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Developmental ability of Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) was examined on six different sorghum milling fractions: Bran, Shorts, Cgrits, Fgrits, Red dogs, Flour, and also on a standard diet of Oat Flakes. For this purpose, a 1-day-old egg was placed in a vial containing 1 g of one of the sorghum fractions and exposed to three temperatures: 25, 30, or 32°C. All vials were checked daily for pupal and adult emergence and mortality of immatures. The developmental time was significantly affected by the type of sorghum fraction. After two weeks, for most of the temperatures examined, the longest developmental times in most cases for both pupation and adult emergence were observed in Flour and Oat flakes. Increasing the temperature from 25 to 30 accelerated the development, while adult emergence time did not differ between 30 and 32°C for all fractions except Flour. Egg mortality ranged from 11 to 78%, while larval and pupal mortality ranged from 0 to 22 and 0 to 45%, respectively for all sorghum fractions and different temperatures tested. Moreover, the mean overall immature mortality occurred at 30°C was 49.2, 39.7, and 65.1% at 25, 30, and 32°C for all the diets examined. The findings of the present work show that O. surinamensis can develop and survive in sorghum milling fractions and that the optimal temperatures for growth enhancement are 30 and 32°C. The temperatures within sorghum milling facilities could support O. surinamensis development on milling fractions if they are not addressed through phytosanitary measures.

Insects and microbes are known to interact in a variety of ways at food facilities, compounding damage. However, little research has explicated how specific common fungal species affect the behavior of the cosmopolitan secondary stored product pest, Lasioderma serricorne. Enhanced knowledge about attraction to microbially-produced volatile organic compounds (MVOCs) may be used to manipulate insect behavior. Aspergillus flavus and Fusarium verticillioides are two common, widespread pre- and postharvest fungi on small cereals that produce aflatoxins and fumonisins, respectively, while directly competing with each other for nutrients. Our goals were to (1) characterize the volatile emissions from grain inoculated by A. flavus or F. verticillioides derived from the cuticle of L. serricorne compared to uninoculated and sanitized grain, and (2) understand how MVOCs from each fungal species affects mobility, attraction, and preference by L. serricorne. Headspace collection revealed that the F. verticillioides- and A. flavus-inoculated grain produced significantly different volatiles compared to sanitized grain or the positive control. Changes in MVOC emissions affected close-range foraging during an Ethovision movement assay, with a greater frequency of entering and spending time in a small zone with kernels inoculated with A. flavus compared to other treatments. In the release-recapture assay, MVOCs were found to be attractive to L. serricorne at longer distances in commercial pitfall traps. There was no preference shown among semiochemical stimuli in a still-air, four-way olfactometer. Overall, our study suggests that MVOCs are important for close- and long-range orientation of L. serricorne during foraging, and that MVOCs may have the potential for inclusion in behaviorally-based tactics for this species.

Background As effects of global climate change intensify, the interaction of biotic and abiotic stresses increasingly threatens current agricultural practices. The secondary cell wall is a vanguard of resistance to these stresses. Fusarium thapsinum (Fusarium stalk rot) and Macrophomina phaseolina (charcoal rot) cause internal damage to the stalks of the drought tolerant C4 grass, sorghum (Sorghum bicolor (L.) Moench), resulting in reduced transpiration, reduced photosynthesis, and increased lodging, severely reducing yields. Drought can magnify these losses. Two null alleles in monolignol biosynthesis of sorghum (brown midrib 6-ref, bmr6-ref; cinnamyl alcohol dehydrogenase, CAD; and bmr12-ref; caffeic acid O-methyltransferase, COMT) were used to investigate the interaction of water limitation with F. thapsinum or M. phaseolina infection. Results The bmr12 plants inoculated with either of these pathogens had increased levels of salicylic acid (SA) and jasmonic acid (JA) across both watering conditions and significantly reduced lesion sizes under water limitation compared to adequate watering, which suggested that drought may prime induction of pathogen resistance. RNA-Seq analysis revealed coexpressed genes associated with pathogen infection. The defense response included phytohormone signal transduction pathways, primary and secondary cell wall biosynthetic genes, and genes encoding components of the spliceosome and proteasome. Conclusion Alterations in the composition of the secondary cell wall affect immunity by influencing phenolic composition and phytohormone signaling, leading to the action of defense pathways. Some of these pathways appear to be activated or enhanced by drought. Secondary metabolite biosynthesis and modification in SA and JA signal transduction may be involved in priming a stronger defense response in water-limited bmr12 plants.

