Phenylpropanoids Following Wounding and Infection of Sweet Sorghum Lines Differing in Responses to Stalk Pathogens.

Sweet sorghum (Sorghum bicolor) lines M81-E and Colman were previously shown to differ in responses to Fusarium thapsinum and Macrophomina phaseolina, stalk rot pathogens that can reduce the yields and quality of biomass and extracted sugars. Inoculated tissues were compared for transcriptomic, phenolic metabolite, and enzymatic activity during disease development 3 and 13 days after inoculation (DAI). At 13 DAI, M81-E had shorter mean lesion lengths than Colman when inoculated with either pathogen. Transcripts encoding monolignol biosynthetic and modification enzymes were associated with transcriptional wound (control) responses of both lines at 3 DAI. Monolignol biosynthetic genes were differentially coexpressed with transcriptional activator SbMyb76 in all Colman inoculations, but only following M. phaseolina inoculation in M81-E, suggesting that SbMyb76 is associated with lignin biosynthesis during pathogen responses. In control inoculations, defense-related genes were expressed at higher levels in M81-E than Colman. Line, treatment, and timepoint differences observed in phenolic metabolite and enzyme activities did not account for observed differences in lesions. However, generalized additive models were able to relate metabolites, but not enzyme activities, to lesion length for quantitatively modeling disease progression: in M81-E, but not Colman, sinapic acid levels positively predicted lesion length at 3 DAI when cell wall-bound syringic acid was low, soluble caffeic acid was high, and lactic acid was high, suggesting that sinapic acid may contribute to responses at 3 DAI. These results provide potential gene targets for development of sweet sorghum varieties with increased stalk rot resistance to ensure biomass and sugar quality.

The evolution and genomic basis of beetle diversity.

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.

Pathogen and drought stress affect cell wall and phytohormone signaling to shape host responses in a sorghum COMT bmr12 mutant.

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.

Wheat streak mosaic virus alters the transcriptome of its vector, wheat curl mite (Aceria tosichella Keifer), to enhance mite development and population expansion.

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.

Bacterial and Fungal Midgut Community Dynamics and Transfer Between Mother and Brood in the Asian Longhorned Beetle (Anoplophora glabripennis), an Invasive Xylophage.

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.

Seasonal below-ground metabolism in switchgrass.

Switchgrass (Panicum virgatum), a perennial, polyploid, C4 warm-season grass is among the foremost herbaceous species being advanced as a source of biomass for biofuel end uses. At the end of every growing season, the aerial tissues senesce, and the below-ground rhizomes become dormant. Future growth is dependent on the successful over-wintering of the rhizomes. Although the importance of rhizome health to overall year-upon-year plant productivity has been long recognized, there is limited information on seasonal changes occurring during dormancy at both the transcriptome and metabolite levels. Here, global changes in transcriptomes and metabolites were investigated over two growing seasons in rhizomes harvested from field-grown plants. The objectives were: (a) synthesize information on cellular processes that lead to dormancy; and (b) provide models that could account for major metabolic pathways present in dormant switchgrass rhizomes. Overall, metabolism during dormancy appeared to involve discrete but interrelated events. One was a response to abscisic acid that resulted in dehydration, increases in osmolytes and upregulation of autophagic processes, likely through the target of rapamycin complex and sucrose non-fermentative-related kinase-based signaling cascades. Another was a recalibration of energy transduction through apparent reductions in mitochondrial oxidative phosphorylation, increases in substrate level generation of ATP and reducing equivalents, and recycling of N and possibly CO2 through refixation. Lastly, transcript abundances indicated that cold-related signaling was also occurring. Altogether, these data provide a detailed overview of rhizome metabolism, especially during dormancy, which can be exploited in the future to improve winter survival in switchgrass.

Overexpression of SbMyb60 in Sorghum bicolor impacts both primary and secondary metabolism.

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.

Characterization of Class III Peroxidases from Switchgrass.

