pupal weight; survivors inoculated with AcMNPV grew slower than the control larvae, while survivors of HzSNPV pupated earlier than control larvae. Our results highlight the complexity of immune responses and fitness costs associated with combating different baculoviruses.

Management of the European gypsy moth [Lymantria dispar dispar (Linnaeus)] in North America has benefited from more than a century of research. The East Asian strains of the gypsy moth, however, bring new challenges including multiple subspecies (Lymantria dispar asiatica Vnukovskij and Lymantria dispar japonica Motschulsky), broad distributions across heterogeneous habitats, and a lack of data on the variation in the phenology of source populations, which may affect risk. To address these issues, published phenology parameters for eight populations of Asian gypsy moth were used to develop eight strain-specific agent-based phenological models. These models were applied to 47 ports in East Asia where the Asian gypsy moth is native, and output was compared with available trap data to assess the role of interpopulation variation in phenological parameters in predicting moth flight among varied locations, assess variation in the performance of models among years, and assess the importance of modeling phenology using parameters from a ‘local’ moth population. Variation in phenological parameters among the eight populations yielded variation in predicted flight times among the 47 ports analyzed, and the use of ‘local’ populations did not generally improve model fit. Model accuracy varied substantially among ports and among years within some ports. The larva-to-adult agent-based models described here have utility in estimating flight periods for some ports in their current form, but variation in model quality across the landscape suggests that there is potential for unsampled and unparameterized moth populations and factors that remain to be quantified.

Baculoviruses can induce climbing behaviour in their caterpillar hosts to ensure they die at elevated positions to enhance virus transmission, providing an excellent model to study parasitic manipulation of host behaviour. Here, we demonstrate that climbing behaviour occurred mostly during daylight hours, and that the height at death of Helicoverpa armigera single nucleopolyhedrovirus (HearNPV)-infected larvae increases with the height of the light source. Phototaxic and electroretinogram (ERG) responses were enhanced after HearNPV-infection in host larvae, and ablation of stemmata in infected larvae prevented both phototaxis and climbing behaviour. Through transcriptome and quantitative PCR, we confirmed that two opsin genes (a blue light-sensitive gene, HaBL; and a long wave-sensitive gene, HaLW) as well as the TRPL (transient receptor potential-like channel protein) gene, all integral to the host's visual perception pathway, were significantly upregulated after HearNPV infection. Knockout of HaBL, HaLW, or TRPL genes using the CRISPR/Cas9 system resulted in significantly reduced ERG responses, phototaxis, and climbing behaviour in HearNPV-infected larvae. These results reveal that HearNPV alters the expression of specific genes to hijack host visual perception at fundamental levels—photoreception and phototransduction—in order to induce climbing behaviour in host larvae.

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.

Significance Many plant defenses that deter insect herbivory target the attacker’s digestive system. We found that plant defenses against the fall armyworm created opportunities for resident gut microbes to penetrate protective gut barriers, invading the body cavity and exacerbating the negative impacts of plant defenses on the insect. These interactions triggered insect immune responses and collectively overwhelmed the insect’s ability to cope with multiple stressors. However, the effects varied between bacterial taxa, indicating that variation in the caterpillar microbiome can alter their phenotype. Our results reveal a previously unrecognized, and likely widespread, mechanism allowing the plant to use the insect’s gut microbiota against it in collaboration with the plant’s own defenses., Plants produce suites of defenses that can collectively deter and reduce herbivory. Many defenses target the insect digestive system, with some altering the protective peritrophic matrix (PM) and causing increased permeability. The PM is responsible for multiple digestive functions, including reducing infections from potential pathogenic microbes. In our study, we developed axenic and gnotobiotic methods for fall armyworm (Spodoptera frugiperda) and tested how particular members present in the gut community influence interactions with plant defenses that can alter PM permeability. We observed interactions between gut bacteria with plant resistance. Axenic insects grew more but displayed lower immune-based responses compared with those possessing Enterococcus, Klebsiella, and Enterobacter isolates from field-collected larvae. While gut bacteria reduced performance of larvae fed on plants, none of the isolates produced mortality when injected directly into the hemocoel. Our results strongly suggest that plant physical and chemical defenses not only act directly upon the insect, but also have some interplay with the herbivore’s microbiome. Combined direct and indirect, microbe-mediated assaults by maize defenses on the fall armyworm on the insect digestive and immune system reduced growth and elevated mortality in these insects. These results imply that plant–insect interactions should be considered in the context of potential mediation by the insect gut microbiome.