BACKGROUND Prevention is the first line of defense in mitigating losses of post-harvest crops. Long-lasting insecticide treated (LLIN) could be used in food facilities to expose insects to insecticide at different areas within a facility. Prior research has shown that single short exposures reduce movement and longer exposures increase mortality for stored-product insect pests, but we do not know how multiple short duration exposures and biotic and abiotic conditions affect insects exposed to LLIN. Here, we repeatedly exposed red flour beetles, Tribolium castaneum, to LLIN to assess the cumulative effects. We also examined the effects of beetle age and time of day during exposure, and temperature, humidity and food availability during recovery after a single exposure to LLIN. RESULTS We found that four repeated 10-min exposures had similar knockdown effects as a single 30-min exposure. We also found that beetles were more affected when aged 1-6 days versus 14-20 days or were exposed at mid- or late in the day versus earlier in the day. Higher recovery levels were observed with food and at higher relative humidity. In addition, older beetles were more active than younger beetles during exposure, which could reduce time in contact with netting and partially explain why older beetles tended to be less affected. CONCLUSION Some individuals can recover after exposure to LLIN, dependent on exposure duration and environmental factors, but our study shows that sublethal effects likely persist and future work should consider the physiology of T. castaneum before, during, and after exposure to LLIN. Published 2020. This article is a U.S. Government work and is in the public domain in the USA.

Dermatophagoides farinae is inhabited by an intracellular bacterium, Cardinium. Using correlations between host and symbiont gene expression profiles, we identified several important molecular pathways that potentially regulate/facilitate their interactions. The expression of Cardinium genes collectively explained 95% of the variation in the expression of mite genes assigned to pathways for phagocytosis, apoptosis, the MAPK signaling cascade, endocytosis, the tumor necrosis factor (TNF) pathway, the transforming growth factor beta (TGF-β) pathway, lysozyme, and the Toll/Imd pathway. In addition, expression of mite genes explained 76% of the variability in Cardinium gene expression. In particular, the expression of the Cardinium genes encoding the signaling molecules BamD, LepA, SymE, and VirD4 was either positively or negatively correlated with the expression levels of mite genes involved in endocytosis, phagocytosis, and apoptosis. We also found that Cardinium possesses a complete biosynthetic pathway for lipoic acid and may provide lipoate, but not biotin, to mites. Cardinium gene expression collectively explained 84% of the variation in expression related to several core mite metabolic pathways, and, most notably, a negative correlation was observed between bacterial gene expression and expression of mite genes assigned to the glycolysis and citric acid cycle pathways. Furthermore, we showed that Cardinium gene expression is correlated with expression levels of genes associated with terpenoid backbone biosynthesis. This pathway is important for the synthesis of pheromones, thus providing an opportunity for Cardinium to influence mite reproductive behavior to facilitate transmission of the bacterium. Overall, our study provided correlational gene expression data that can be useful for future research on mite-Cardinium interactions. IMPORTANCE The molecular mechanisms of mite-symbiont interactions and their impacts on human health are largely unknown. Astigmatid mites, such as house dust and stored-product mites, are among the most significant allergen sources worldwide. Although mites themselves are the main allergen sources, recent studies have indicated that mite-associated microbiomes may have implications for allergen production and human health. The major medically important house dust mite, D. farinae, is known to harbor a highly abundant intracellular bacterium belonging to the genus Cardinium. Expression analysis of the mite and symbiont genes can identify key mite molecular pathways that facilitate interactions with this endosymbiont and possibly shed light on how this bacterium affects mite allergen production and physiology in general.

HighlightsNIR spectroscopy detects quantitative and qualitative levels of chlorpyrifos-methyl residues in bulk rice.Levels of chlorpyrifos-methyl residues in bulk rice can be differentiated at 78% to 100% correct classification.Important NIR wavelengths for chlorpyrifos-methyl residue detection were identified.NIR spectroscopy can be used to detect maximum residue levels of chlorpyrifos-methyl pesticide in rice.Abstract. A rapid technique that uses near-infrared reflectance (NIR) spectroscopy for simultaneous qualitative and quantitative determination of the presence of varying concentrations of chlorpyrifos-methyl in bulk samples of rough, brown, and milled rice was established. Five rice varieties, free of pesticides, obtained from RiceTec Inc. and USDA-ARS Arkansas experimental field were used as rough rice samples and also processed to obtain corresponding brown and milled rice. Rice samples were treated with StorcideTM II containing varying levels of the active ingredient, chlorpyrifos-methyl: 0, 1.5, 3, 6, 9, and 12 ppm for rough rice, 0, 0.75, 1.5, 3, 4.5, and 6 ppm for brown rice, and 0, 0.1, 0.2, 0.4, 0.6, and 0.8 ppm for milled rice. Concentrations of chlorpyrifos-methyl were verified using gas chromatography-mass spectrometry analyses. A commercial NIR spectrometer (950-1650 nm wavelength range) was used to obtain spectra of bulk samples. Using partial least squares analysis for quantitative analysis, independent validation showed that chlorpyrifos-methyl residues in rough, brown, and milled rice are predictable with R2 ranging from 0.702 to 0.839 and standard error of prediction (SEP) of 1.763 to 2.374 for rough rice, R2 ranging from 0.722 to 0.800 and SEP of 0.953 to 1.168 for brown rice, and R2 ranging from 0.693 to 0.789 and SEP of 0.131 to 0.164 for milled rice. For qualitative analysis obtained using discriminant analysis, rough rice samples with concentrations of 0, 1.5, and 3 ppm pooled as low pesticide level (LPL) is distinguishable to 6, 9, and 12 ppm which were pooled as high pesticide level (HPL). Similarly, for brown and milled rice, the lower three concentrations pooled as LPL is distinguishable from the higher three concentrations pooled as HPL. Independent validation showed overall correct classifications ranging from 77.8% to 92.6% for rough rice, 79.6% to 88.9% for brown rice, and 94.4% to 100% for milled rice. Keywords: Food safety, Grain quality, NIR spectroscopy, Pesticide residue, Rice.