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.

Overexpression of SbMyb60 impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in Sorghum bicolor.

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.

Transcriptional analysis of defense mechanisms in upland tetraploid switchgrass to greenbugs.

BACKGROUND: Aphid infestation of switchgrass (Panicum virgatum) has the potential to reduce yields and biomass quality. Although switchgrass-greenbug (Schizaphis graminum; GB) interactions have been studied at the whole plant level, little information is available on plant defense responses at the molecular level. RESULTS: The global transcriptomic response of switchgrass cv Summer to GB was monitored by RNA-Seq in infested and control (uninfested) plants harvested at 5, 10, and 15 days after infestation (DAI). Differentially expressed genes (DEGs) in infested plants were analyzed relative to control uninfested plants at each time point. DEGs in GB-infested plants induced by 5-DAI included an upregulation of reactive burst oxidases and several cell wall receptors. Expression changes in genes linked to redox metabolism, cell wall structure, and hormone biosynthesis were also observed by 5-DAI. At 10-DAI, network analysis indicated a massive upregulation of defense-associated genes, including NAC, WRKY, and MYB classes of transcription factors and potential ancillary signaling molecules such as leucine aminopeptidases. Molecular evidence for loss of chloroplastic functions was also detected at this time point. Supporting these molecular changes, chlorophyll content was significantly decreased, and ROS levels were elevated in infested plants 10-DAI. Total peroxidase and laccase activities were elevated in infested plants at 10-DAI relative to control uninfested plants. The net result appeared to be a broad scale defensive response that led to an apparent reduction in C and N assimilation and a potential redirection of nutrients away from GB and towards the production of defensive compounds, such as pipecolic acid, chlorogenic acid, and trehalose by 10-DAI. By 15-DAI, evidence of recovery in primary metabolism was noted based on transcript abundances for genes associated with carbon, nitrogen, and nutrient assimilation. CONCLUSIONS: Extensive remodeling of the plant transcriptome and the production of ROS and several defensive metabolites in an upland switchgrass cultivar were observed in response to GB feeding. The early loss and apparent recovery in primary metabolism by 15-DAI would suggest that these transcriptional changes in later stages of GB infestation could underlie the recovery response categorized for this switchgrass cultivar. These results can be exploited to develop switchgrass lines with more durable resistance to GB and potentially other aphids.

Differences in Fusarium Species in brown midrib Sorghum and in Air Populations in Production Fields.

Several Fusarium spp. cause sorghum (Sorghum bicolor) grain mold, resulting in deterioration and mycotoxin production in the field and during storage. Fungal isolates from the air (2005 to 2006) and from leaves and grain from wild-type and brown midrib (bmr)-6 and bmr12 plants (2002 to 2003) were collected from two locations. Compared with the wild type, bmr plants have reduced lignin content, altered cell wall composition, and different levels of phenolic intermediates. Multilocus maximum-likelihood analysis identified two Fusarium thapsinum operational taxonomic units (OTU). One was identified at greater frequency in grain and leaves of bmr and wild-type plants but was infrequently detected in air. Nine F. graminearum OTU were identified: one was detected at low levels in grain and leaves while the rest were only detected in air. Wright's F statistic (FST) indicated that Fusarium air populations differentiated between locations during crop anthesis but did not differ during vegetative growth, grain development, and maturity. FST also indicated that Fusarium populations from wild-type grain were differentiated from those in bmr6 or bmr12 grain at one location but, at the second location, populations from wild-type and bmr6 grain were more similar. Thus, impairing monolignol biosynthesis substantially effected Fusarium populations but environment had a strong influence.

Comprehensive annotation of Glossina pallidipes salivary gland hypertrophy virus from Ethiopian tsetse flies: a proteogenomics approach.

Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) can establish asymptomatic and symptomatic infection in its tsetse fly host. Here, we present a comprehensive annotation of the genome of an Ethiopian GpSGHV isolate (GpSGHV-Eth) compared with the reference Ugandan GpSGHV isolate (GpSGHV-Uga; GenBank accession number EF568108). GpSGHV-Eth has higher salivary gland hypertrophy syndrome prevalence than GpSGHV-Uga. We show that the GpSGHV-Eth genome has 190 291 nt, a low G+C content (27.9 %) and encodes 174 putative ORFs. Using proteogenomic and transcriptome mapping, 141 and 86 ORFs were mapped by transcripts and peptides, respectively. Furthermore, of the 174 ORFs, 132 had putative transcriptional signals [TATA-like box and poly(A) signals]. Sixty ORFs had both TATA-like box promoter and poly(A) signals, and mapped by both transcripts and peptides, implying that these ORFs encode functional proteins. Of the 60 ORFs, 10 ORFs are homologues to baculovirus and nudivirus core genes, including three per os infectivity factors and four RNA polymerase subunits (LEF4, 5, 8 and 9). Whereas GpSGHV-Eth and GpSGHV-Uga are 98.1 % similar at the nucleotide level, 37 ORFs in the GpSGHV-Eth genome had nucleotide insertions (n = 17) and deletions (n = 20) compared with their homologues in GpSGHV-Uga. Furthermore, compared with the GpSGHV-Uga genome, 11 and 24 GpSGHV ORFs were deleted and novel, respectively. Further, 13 GpSGHV-Eth ORFs were non-canonical; they had either CTG or TTG start codons instead of ATG. Taken together, these data suggest that GpSGHV-Eth and GpSGHV-Uga represent two different lineages of the same virus. Genetic differences combined with host and environmental factors possibly explain the differential GpSGHV pathogenesis observed in different G. pallidipes colonies.

Herbivore exploits orally secreted bacteria to suppress plant defenses.

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

Characterization of novel Brown midrib 6 mutations affecting lignin biosynthesis in sorghum.

The presence of lignin reduces the quality of lignocellulosic biomass for forage materials and feedstock for biofuels. In C4 grasses, the brown midrib phenotype has been linked to mutations to genes in the monolignol biosynthesis pathway. For example, the Bmr6 gene in sorghum (Sorghum bicolor) has been previously shown to encode cinnamyl alcohol dehydrogenase (CAD), which catalyzes the final step of the monolignol biosynthesis pathway. Mutations in this gene have been shown to reduce the abundance of lignin, enhance digestibility, and improve saccharification efficiencies and ethanol yields. Nine sorghum lines harboring five different bmr6 alleles were identified in an EMS-mutagenized TILLING population. DNA sequencing of Bmr6 revealed that the majority of the mutations impacted evolutionarily conserved amino acids while three-dimensional structural modeling predicted that all of these alleles interfered with the enzyme's ability to bind with its NADPH cofactor. All of the new alleles reduced in vitro CAD activity levels and enhanced glucose yields following saccharification. Further, many of these lines were associated with higher reductions in acid detergent lignin compared to lines harboring the previously characterized bmr6-ref allele. These bmr6 lines represent new breeding tools for manipulating biomass composition to enhance forage and feedstock quality.

The WRKY transcription factor family and senescence in switchgrass.