The spotted lanternfly, Lycorma delicatula (White), is a new invasive pest in the United States. To quantify spotted lanternfly population abundance, one must understand this pest’s dispersion pattern, that is, the spatial arrangement of individuals within a population. Spotted lanternflies overwinter in egg masses from late fall to May, making this life stage suitable for population assessments. We measured the dispersion pattern of egg masses at two types of sites: a suburban housing development, where we used individual trees as the sampling unit, and rural woodlots, where we used individual trees and also plots with 5.64 m radius as sampling units. Plots were the same size as those recommended for monitoring the gypsy moth, a well-studied pest with similar egg laying habit to the spotted lanternfly. Egg masses in both sampling units were counted up to a height of 3 m. With trees as the sampling unit, egg masses were aggregated in 12 of 20 rural sampling universes, randomly dispersed at 6, and completely absent at 2. Similar patterns were seen when using the 5.64-m radius rural sampling units and for suburban sampling universes. We calculated sample size requirements for a range of mean densities at a precision of 25 and 30%. Additionally, the vertical distribution of egg masses was characterized on the invasive tree of heaven [Ailanthus altissima (Mill.) Swingle], a preferred host for spotted lanternflies. For small trees, there was a positive relationship between number of egg masses in the bottom 3 m of the tree and the total count.

Spotted lanternfly (Lycorma delicatula) is a recently introduced pest in the United States, where it threatens the wine, timber, and ornamentals industries. Knowledge of the dispersal ability of L. delicatula is key to developing effective management strategies for this invasive pest. We conducted a mark, release, re-sight study, marking nymphs with fluorescent powders and observing dispersal distances from a central release point at three time points over 7 d following release. To examine how dispersal patterns changed over the course of nymphal development, we repeated this process for each of L. delicatula’s four instars. All releases were conducted in contiguous, deciduous forest, which is a widespread habitat type within L. delicatula’s invaded range and a habitat where this pest may have negative ecological and economic impacts. We found that nymphs displayed clear directionality in their movement following release, apparently preferring to move uphill on the modest 6° grade at our release site. Most nymphs remained near the release location, while some moved tens of meters. The maximum displacement we observed was 65 m from the release point, 10 d after release. Nymphs were re-sighted singly and in small groups on a variety of trees, shrubs, and understory vegetation. All four instars had similar dispersal distances over time, though third instar nymphs moved farthest on average, with estimated median displacement of 16.9 m 7 d after release. Further studies are needed to provide additional information on what factors influence spotted lanternfly dispersal.

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.

Insect herbivores possess a diverse and abundant gut microbiota that may influence plant growth in nature. The application of plant beneficial bacteria to improve agricultural production and soil quality has long been of interest. Thus, these insect-associated microbiota have the potential to be developed into effective bio-fertilizers. The bacterium, Enterobacter ludwigii, was isolated from the regurgitant of field-collected tomato fruitworm, Helicoverpa zea. The bacterium can be secreted by the insect onto tomato seeds during fruit feeding and is also commonly found in the soil. We applied E. ludwigii to germinated tomato seeds and measured tomato plant growth and productivity under controlled greenhouse conditions. Since there are often trade-offs between plant growth and plant defenses, we examined whether the E. ludwigii-mediated faster growth corresponds with weaker anti-herbivore defenses. When E. ludwigii was applied to germinated tomato seeds, the plants exhibited faster root, shoot and hypocotyl growth, and produced more fruits and seeds than untreated control plants. The plants treated with bacteria exhibited the same activity levels of two key enzymes involved in anti-herbivore defenses, polyphenol oxidase and peroxidase, and induced the same levels of mortality and growth inhibition in H. zea larvae as untreated plants. Thus, our results demonstrate that the application of E. ludwigii to seeds can promote tomato plant growth and yield without compromising anti-herbivore defenses.