Interspecific competition between agricultural pests may affect the species that can establish, and may also affect food production. Prostephanus truncatus (Horn), the larger grain borer, is endemic to Central America, but invaded Africa with disastrous consequences for maize production. Its main competitor is Sitophilus zeamais Motschulsky, the maize weevil, which is cosmopolitan. These insects co-occur in many regions of the world and both are threats to maize. However, the impact of competition between these two species is not well-understood, nor is its effect on grain quality or potential to limit P. truncatus invasion in new areas. The aims of our study were to evaluate the outcome of interspecific competition between P. truncatus and S. zeamais at four different temperatures on a fixed quantity of grain, and determine effects on progeny production, grain damage, and mold growth. We found that coexistence may be possible at a range of 25–30°C, but mixed colonies experienced a direct competitive cost compared to single-species colonies. Prostephanus truncatus performed better at warmer temperatures, while S. zeamais favored cooler temperatures. The majority of grain damage was the result of P. truncatus activity as opposed to S. zeamais. Finally, mold growth was greater where both species were present, and species of mold that produce aflatoxin were identified. Although there are an increasing number of areas where both of these species occur, our results suggest P. truncatus will be capable of destroying much more maize in a shorter period compared to S. zeamais at temperatures greater than 25°C.

With less emphasis on fumigation after harvest, due to the phase-out of methyl bromide and increasing phosphine resistance, diversified postharvest integrated pest management (IPM) programs are needed. Here, we synthesize knowledge on semiochemical-mediated, behaviorally-based tactics, wherein semiochemicals are deployed to manipulate pest behavior to protect commodities. We note that beyond monitoring, commercial use is limited to mating disruption targeting mostly moths. In total, behaviorally-based tactics have been attempted for eight species of stored product insects from two orders and six families. Eighteen challenges were identified that may have prevented robust implementation of semiochemicals for behaviorally-based management in stored products, including direct competition with ubiquitous food cues, and the diverse insect assemblages that colonize food facilities. Further, we discuss the scientific data and methods required to support stakeholder acceptance of semiochemicals at food facilities, including demonstrating that pests are not attracted from the landscape and minimal spillover around pheromones. We sketch a robust areawide behaviorally-based IPM program after harvest, and clarify properties for improving semiochemicals, including incorporating those that are broad spectrum, competitive with food cues, potent at low concentration, and exhibit dose-dependent attraction. The research gaps and testable hypotheses described here will speed developing behaviorally-based tactics at food facilities. © 2021 Society of Chemical Industry.

Aphid herbivory elicits plant defense-related networks that are influenced by host genetics. Plants of the upland switchgrass (Panicum virgatum) cultivar Summer can be a suitable host for greenbug aphids (Schizaphis graminum; GB), and yellow sugarcane aphids (Sipha flava, YSA), whereas the lowland cultivar Kanlow exhibited multi-species resistance that curtails aphid reproduction. However, stabilized hybrids of Summer (♀) x Kanlow (♂) (SxK) with improved agronomics can be damaged by both aphids. Here, hormone and metabolite analyses, coupled with RNA-Seq analysis of plant transcriptomes, were utilized to delineate defense networks induced by aphid feeding in SxK switchgrass and pinpoint plant transcription factors (TFs), such as WRKYs that potentially regulate these responses. Abscisic acid (ABA) levels were significantly higher in GB infested plants at 5 and 10 days after infestation (DAI). ABA levels were highest at 15DAI in YSA infested plants. Jasmonic acid levels were significantly elevated under GB infestation, while salicylic acid levels were signifi40cantly elevated only at 15 DAI in YSA infested plants. Similarly, levels of several metabolites were altered in common or specifically to each aphid. YSA infestation induced a significant enrichment of flavonoids consistent with an upregulation of many genes associated with flavonoid biosynthesis at 15DAI. Gene co-expression modules that responded singly to either aphid or in common to both aphids were differentiated and linked to specific TFs. Together, these data provide important clues into the interplay of metabolism and transcriptional remodeling accompanying defense responses to aphid herbivory in hybrid switchgrass.

