A draft Diabrotica virgifera virgifera genome: insights into control and host plant adaption by a major maize pest insect.

BACKGROUND: Adaptations by arthropod pests to host plant defenses of crops determine their impacts on agricultural production. The larval host range of western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is restricted to maize and a few grasses. Resistance of D. v. virgifera to crop rotation practices and multiple insecticides contributes to its status as the most damaging pest of cultivated maize in North America and Europe. The extent to which adaptations by this pest contributes to host plant specialization remains unknown. RESULTS: A 2.42 Gb draft D. v. virgifera genome, Dvir_v2.0, was assembled from short shotgun reads and scaffolded using long-insert mate-pair, transcriptome and linked read data. K-mer analysis predicted a repeat content of ≥ 61.5%. Ortholog assignments for Dvir_2.0 RefSeq models predict a greater number of species-specific gene duplications, including expansions in ATP binding cassette transporter and chemosensory gene families, than in other Coleoptera. A majority of annotated D. v. virgifera cytochrome P450s belong to CYP4, 6, and 9 clades. A total of 5,404 transcripts were differentially-expressed between D. v. virgifera larvae fed maize roots compared to alternative host (Miscanthus), a marginal host (Panicum virgatum), a poor host (Sorghum bicolor) and starvation treatments; Among differentially-expressed transcripts, 1,908 were shared across treatments and the least number were between Miscanthus compared to maize. Differentially-expressed transcripts were enriched for putative spliceosome, proteosome, and intracellular transport functions. General stress pathway functions were unique and enriched among up-regulated transcripts in marginal host, poor host, and starvation responses compared to responses on primary (maize) and alternate hosts. CONCLUSIONS: Manual annotation of D. v. virgifera Dvir_2.0 RefSeq models predicted expansion of paralogs with gene families putatively involved in insecticide resistance and chemosensory perception. Our study also suggests that adaptations of D. v. virgifera larvae to feeding on an alternate host plant invoke fewer transcriptional changes compared to marginal or poor hosts. The shared up-regulation of stress response pathways between marginal host and poor host, and starvation treatments may reflect nutrient deprivation. This study provides insight into transcriptomic responses of larval feeding on different host plants and resources for genomic research on this economically significant pest of maize.

Up-regulation of apoptotic- and cell survival-related gene pathways following exposures of western corn rootworm to B. thuringiensis crystalline pesticidal proteins in transgenic maize roots.

BACKGROUND: Resistance of pest insect species to insecticides, including B. thuringiensis (Bt) pesticidal proteins expressed by transgenic plants, is a threat to global food security. Despite the western corn rootworm, Diabrotica virgifera virgifera, being a major pest of maize and having populations showing increasing levels of resistance to hybrids expressing Bt pesticidal proteins, the cell mechanisms leading to mortality are not fully understood. RESULTS: Twenty unique RNA-seq libraries from the Bt susceptible D. v. virgifera inbred line Ped12, representing all growth stages and a range of different adult and larval exposures, were assembled into a reference transcriptome. Ten-day exposures of Ped12 larvae to transgenic Bt Cry3Bb1 and Gpp34/Tpp35Ab1 maize roots showed significant differential expression of 1055 and 1374 transcripts, respectively, compared to cohorts on non-Bt maize. Among these, 696 were differentially expressed in both Cry3Bb1 and Gpp34/Tpp35Ab1 maize exposures. Differentially-expressed transcripts encoded protein domains putatively involved in detoxification, metabolism, binding, and transport, were, in part, shared among transcripts that changed significantly following exposures to the entomopathogens Heterorhabditis bacteriophora and Metarhizium anisopliae. Differentially expressed transcripts in common between Bt and entomopathogen treatments encode proteins in general stress response pathways, including putative Bt binding receptors from the ATP binding cassette transporter superfamily. Putative caspases, pro- and anti-apoptotic factors, as well as endoplasmic reticulum (ER) stress-response factors were identified among transcripts uniquely up-regulated following exposure to either Bt protein. CONCLUSIONS: Our study suggests that the up-regulation of genes involved in ER stress management and apoptotic progression may be important in determining cell fate following exposure of susceptible D. v. virgifera larvae to Bt maize roots. This study provides novel insights into insect response to Bt intoxication, and a possible framework for future investigations of resistance mechanisms.