BACKGROUND: Early aerial senescence in switchgrass (Panicum virgatum) can significantly limit biomass yields. WRKY transcription factors that can regulate senescence could be used to reprogram senescence and enhance biomass yields. METHODS: All potential WRKY genes present in the version 1.0 of the switchgrass genome were identified and curated using manual and bioinformatic methods. Expression profiles of WRKY genes in switchgrass flag leaf RNA-Seq datasets were analyzed using clustering and network analyses tools to identify both WRKY and WRKY-associated gene co-expression networks during leaf development and senescence onset. RESULTS: We identified 240 switchgrass WRKY genes including members of the RW5 and RW6 families of resistance proteins. Weighted gene co-expression network analysis of the flag leaf transcriptomes across development readily separated clusters of co-expressed genes into thirteen modules. A visualization highlighted separation of modules associated with the early and senescence-onset phases of flag leaf growth. The senescence-associated module contained 3000 genes including 23 WRKYs. Putative promoter regions of senescence-associated WRKY genes contained several cis-element-like sequences suggestive of responsiveness to both senescence and stress signaling pathways. A phylogenetic comparison of senescence-associated WRKY genes from switchgrass flag leaf with senescence-associated WRKY genes from other plants revealed notable hotspots in Group I, IIb, and IIe of the phylogenetic tree. CONCLUSIONS: We have identified and named 240 WRKY genes in the switchgrass genome. Twenty three of these genes show elevated mRNA levels during the onset of flag leaf senescence. Eleven of the WRKY genes were found in hotspots of related senescence-associated genes from multiple species and thus represent promising targets for future switchgrass genetic improvement. Overall, individual WRKY gene expression profiles could be readily linked to developmental stages of flag leaves.

Functional genomics and microbiome profiling of the Asian longhorned beetle (Anoplophora glabripennis) reveal insights into the digestive physiology and nutritional ecology of wood feeding beetles.

BACKGROUND: Wood-feeding beetles harbor an ecologically rich and taxonomically diverse assemblage of gut microbes that appear to promote survival in woody tissue, which is devoid of nitrogen and essential nutrients. Nevertheless, the contributions of these apparent symbionts to digestive physiology and nutritional ecology remain uncharacterized in most beetle lineages. RESULTS: Through parallel transcriptome profiling of beetle- and microbial- derived mRNAs, we demonstrate that the midgut microbiome of the Asian longhorned beetle (Anoplophora glabripennis), a member of the beetle family Cerambycidae, is enriched in biosynthetic pathways for the synthesis of essential amino acids, vitamins, and sterols. Consequently, the midgut microbiome of A. glabripennis can provide essential nutrients that the beetle cannot obtain from its woody diet or synthesize itself. The beetle gut microbiota also produce their own suite of transcripts that can enhance lignin degradation, degrade hemicellulose, and ferment xylose and wood sugars. An abundance of cellulases from several glycoside hydrolase families are expressed endogenously by A. glabripennis, as well as transcripts that allow the beetle to convert microbe-synthesized essential amino acids into non-essential amino acids. A. glabripennis and its gut microbes likely collaborate to digest carbohydrates and convert released sugars and amino acid intermediates into essential nutrients otherwise lacking from their woody host plants. CONCLUSIONS: The nutritional provisioning capabilities of the A. glabripennis gut microbiome may contribute to the beetles' unusually broad host range. The presence of some of the same microbes in the guts of other Cerambycidae and other wood-feeding beetles suggests that partnerships with microbes may be a facilitator of evolutionary radiations in beetles, as in certain other groups of insects, allowing access to novel food sources through enhanced nutritional provisioning.

Microbial vectoring capacity by internal- and external-infesting stored product insects after varying dispersal periods between novel food patches: An underestimated risk.

Understanding the ability of internal- and external-infesting stored product insects to vector microbes is important for estimating the relative risk that insects pose to postharvest commodities as they move between habitat patches and in the landscape. Thus, the aim of the current study was to evaluate and compare the microbial growth in novel food patches at different dispersal periods by different populations of Sitophilus oryzae (e.g., internal-infesting) and Lasioderma serricorne (e.g., external-infesting). Adults of both species collected from laboratory colonies or field-captured populations were either placed immediately in a novel food patch, or given a dispersal period of 24 or 72 h in a sterilized environment before entering a surrogate food patch. Vectored microbes in new food patches were imaged after 3 or 5 days of foraging, and microbial growth was processed using ImageJ while fungal species were identified through sequencing the ITS4/5 ribosomal subunit. We found that increasing dispersal time resulted in multiple-fold reductions in microbial growth surrogate food patches by L. serricorne but not S. oryzae. This was likely attributable to higher mobility by S. oryzae than L. serricorne. A total of 20 morphospecies were identified from 13 genera among the 59 sequences, with a total of 23% and 16% classified as Aspergillus and Penicillium spp. Our data suggest that there is a persistent risk of microbial contamination by both species, which has important food safety implications at food facilities.