The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a major pest of facilities where grain is processed because of its ability to find and colonize food resource patches. Traps baited with pheromone and kairomone lures are commonly used to monitor for the presence of insects in warehouses or flour mills, for example. However, two nonmutually exclusive components, environment and genetics, could influence insect responsiveness to volatiles, impacting the efficacy of monitoring. Intraspecific variation in attraction behavior to food and mates is largely unexplored in stored-product insects, but tapping into natural genetic variation could provide a baseline for identifying genetic mechanisms associated with finding resources. Here, we assess eight strains of T. castaneum for variation in response to kairomone- and pheromone-based lures using three behavioral assays: paired choice with no forced air flow, upwind attraction with forced air flow, and movement pattern in an arena with a single odor source. We find strain-specific responses to kairomones and pheromones and evidence for heritability in behavioral responses. However, environmental coefficients for behavioral responses to both lures are high, suggesting that environment, and its potential interaction with genotype, strongly influences behavioral outcomes in these assays. Furthermore, despite the different environmental conditions among the different behavioral assays, we find a correlation for volatile preference among the assays. Our results provide a baseline assessment of natural variation for preference to kairomone and pheromone lures and suggest that careful consideration of behavioral assay is key to understanding the mechanisms of attraction in these stored-product pests.

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 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.

Few transcription factors have been identified in C4 grasses that either positively or negatively regulate monolignol biosynthesis. Previously, the overexpression of SbMyb60 in sorghum (Sorghum bicolor) has been shown to induce monolignol biosynthesis, which leads to elevated lignin deposition and altered cell wall composition. To determine how SbMyb60 overexpression impacts other metabolic pathways, RNA-Seq and metabolite profiling were performed on stalks and leaves. 35S::SbMyb60 was associated with the transcriptional activation of genes involved in aromatic amino acid, S-adenosyl methionine (SAM) and folate biosynthetic pathways. The high coexpression values between SbMyb60 and genes assigned to these pathways indicate that SbMyb60 may directly induce their expression. In addition, 35S::SbMyb60 altered the expression of genes involved in nitrogen (N) assimilation and carbon (C) metabolism, which may redirect C and N towards monolignol biosynthesis. Genes linked to UDP-sugar biosynthesis and cellulose synthesis were also induced, which is consistent with the observed increase in cellulose deposition in the internodes of 35S::SbMyb60 plants. However, SbMyb60 showed low coexpression values with these genes and is not likely to be a direct regulator of cell wall polysaccharide biosynthesis. These findings indicate that SbMyb60 can activate pathways beyond monolignol biosynthesis, including those that synthesize the substrates and cofactors required for lignin biosynthesis.

Significance We inferred the phylogeny and evolution of beetles using genomic data of an unprecedented scale. Moreover, we documented the diversification of plant-feeding (herbivorous) beetles, which account for nearly half of all beetle species and a similar proportion of herbivorous insects, following convergent horizontal transfers of bacterial and fungal genes enabling the digestion of lignocellulose in plant cell walls. Our findings clarify beetle phylogenetic relationships and reveal new insights into the evolution of specialized herbivory and why there are so many species of beetles. Furthermore, they underscore the intimacy and complexity of the evolutionary relationships between insects, plants, and microorganisms and show how analyses of large-scale genomic data are revealing the evolution and genomic basis of insect biodiversity., The order Coleoptera (beetles) is arguably the most speciose group of animals, but the evolutionary history of beetles, including the impacts of plant feeding (herbivory) on beetle diversification, remain poorly understood. We inferred the phylogeny of beetles using 4,818 genes for 146 species, estimated timing and rates of beetle diversification using 89 genes for 521 species representing all major lineages and traced the evolution of beetle genes enabling symbiont-independent digestion of lignocellulose using 154 genomes or transcriptomes. Phylogenomic analyses of these uniquely comprehensive datasets resolved previously controversial beetle relationships, dated the origin of Coleoptera to the Carboniferous, and supported the codiversification of beetles and angiosperms. Moreover, plant cell wall-degrading enzymes (PCWDEs) obtained from bacteria and fungi via horizontal gene transfers may have been key to the Mesozoic diversification of herbivorous beetles—remarkably, both major independent origins of specialized herbivory in beetles coincide with the first appearances of an arsenal of PCWDEs encoded in their genomes. Furthermore, corresponding (Jurassic) diversification rate increases suggest that these novel genes triggered adaptive radiations that resulted in nearly half of all living beetle species. We propose that PCWDEs enabled efficient digestion of plant tissues, including lignocellulose in cell walls, facilitating the evolution of uniquely specialized plant-feeding habits, such as leaf mining and stem and wood boring. Beetle diversity thus appears to have resulted from multiple factors, including low extinction rates over a long evolutionary history, codiversification with angiosperms, and adaptive radiations of specialized herbivorous beetles following convergent horizontal transfers of microbial genes encoding PCWDEs.