We performed single-sensillum recordings from male and female antennae of the Asian longhorned beetle, Anoplophora glabripennis, that included as stimuli the two components of this species' aggregation-sex pheromone in addition to various general odorants. We compared the aggregation-sex-pheromone-component responses of olfactory sensory neurons (OSNs) to those of OSNs that responded to a variety of plant-related odorants. In the smooth-tipped, tapered, trichoid sensilla on the most distal antennal flagellomeres nos. 10 or 11 of both males and females, we found OSNs with high-amplitude action potentials that were tuned to the aldehyde and alcohol pheromone components and that did not respond to various plant-related volatiles. Because this OSN type responded to both the alcohol and aldehyde components it cannot be considered to be specifically tuned to either component. These large-spiking OSNs were co-compartmentalized in these sensilla with a second, smaller-spiking OSN responding to plant-related volatiles such as geraniol, citronellal, limonene, 1-octanol, nerol and citral. The large-spiking OSNs thus appear to be a type that will be involved in aggregation-sex pheromone pathways targeting a specific glomerulus in the antennal lobe and in generating pheromone-related behavioral responses in A. glabripennis. In other sensilla located in these distal antennal flagellomeres as well as those located more proximally, i.e., mid-length along the antenna on flagellomere nos. 4-7, we found OSNs in blunt-tipped basiconic sensilla that were responsive to other plant-related volatiles, especially the terpenoids, (E,E)-alpha farnesene, (E)-β-farnesene, β-caryophyllene, and eugenol. Some of these terpenoids have been implicated in improving attraction to pheromone-baited traps. Some of these same OSNs responded additionally to either of the two sex pheromone components, but because these OSNs also responded to some of the above plant volatiles as shown by cross-adaptation experiments, these OSNs will not be the types that convey sex-pheromone-specific information to the antennal lobe.

Microbial symbionts play pivotal roles in the ecology and physiology of insects feeding in woody plants. Both eukaryotic and bacterial members occur in these systems where they facilitate digestive and nutrient provisioning. The larval gut of the Asian longhorned beetle (Anoplophora glabripennis) is associated with a microbial consortium that fulfills these metabolic roles. While members of the community vary in presence and abundance among individuals from different hosts, A. glabripennis is consistently associated with a fungus in the Fusarium solani species complex (FSSC). We used amplicon sequencing, taxon-specific PCR, culturing, and imaging to determine how bacterial and fungal communities differ between life stages and possible modes of symbiont transfer. The bacterial and fungal communities of adult guts were more diverse than those from larvae and eggs. The communities of larvae and eggs were more similar to those from oviposition sites than from adult female guts. FSSC isolates were not detected in the reproductive tissues of adult females, but were consistently detected on egg surfaces after oviposition and in frass. These results demonstrate that frass can serve as a vehicle of transmission of a subset for the beetle gut microbiota. Vertically transmitted symbionts are often beneficial to their host, warranting subsequent functional studies.

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.