Flight capacity and behavior of Ephestia kuehniella (Lepidoptera: Pyralidae) in response to kairomonal and pheromonal stimuli.

Flight behavior is an important component to understand in the context of pest management. However, because of their small size, little is known about the flight capacity of most stored-product insects, and when a flight has been assessed, it usually consists of a propensity for initiating flight. Despite a priori expectations of the importance of flight for moths, there are no data about the flight capacity and little on the flight behavior of the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). As a result, the objective of the current study was to (i) characterize the baseline flight capacity of E. kuehniella and (ii) determine how flight capacity is affected by the presence of kairomonal, pheromonal, or no stimuli. We found adult E. kuehniella flew a mean of 24-34 km in a 24-h period, and the distance flown per bout increased from 91 to 207 m in the presence of pheromones but decreased to 41 m when food was nearby compared to a negative control. The total number of flight bouts was 1.6-fold higher in the presence of pheromone compared to the negative control, but E. kuehniella flew significantly slower with pheromone and food cues present, suggesting they may be exhibiting an optimal foraging strategy. Our data on flight capacity results in qualitatively and quantitatively different conclusions about flight than those conclusions formed if only flight initiation is considered. Overall, this novel information is useful for understanding the spread within facilities and in the landscape (between facilities), as well as parameterizing ecological modeling.

Midgut transcriptome profiling of Anoplophora glabripennis, a lignocellulose degrading cerambycid beetle.

BACKGROUND: Wood-feeding insects often work in collaboration with microbial symbionts to degrade lignin biopolymers and release glucose and other fermentable sugars from recalcitrant plant cell wall carbohydrates, including cellulose and hemicellulose. Here, we present the midgut transcriptome of larval Anoplophora glabripennis, a wood-boring beetle with documented lignin-, cellulose-, and hemicellulose- degrading capabilities, which provides valuable insights into how this insect overcomes challenges associated with feeding in woody tissue. RESULTS: Transcripts from putative protein coding regions of over 9,000 insect-derived genes were identified in the A. glabripennis midgut transcriptome using a combination of 454 shotgun and Illumina paired-end reads. The most highly-expressed genes predicted to encode digestive-related enzymes were trypsins, carboxylesterases, ß-glucosidases, and cytochrome P450s. Furthermore, 180 unigenes predicted to encode glycoside hydrolases (GHs) were identified and included several GH 5, 45, and 48 cellulases, GH 1 xylanases, and GH 1 ß-glucosidases. In addition, transcripts predicted to encode enzymes involved in detoxification were detected, including a substantial number of unigenes classified as cytochrome P450s (CYP6B) and carboxylesterases, which are hypothesized to play pivotal roles in detoxifying host tree defensive chemicals and could make important contributions to A. glabripennis' expansive host range. While a large diversity of insect-derived transcripts predicted to encode digestive and detoxification enzymes were detected, few transcripts predicted to encode enzymes required for lignin degradation or synthesis of essential nutrients were identified, suggesting that collaboration with microbial enzymes may be required for survival in woody tissue. CONCLUSIONS: A. glabripennis produces a number of enzymes with putative roles in cell wall digestion, detoxification, and nutrient extraction, which likely contribute to its ability to thrive in a broad range of host trees. This system is quite different from the previously characterized termite fermentation system and provides new opportunities to discover enzymes that could be exploited for cellulosic ethanol biofuel production or the development of novel methods to control wood-boring pests.

Development and Immature Mortality of the Sawtoothed Grain Beetle (Coleoptera: Silvanidae), on Different Sorghum Fractions and Different Temperatures.

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.