Extreme temperature has been used as an alternative to chemical treatments for stored product pests for years. Resistance to heat or cold treatments has not been documented in stored product insects, but repeated use of ineffective treatments could lead to adaptive tolerance. Trogoderma variabile (Dermestidae) is a common pest of stored products, and the larval stage is highly resistant to cold and destructive. We artificially selected populations by inducing chill coma at four different cold temperature treatments: 3 and 5 h at −10°C and 3 and 5 h at 0°C. Recovery time was highly heritable after selection for seven generations for decreased recovery time (cold tolerance) and increased recovery time (cold susceptibility) at all time and temperature combinations. Three replicate populations for each time and temperature combination varied substantially, suggesting different mutations in each population were probably responsible for selected phenotypes. Body size decreased in populations selected for cold susceptibility compared with those selected for cold tolerance and survivorship to long-term cold stress increased in the cold-tolerant populations compared with the susceptible populations. After the cessation of the selection experiment, cold tolerance dissipated within four generations from the populations at −10°C, but was maintained in populations exposed to 0°C. Our results suggest that warehouse beetles can adapt to cold stress quickly, but in the absence of cold stress, the proportion of cold-tolerant/susceptible individuals is quickly reduced, suggesting that some of the mutations responsible for these phenotypes may be associated with fitness costs under normal conditions.

The molecular bases of aphid virulence to aphid crop plant resistance genes are poorly understood. The Russian wheat aphid, Diuraphis noxia, (Kurdjumov), and the greenbug, Schizaphis graminum (Rondani), are global pest of cereal crops. Each species damages barley, oat, rye and wheat, but S. graminum includes fescue, maize, rice and sorghum in its host range. This study was conducted to compare and contrast the transcriptomes of S. graminum biotype I and D. noxia biotype 1 when each ingested phloem from leaves of varieties of bread wheat, Triticum aestivum L., containing no aphid resistance (Dn0), resistance to D. noxia biotype 1 (Dn4), or resistance to both D. noxia biotype 1 and S. graminum biotype I (Dn7, wheat genotype 94M370). Gene ontology enrichments, k-means analysis and KEGG pathway analysis indicated that 94M370 plants containing the Dn7 D. noxia resistance gene from rye had stronger effects on the global transcriptional profiles of S. graminum and D. noxia relative to those fed Dn4 plants. S. graminum responds to ingestion of phloem sap from 94M370 plants by expression of unigenes coding for proteins involved in DNA and RNA repair, and delayed tissue and structural development. In contrast, D. noxia displays a completely different transcriptome after ingesting phloem sap from Dn4 or 94M370 plants, consisting of unigenes involved primarily in detoxification, nutrient acquisition and structural development. These variations in transcriptional responses of D. noxia and S. graminum suggest that the underlying evolutionary mechanism(s) of virulence in these aphids are likely species specific, even in cases of cross resistance.

Insects, like all animals, are exposed to diverse environmental microbes throughout their life cycle. Yet, we know little about variation in the microbial communities associated with the majority of wild, unmanaged insect species. Here, we use a 16S rRNA gene metabarcoding approach to characterize temporal and geographic variation in the gut bacterial communities of herbivores (Acalymma vittatum and A. trivittatum) and pollinators (Eucera (Peponapis) pruinosa) that have co-evolved with the plant genus Cucurbita (pumpkin, squash, zucchini and gourds). Overall, we find high variability in the composition of bacterial communities in squash bees and beetles collected from different geographic locations and different time points throughout a growing season. Still, many of the most common OTUs are shared in E. (P.) pruinosa, A. vittatum and A. trivittatum. This suggests these insects may be exposed to similar environmental microbial sources while foraging on the same genus of host plants, and that similar microbial taxa may aid in digestion of Cucurbita plant material. The striped cucumber beetle A. vittatum can also transmit Erwinia tracheiphila, the causal agent of bacterial wilt of cucurbits. We find that few field-collected A. vittatum individuals have detectable E. tracheiphila, and when this plant pathogen is detected, it comprises less than 1% of the gut bacterial community. Together, these results are consistent with previous studies showing that plant feeding insects have highly variable gut bacterial communities, and provides a first step towards understanding the spatiotemporal variation in the microbial communities associated with herbivores and pollinators that depend on Cucurbita host plants.

Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae) is an economically important wheat virus that is transmitted by the wheat curl mite (WCM; Aceria tosichella Keifer) in a persistent manner. Virus–vector coevolution may potentially influence vector gene expression to prolong viral association and thus increase virus transmission efficiency and spread. To understand the transcriptomic responses of WCM to WSMV, RNA sequencing was performed to assemble and analyse transcriptomes of WSMV viruliferous and aviruliferous mites. Among 7291 de novo-assembled unigenes, 1020 were differentially expressed between viruliferous and aviruliferous WCMs using edgeR at a false discovery rate ≤0.05. Differentially expressed unigenes were enriched for 108 gene ontology terms, with the majority of the unigenes showing downregulation in viruliferous mites in comparison to only a few unigenes that were upregulated. Protein family and metabolic pathway enrichment analyses revealed that most downregulated unigenes encoded enzymes and proteins linked to stress response, immunity and development. Mechanistically, these predicted changes in mite physiology induced by viral association could be suggestive of pathways needed for promoting virus–vector interactions. Overall, our data suggest that transcriptional changes in viruliferous mites facilitate prolonged viral association and alter WCM development to expedite population expansion, both of which could enhance viral transmission.