Complex Fistula Formations After Orbital Fracture Repair With Teflon: A Review of 3 Case Reports.

ABSTRACT: The authors present 3 unique cases of complex fistula formations because of orbital fracture repair with a Teflon (polytetrafluroethylene) implant. A 26-year-old man presented with dacryocystitis and a cutaneous fistula 8 years after left orbital floor and medial wall fracture repair with a Teflon implant. A 46-year-old woman suffered orbital trauma after a motor vehicle accident as a teenager and the fracture was repaired with Teflon implant. Thirty-two years later, she presented with lower eyelid fistula, ectropion, and retraction. A 65-year-old woman also previously had Teflon implants for the repair of her left inferior and lateral orbital rim after a motor vehicle accident. Twenty-five years later, she presented with chronic infections involving the repaired areas, as well as left lower lid ectropion and fistula formation. The woven material nature of Teflon acted as a nidus for infection, inflammation, and led to complex cutaneous fistula formations in these patients.

Enhanced metabolism and selection of pyrethroid-resistant western corn rootworms (Diabrotica virgifera virgifera LeConte).

Western corn rootworm (WCR) pyrethroid resistance has been previously reported in the United States (US) western Corn Belt, and cross-resistance and synergism studies suggested that both target site insensitivity and enhanced metabolism may be conferring WCR resistance to pyrethroids. The present study aimed to investigate the potential mechanisms of WCR pyrethroid resistance and to estimate the heritability of the resistance trait. Biochemical assays using model substrates and spectrophotometry revealed 2-4-fold higher activity of P450s and esterases in pyrethroid-resistant WCR populations, whereas the biological activity of glutathione S-transferase was similar between populations tested. No mutation in the voltage-gated sodium channel was detected in pyrethroid-resistant WCR individuals by sequencing PCR products containing the para-homologous L1014, T929, and M918 amino acid positions that are commonly associated with target site mutations in other pyrethroid-resistant insects. A pilot estimation of pyrethroid resistance heritability obtained during laboratory selection of a WCR population suggested a major genetic component of the resistance trait and predicted a 10-fold increase in WCR bifenthrin resistance within ~7 generations of insecticide lethal exposure. Results support earlier indirect evidence that enhanced metabolism may be contributing to WCR resistance to pyrethroids and illustrates the potential of WCR pyrethroid resistance evolution.

The Glutamine-Alanine Repeat Domain of TCERG1 is Required for the Inhibition of the Growth Arrest Activity of C/EBPα.

TCERG1 was characterized previously as a repressor of the transcription factor C/EBPα through a mechanism that involved relocalization of TCERG1 from nuclear speckles to pericentromeric regions. The inhibitory activity as well as the relocalization activity has been demonstrated to lie in the amino terminal half of the protein, which contains several discrete motifs including an imperfect glutamine-alanine (QA) repeat. In the present study, we showed that deletion of this domain completely abrogated the ability of TCERG1 to inhibit the growth arrest activity of C/EBPα. Moreover, the QA repeat deletion mutant of TCERG1 lost the ability to be relocalized from nuclear speckles to pericentromeric regions, and caused an increase in the average size of individual speckles. We also showed that deletion of the QA repeat abrogated the complex formation between TCERG1 and C/EBPα. Examination of mutants with varying numbers of QA repeats indicated that a minimal number of repeats are required for inhibitory activity as well as relocalization ability. These data contribute to our overall understanding of how TCERG1 can have gene-specific effects in addition to its more general roles in coordinating transcription elongation and splicing.

Transcriptional analysis of susceptible and resistant European corn borer strains and their response to Cry1F protoxin.