Significance The role of herbivore-associated microbes in mediating plant–herbivore interactions has gained recent attention. We show that a parasitoid associated with its caterpillar host not only suppresses the immune system of the caterpillar but also suppresses the induced defenses of the caterpillar’s host plant. Parasitoids inject eggs into their hosts but also inject polydnaviruses that suppress the caterpillar’s immunity. Immunosuppression enables eggs to hatch and develop as larvae within caterpillars. Additionally, the polydnavirus reduces salivary glucose oxidase, the primary elicitor found in the caterpillar’s oral secretions. Caterpillars injected with polydnavirus induce lower plant defenses than untreated caterpillars. Our results reveal a dimension to the complexity of plant–herbivore interactions indicating that polydnaviruses mediate the phenotypes of the parasitoid, herbivore, and plant., Obligate symbioses occur when organisms require symbiotic relationships to survive. Some parasitic wasps of caterpillars possess obligate mutualistic viruses called “polydnaviruses.” Along with eggs, wasps inject polydnavirus inside their caterpillar hosts where the hatching larvae develop inside the caterpillar. Polydnaviruses suppress the immune systems of their caterpillar hosts, which enables egg hatch and wasp larval development. It is unknown whether polydnaviruses also manipulate the salivary proteins of the caterpillar, which may affect the elicitation of plant defenses during feeding by the caterpillar. Here, we show that a polydnavirus of the parasitoid Microplitis croceipes, and not the parasitoid larva itself, drives the regulation of salivary enzymes of the caterpillar Helicoverpa zea that are known to elicit tomato plant-defense responses to herbivores. The polydnavirus suppresses glucose oxidase, which is a primary plant-defense elicitor in the saliva of the H. zea caterpillar. By suppressing plant defenses, the polydnavirus allows the caterpillar to grow at a faster rate, thus improving the host suitability for the parasitoid. Remarkably, polydnaviruses manipulate the phenotypes of the wasp, caterpillar, and host plant, demonstrating that polydnaviruses play far more prominent roles in shaping plant–herbivore interactions than ever considered.

Baculoviruses manipulate host climbing behaviour to ensure that the hosts die at elevated positions on host plants to facilitate virus proliferation and transmission, which is a process referred to as tree-top disease. However, the detailed molecular mechanism underlying tree-top disease has not been elucidated. Using transcriptome analysis, we showed that two hormone signals, juvenile hormone (JH) and 20-hydroxyecdysone (20E), are key components involved in HaSNPV-induced tree-top disease in Helicoverpa armigera larvae. RNAi-mediated knockdown and exogenous hormone treatment assays demonstrated that 20E inhibits virus-induced tree-top disease, while JH mediates tree-top disease behaviour. Knockdown of BrZ2, a downstream signal of JH and 20E, promoted HaSNPV-induced tree-top disease. We also found that two miRNAs target BrZ2 and are involved in the cross-talk regulation between 20E and JH manipulating HaSNPV replication, time to death and HaSNPV-induced tree-top disease.

Insect guts harbor diverse microbial assemblages that can be influenced by multiple factors, including gut physiology and interactions by the host with its environment. The Asian longhorned beetle (A. glabripennis; Cerambycidae: Lamiinae) is an invasive tree-killing insect that harbors a diverse consortium of fungal and bacterial gut associates that provision nutrients and facilitate lignocellulose digestion. The physicochemical conditions of the A. glabripennis gut and how these conditions may influence the microbial composition across gut regions are unknown. In this study, we used microsensors to measure in situ oxygen concentrations, pH, and redox potential along the length of the A. glabripennis larval gut from two North American populations. We then analyzed and compared bacterial and fungal gut communities of A. glabripennis within individual hosts along the length of the gut using 16S and ITS1 amplicon sequencing. The A. glabripennis midgut lumen was relatively anoxic (0.01kPa) with a pH gradient from 5.5 to 9, moving anterior to posterior. Redox potential was higher in the anterior midgut relative to posterior regions. No differences in physicochemistry were measured between the two populations of the beetle, but the two populations harbored different communities of bacteria and fungi. However, microbial composition of the A. glabripennis gut microbiota did not differ among gut regions despite physicochemical differences. Unlike other insect systems that have distinct gut compartmentalization and corresponding microbial assemblages, the A. glabripennis gut lacks dramatic morphological modifications, which may explain why discrete microbial community structures were not found along the digestive system.