The sugarcane aphid (Melanaphis sacchari) has emerged as a significant pest for sorghum. The use of sugarcane aphid-resistant sorghum germplasm with integrated pest management strategies appears to be an excellent solution to this problem. In this study, a resistant line (RTx2783) and a susceptible line (A/BCK60) were used to characterize the differences in plant responses to the sugarcane aphid through a series of experiments, which examined global sorghum gene expression, aphid feeding behavior and inheritance of aphid resistance. The global transcriptomic responses to sugarcane aphids in resistant and susceptible plants were identified using RNA-seq and compared to the expression profiles of uninfested plants at 5, 10, and 15 days post-infestation. The expression of genes from several functional categories were altered in aphid-infested susceptible plants, which included genes related to cell wall modification, photosynthesis and phytohormone biosynthesis. In the resistant line, only 31 genes were differentially expressed in the infested plants relative to uninfested plants over the same timecourse. However, network analysis of these transcriptomes identified a co-expression module where the expression of multiple sugar and starch associated genes were repressed in infested resistant plants at 5 and 10 days. Several nucleotide-binding-site, leucine-rich repeat (NBS-LRR) and disease resistance genes similar to aphid resistance genes identified in other plants are identified in the current study which may be involved in sugarcane aphid resistance. The electrical penetration graph (EPG) results indicated that sugarcane aphid spent approximately twice as long in non-probing phase, and approximately a quarter of time in phloem ingestion phase on the resistant and F1 plants compared to susceptible plant. Additionally, network analysis identified a phloem protein 2 gene expressed in both susceptible and resistant plants early (day 5) of infestation, which may contribute to defense against aphid feeding within sieve elements. The resistant line RTx2783 displayed both antixenosis and antibiosis modes of resistance based on EPG and choice bioassays between susceptible, resistant and F1 plants. Aphid resistance from RTx2783 segregated as a single dominant locus in the F2 generation, which will enable breeders to rapidly develop sugarcane aphid-resistant hybrids using RTx2783 as the male parent.

Bacteria are essential for stable fly (Stomoxys calcitrans (L.)) larval survival and development, but little is known about the innate microbial communities of stable flies, and it is not known if their varied dietary substrates influence their gut microbial communities. This investigation utilized 454 sequencing of 16S and 18S amplicons to characterize and compare the bacterial and eukaryotic microbial communities in stable fly larvae and their developmental substrates. The microbial community of the third-instar stable fly larvae is unambiguously distinct from the microbial community of the supporting substrate, with bacterial communities from larvae reared on different substrates more similar to each other than to the communities from their individual supporting substrates. Bacterial genera that were more abundant proportionally in larvae compared to their substrates were Erysipelothrix, Dysgonomonas, Ignatzschineria (Gammaproteobacteria), and Campylobacter (Epsilonprotobacteria). The alphaproteobacteria Devosia, Brevundimonas, Sphingopyxix, and Paracoccus were more abundant proportionally in field substrates compared to their larvae. The main genera responsible for differences between the positive and negative field substrates were Dysgonomonas and Proteiniphilum. In contrast to Dysgonomonas, Proteiniphilum was more abundant in substrate than in the larvae. A large number of sequences were assigned to an unclassified protest of the superphylum Alveolata in larvae and their substrate. Microscopy validated these findings and a previously undescribed gregarine (phylum Apicomplexa, class Conoidasida) was identified in stable fly larvae and adults.

Schizaphis graminum (green bug; GB) and Sipha flava (yellow sugarcane aphid; YSA) are two cereal aphid species with broad host ranges capable of establishing on sorghum (Sorghum bicolor) and several switchgrass (Panicum virgatum) cultivars. Switchgrass and sorghum are staple renewable bioenergy crops that are vulnerable to damage by aphids, therefore, identifying novel targets to control aphids has the potential to drastically improve yields and reduce losses in these bioenergy crops. Despite the wealth of genomic and transcriptomic information available from a closely related model aphid species, the pea aphid (Acyrthosiphon pisum), similar genomic information, including the identification of small RNAs, is still limited for GB and YSA. Deep sequencing of miRNAs expressed in GB and YSA was conducted and 72 and 56 miRNA candidates (including 14 and eight novel) were identified, respectively. Of the identified miRNAs, 45 were commonly expressed in both aphid species. Further, plant derived miRNAs were also detected in both aphid samples, including 13 (eight known and five novel) sorghum miRNAs and three (novel) barley miRNAs. In addition, potential aphid gene targets for the host plant-derived miRNAs were predicted. The establishment of miRNA repertoires in these two aphid species and the detection of plant-derived miRNA in aphids will ultimately lead to a better understanding of the role of miRNAs in regulating gene expression networks in these two aphids and the potential roles of plant miRNAs in mediating plant-insect interactions.

Class III peroxidases (CIIIPRX) catalyze the oxidation of monolignols, generate radicals, and ultimately lead to the formation of lignin. In general, CIIIPRX genes encode a large number of isozymes with ranges of in vitro substrate specificities. In order to elucidate the mode of substrate specificity of these enzymes, we characterized one of the CIIIPRXs (PviPRX9) from switchgrass (Panicum virgatum), a strategic plant for second-generation biofuels. The crystal structure, kinetic experiments, molecular docking, as well as expression patterns of PviPRX9 across multiple tissues and treatments, along with its levels of coexpression with the majority of genes in the monolignol biosynthesis pathway, revealed the function of PviPRX9 in lignification. Significantly, our study suggested that PviPRX9 has the ability to oxidize a broad range of phenylpropanoids with rather similar efficiencies, which reflects its role in the fortification of cell walls during normal growth and root development and in response to insect feeding. Based on the observed interactions of phenylpropanoids in the active site and analysis of kinetics, a catalytic mechanism involving two water molecules and residues histidine-42, arginine-38, and serine-71 was proposed. In addition, proline-138 and gluntamine-140 at the 137P-X-P-X140 motif, leucine-66, proline-67, and asparagine-176 may account for the broad substrate specificity of PviPRX9. Taken together, these observations shed new light on the function and catalysis of PviPRX9 and potentially benefit efforts to improve biomass conservation properties in bioenergy and forage crops.

Protectants applied to grain can reduce damage caused by insect feeding during storage. Although these protectants are effective against many external feeders, they may also reduce damage caused by internal feeders as they often interact with the exterior surface of the grain during their larval or adult stages to feed or oviposit. For this study, we investigated impacts of Storicide® II applied to brown rice on three different internal feeders: Rhyzopertha dominica (Fauvel), Sitophilus oryzae (L.), and Sitotroga cerealella (Olivier). We also investigated the effects of this protectant at three different temperatures and when it was combined with different percentages of untreated brown rice. Time-series clustering was also performed to determine whether treatments caused disruptions to the timing of progeny emergence. Overall, R. dominica was the most susceptible as mortality and knockdown were observed in mixtures containing 10% treated brown rice. In contrast, S. cerealella was the least susceptible as mixtures containing at least 50–75% treated brown rice were required to reduce progeny production. However, lowering the temperature to 22 °C did reduce the amount of treated brown rice required to reduce progeny emergence and also reduced the number of progeny that emerged synchronously, which would likely reduce mating and reduce population levels over time. Similar effects on progeny were observed for S. oryzae and R. dominica. Overall, these findings suggest that Storicide II can reduce population levels of internal feeders and that combining this protectant with cooler temperatures can provide additional protection.

A series of tests was conducted to determine if Tribolium castaneum (Herbst), the red flour beetle, could survive on six milled sorghum fractions: Bran, Coarse Grits, Fine Grits, Flour, Red Dogs, and Shorts. In the first test, parental adults were exposed on the fractions, removed, and then the fractions were held for six-seven weeks at 27 °C. Late instar larvae and progeny adults were present in all fractions. In the second test, at least 80% of single neonates (1-2-day-old larvae) held on 1 g of a fraction at 27 °C for seven weeks were able to complete development to the adult stage. In the final test, individual neonates were held on 1 g of a fraction at 37 °C, 32 °C, 27 °C, or 22 °C. Time to adult emergence at each temperature ranged from 17 to 23 days, 21–27 days, 28–50 days, and 67–113 days, respectively, depending on the specific fraction. Logistic functions were compared for mean developmental times for each temperature-fraction combination. The six fractions were also analyzed for ash, fat, fiber, moisture, protein, and starch content. The fractions varied with respect to these chemical constituents; fat and moisture content were negatively correlated with development in some comparisons, though overall there was no correlation between these chemical components and neonate development on the fractions. Temperature had an obvious effect on neonate development, which has implications for assessing risk of pest infestations inside sorghum mills during warmer months of the calendar year. In addition, T. castaneum will reproduce and develop on all of the sorghum fractions included in this study, which are commonly generated during the sorghum milling process. Sanitation and removal of residual materials such as the measured fractions could also help with overall pest management of T. castaneum.

Erwinia tracheiphila is a bacterial plant pathogen emerging in eastern North America. To aid in understanding genetic variation within E. tracheiphila, here we sequence the first reference genome of an infected muskmelon (Cucumis melo)., Erwinia tracheiphila is a bacterial plant pathogen emerging in eastern North America. To aid in understanding genetic variation within E. tracheiphila, here we sequence the first reference genome of an infected muskmelon (Cucumis melo). The genome assembles into a single chromosomal contig, three plasmid contigs, and one bacteriophage contig.