Periodic introductions of the Asian subspecies of gypsy moth, Lymantria dispar asiatica Vnukovskij and Lymantria dispar japonica Motschulsky, in North America are threatening forests and interrupting foreign trade. Although Asian gypsy moth has similar morphology to that of European and North American gypsy moth, it has several traits that make it a greater threat, the most important being the flight capability of females. Asian gypsy moth is not yet established in North America; however, infestations have been detected multiple times in Canada and the United States. To facilitate detection and eradication efforts, we evaluated the effect of a range of temperatures on development time, survivorship, and fertility of eight populations of Asian gypsy moth. There were significant impacts of temperature and population on these life history characteristics. The larval developmental rate increased with temperature until it reached an optimum at 29 °C. Larvae experienced significant molting problems at the highest and lowest temperatures tested (10 °C and 30 °C). At 30 °C, female fitness was markedly compromised, as evidenced by reduced fecundity and fertility. This suggests that development and survival of Asian gypsy moth may be limited by summer temperature extremes in the Southern United States. We also determined the degree-day requirements for two critical life stages and two populations of Asian gypsy moth, which represent the extremes in latitude, to predict the timing for biopesticide application and adult trap deployment. Our data will benefit pest managers in developing management strategies, pest risk assessments, and timing for implementation of management tactics.

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.

Baculoviruses are food-borne microbial pathogens that are ingested by insects on contaminated foliage. Oxidation of plant-derived phenolics, activated by insect feeding, can directly interfere with infections in the gut. Since phenolic oxidation is an important component of plant resistance against insects, baculoviruses are suggested to be incompatible with plant defences. However, plants among and within species invest differently in a myriad of chemical and physical defences. Therefore, we hypothesized that among eight soybean genotypes, some genotypes would be able to maintain both high resistance against an insect pest and high efficacy of a baculovirus. Soybean constitutive (non-induced) and jasmonic acid (JA)-induced (anti-herbivore response) resistance was measured against the fall armyworm Spodoptera frugiperda (weight gain, leaf consumption and utilization). Indicators of phenolic oxidation were measured as foliar phenolic content and peroxidase activity. Levels of armyworm mortality inflicted by baculovirus (SfMNPV) did not vary among soybean genotypes when the virus was ingested with non-induced foliage. Ingestion of the virus on JA-induced foliage reduced armyworm mortality, relative to non-induced foliage, on some soybean genotypes. Baculovirus efficacy was lower when ingested with foliage that contained higher phenolic content and defensive properties that reduced armyworm weight gain and leaf utilization. However, soybean genotypes that defended the plant by reducing consumption rate and strongly deterred feeding upon JA-induction did not reduce baculovirus efficacy, indicating that these defences may be more compatible with baculoviruses to maximize plant protection. Differential compatibility of defence traits with the third trophic level highlights an important cost/trade-off associated with plant defence strategies.

Electrophysiological recordings from the labial and maxillary palps of the Asian longhorned beetle, Anoplophora glabripennis, revealed their ability to detect several volatile chemicals, including water vapor and acetic acid. The results indicate that these appendages may play a large role in this beetle's assessment of its immediate environment. A. glabripennis is a highly destructive, invasive pest that feeds preferentially on maple - but accepts many other tree species - in North America, warranting USDA quarantine zones and an eradication program. While control and sampling techniques are being developed for this insect, a better understanding of its sensory capabilities is helpful. Electropalpograms (EPGs) revealed that both the maxillary and labial palps are highly sensitive to changes in humidity, indicating the presence of hygroreceptors and the likely important role of humidity in such things as feeding and finding water or oviposition sites. Strong EPG responses to a narrow set of volatile chemicals indicate that olfactory sensory neurons (OSNs) on the palps may be tuned to a small number of volatile compounds. The types of odorant molecules eliciting responses indicate that there are likely both odorant receptors (ORs) as well as ionotropic receptors (IRs) expressed on the OSNs, enabling palp OSNs to be able to respond to acids and aldehydes such as acetic acid and butyraldehyde. There were no significant EPG responses to this species' trail-sex pheromone components, which may indicate that the trail pheromone is primarily perceived via gustatory receptors contacting the substrate. These results indicate that the palps have a role in the beetle's assessment of its immediate environment underfoot, and that the sampling of surface odors and humidity via mouth parts may be important to this species' success.

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).