Schizaphis graminum Rondani (Hemiptera: Aphididae) and Sipha flava Forbes (Hemiptera: Aphididae) are two common pests of bioenergy grasses. Despite the fact that they are both considered generalists, they differ in their ability to colonize Panicum virgatum cultivars. For example, S. flava colonizes both P. virgatum cv. Summer and P. virgatum cv. Kanlow whereas S. graminum can only colonize Summer. To study the molecular responses of these aphids to these two switchgrass cultivars, we generated de novo transcriptome assemblies and compared the expression profiles of aphids feeding on both cultivars to profiles associated with feeding on a highly susceptible sorghum host and a starvation treatment. Transcriptome assemblies yielded 8,428 and 8,866 high-quality unigenes for S. graminum and S. flava, respectively. Overall, S. graminum responded strongly to all three treatments after 12 h with an upregulation of unigenes coding for detoxification enzymes while major transcriptional changes were not observed in S. flava until 24 h. Additionally, while the two aphids responded to the switchgrass feeding treatment by downregulating unigenes linked to growth and development, their responses to Summer and Kanlow diverged significantly. Schizaphis graminum upregulated more unigenes coding for stress-responsive enzymes in the Summer treatment compared to S. flava; however, many of these unigenes were actually downregulated in the Kanlow treatment. In contrast, S. flava appeared capable of overcoming host defenses by upregulating a larger number of unigenes coding for detoxification enzymes in the Kanlow treatment. Overall, these findings are consistent with previous studies on the interactions of these two cereal aphids to divergent switchgrass hosts.

Erwinia tracheiphila is a virulent phytopathogen that infects two genera of cucurbit crop plants, Cucurbita spp. (pumpkin and squash) and Cucumis spp. (muskmelon and cucumber). One of the unusual ecological traits of this pathogen is that it is limited to temperate eastern North America. Here, we complete the first large-scale sequencing of an E. tracheiphila isolate collection. From phylogenomic, comparative genomic, and empirical analyses, we find that introduced Cucumis spp. crop plants are driving the diversification of E. tracheiphila into multiple lineages. Together, the results from this study show that locally unique biotic (plant population) and abiotic (climate) conditions can drive the evolutionary trajectories of locally endemic pathogens in unexpected ways., Erwinia tracheiphila is the causal agent of bacterial wilt of cucurbits, an economically important phytopathogen affecting an economically important phytopathogen affecting few cultivated Cucurbitaceae few cultivated Cucurbitaceae host plant species in temperate eastern North America. However, essentially nothing is known about E. tracheiphila population structure or genetic diversity. To address this shortcoming, a representative collection of 88 E. tracheiphila isolates was gathered from throughout its geographic range, and their genomes were sequenced. Phylogenomic analysis revealed three genetic clusters with distinct hrpT3SS virulence gene repertoires, host plant association patterns, and geographic distributions. Low genetic heterogeneity within each cluster suggests a recent population bottleneck followed by population expansion. We showed that in the field and greenhouse, cucumber (Cucumis sativus), which was introduced to North America by early Spanish conquistadors, is the most susceptible host plant species and the only species susceptible to isolates from all three lineages. The establishment of large agricultural populations of highly susceptible C. sativus in temperate eastern North America may have facilitated the original emergence of E. tracheiphila into cucurbit agroecosystems, and this introduced plant species may now be acting as a highly susceptible reservoir host. Our findings have broad implications for agricultural sustainability by drawing attention to how worldwide crop plant movement, agricultural intensification, and locally unique environments may affect the emergence, evolution, and epidemic persistence of virulent microbial pathogens.

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.

The phenylpropanoid biosynthetic pathway that generates lignin subunits represents a significant target for altering the abundance and composition of lignin. The global regulators of phenylpropanoid metabolism may include MYB transcription factors, whose expression levels have been correlated with changes in secondary cell wall composition and the levels of several other aromatic compounds, including anthocyanins and flavonoids. While transcription factors correlated with downregulation of the phenylpropanoid biosynthesis pathway have been identified in several grass species, few transcription factors linked to activation of this pathway have been identified in C4 grasses, some of which are being developed as dedicated bioenergy feedstocks. In this study we investigated the role of SbMyb60 in lignin biosynthesis in sorghum (Sorghum bicolor), which is a drought-tolerant, high-yielding biomass crop. Ectopic expression of this transcription factor in sorghum was associated with higher expression levels of genes involved in monolignol biosynthesis, and led to higher abundances of syringyl lignin, significant compositional changes to the lignin polymer and increased lignin concentration in biomass. Moreover, transgenic plants constitutively overexpressing SbMyb60 also displayed ectopic lignification in leaf midribs and elevated concentrations of soluble phenolic compounds in biomass. Results indicate that overexpression of SbMyb60 is associated with activation of monolignol biosynthesis in sorghum. SbMyb60 represents a target for modification of plant cell wall composition, with the potential to improve biomass for renewable uses.