Anoplophora glabripennis has a complex suite of mate-finding behaviors, the functions of which are not entirely understood. These behaviors are elicited by a number of factors, including visual and chemical cues. Chemical cues include a male-produced volatile semiochemical acting as a long-range sex pheromone, a female-produced cuticular hydrocarbon blend serving as a sex-identification contact pheromone, and a recently identified female-produced trail sex pheromone that is followed by mate-seeking males. However, the sensory appendages and sensilla on these appendages used to detect the trail sex pheromone are unknown. We evaluated the ability of virgin male A. glabripennis to follow a sex pheromone trail after removal of the terminal four antennal segments and/or the maxillary and labial palps using a two-choice behavioral bioassay. We also tested the ability of males to follow the trail sex pheromone using volatile pheromone cues only, without physical contact with the pheromone. Results indicate that the palps are primarily responsible for sensing the pheromone, with males lacking palps unable to respond behaviorally to the trail sex pheromone. Under the conditions of this study, males could not follow the sex pheromone trail without direct contact, suggesting that olfaction may not be involved in detection of this pheromone. However, we did not determine to what degree the trail pheromone chemicals can volatilize under our experimental conditions. This work is important in elucidating the behaviors and sensory structures involved in mate-finding by this species on host trees, and these studies may help determine whether the trail sex pheromone has applications for monitoring and management.

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.

Radio-frequency (RF) and microwave (MW) heating for phytosanitary treatment of green wood in compliance with international standards for phytosanitary measures no. 15 (ISPM-15) were evaluated and compared to assess treatment time, depth of electromagnetic wave (EMW) penetration and heating uniformity. White oak (Quercus alba) cants (48 cm long with cross-section dimensions ranging from 10 × 10 cm2 to 25 × 25 cm2) were heated in a 19 MHz RF or 2.45 GHz MW laboratory oven using an equivalent heating power (3.4 kW). In each specimen, temperature was measured at different depths (distance from the upper face). Specimens were held in the treatment chamber for 2 min after the target temperature of 60 °C was achieved through the profile of the specimen to ensure compliance with the ISPM-15 treatment schedule. Thermal image analyses of treated specimens as well as theoretical depth of penetration for dielectric energy were explored. Wood specimens were also heated using RF at high power (9–11 kW) and results were compared with RF heating at 3.4 kW. For wood with cross-section dimensions of 10 × 10 cm2 to 15 × 15 cm2, heating rates for RF and MW were relatively similar. However, above 15 × 15 cm2, RF heating was more than 40 % faster with greater heating uniformity than MW. The theoretical values derived for depth of penetration and thermal image analyses indicate that RF (19 MHz) penetrates wood more uniformly and is better suited than MW (2.45 GHz) for bulk volume treatments of wood.

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.

Plants damaged by herbivore feeding can induce defensive responses that reduce herbivore growth. The slow-growth, high-mortality hypothesis postulates that these non-lethal plant defenses prolong the herbivore's period of susceptibility to natural enemies, such as predators and parasitoids. While many juvenile animals increase their disease resistance as they grow, direct tests of the slow-growth, high-mortality hypothesis in the context of plant-herbivore-pathogen interactions are lacking. Caterpillars increase their resistance to lethal baculoviruses as they develop within and across instars, a phenomenon termed developmental resistance. Progression of developmental resistance can occur through age-related increases in systemic immune functioning and/or midgut-based resistance. Here, we examined the slow-growth, high-mortality hypothesis in the context of developmental resistance of caterpillars to baculoviruses. Intra-stadial (within-instar) developmental resistance of the fall armyworm, Spodoptera frugiperda, to an oral inoculum of the baculovirus SfMNPV increased more rapidly with age when larvae were fed on non-induced foliage than foliage that was induced by jasmonic acid (a phytohormone that up-regulates plant anti-herbivore defenses). The degree of developmental resistance observed was attributable to larval weight at the time of virus inoculation. Thus, slower growth on induced plants prolonged the window of larval susceptibility to the baculovirus. Developmental resistance on induced and non-induced plants was absent when budded virus was injected intrahemocoelically bypassing the midgut, suggesting that developmental resistance was gut-based. Addition of fluorescent brightener, which weakens midgut-based resistance mechanisms to oral virus challenge, abolished developmental resistance. These results highlight the impact of plant defenses on herbivore growth rate and consequences for disease risk.

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 β-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.

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.