BACKGROUND: Despite a number of recent reports of insect resistance to transgenic crops expressing insecticidal toxins from Bacillus thuringiensis (Bt), little is known about the mechanism of resistance to these toxins. The purpose of this study is to identify genes associated with the mechanism of Cry1F toxin resistance in European corn borer (Ostrinia nubilalis Hübner). For this, we compared the global transcriptomic response of laboratory selected resistant and susceptible O. nubilalis strain to Cry1F toxin. We further identified constitutive transcriptional differences between the two strains. RESULTS: An O. nubilalis midgut transcriptome of 36,125 transcripts was assembled de novo from 106 million Illumina HiSeq and Roche 454 reads and used as a reference for estimation of differential gene expression analysis. Evaluation of gene expression profiles of midgut tissues from the Cry1F susceptible and resistant strains after toxin exposure identified a suite of genes that responded to the toxin in the susceptible strain (n = 1,654), but almost 20-fold fewer in the resistant strain (n = 84). A total of 5,455 midgut transcripts showed significant constitutive expression differences between Cry1F susceptible and resistant strains. Transcripts coding for previously identified Cry toxin receptors, cadherin and alkaline phosphatase and proteases were also differentially expressed in the midgut of the susceptible and resistant strains. CONCLUSIONS: Our current study provides a valuable resource for further molecular characterization of Bt resistance and insect response to Cry1F toxin in O. nubilalis and other pest species.

A unified open-source platform for multimodal neural recording and perturbation during naturalistic behavior.

Behavioral neuroscience faces two conflicting demands: long-duration recordings from large neural populations and unimpeded animal behavior. To meet this challenge, we developed ONIX, an open-source data acquisition system with high data throughput (2GB/sec) and low closed-loop latencies (<1ms) that uses a novel 0.3 mm thin tether to minimize behavioral impact. Head position and rotation are tracked in 3D and used to drive active commutation without torque measurements. ONIX can acquire from combinations of passive electrodes, Neuropixels probes, head-mounted microscopes, cameras, 3D-trackers, and other data sources. We used ONIX to perform uninterrupted, long (~7 hours) neural recordings in mice as they traversed complex 3-dimensional terrain. ONIX allowed exploration with similar mobility as non-implanted animals, in contrast to conventional tethered systems which restricted movement. By combining long recordings with full mobility, our technology will enable new progress on questions that require high-quality neural recordings during ethologically grounded behaviors.

Camera phasing in multi-aperture coherent imaging.

The resolution of a diffraction-limited imaging system is inversely proportional to the aperture size. Instead of using a single large aperture, multiple small apertures are used to synthesize a large aperture. Such a multi-aperture system is modular, typically more reliable and less costly. On the other hand, a multi-aperture system requires phasing sub-apertures to within a fraction of a wavelength. So far in the literature, only the piston, tip, and tilt type of inter-aperture errors have been addressed. In this paper, we present an approach to correct for rotational and translational errors as well.

Distribution of genes and repetitive elements in the Diabrotica virgifera virgifera genome estimated using BAC sequencing.

Feeding damage caused by the western corn rootworm, Diabrotica virgifera virgifera, is destructive to corn plants in North America and Europe where control remains challenging due to evolution of resistance to chemical and transgenic toxins. A BAC library, DvvBAC1, containing 109,486 clones with 104 ± 34.5 kb inserts was created, which has an ~4.56X genome coverage based upon a 2.58 Gb (2.80 pg) flow cytometry-estimated haploid genome size. Paired end sequencing of 1037 BAC inserts produced 1.17 Mb of data (~0.05% genome coverage) and indicated ~9.4 and 16.0% of reads encode, respectively, endogenous genes and transposable elements (TEs). Sequencing genes within BAC full inserts demonstrated that TE densities are high within intergenic and intron regions and contribute to the increased gene size. Comparison of homologous genome regions cloned within different BAC clones indicated that TE movement may cause haplotype variation within the inbred strain. The data presented here indicate that the D. virgifera virgifera genome is large in size and contains a high proportion of repetitive sequence. These BAC sequencing methods that are applicable for characterization of genomes prior to sequencing may likely be valuable resources for genome annotation as well as scaffolding.

Genome size evolution in the beetle genus Diabrotica.

Diabrocite corn rootworms are one of the most economically significant pests of maize in the United States and Europe and an emerging model for insect-plant interactions. Genome sizes of several species in the genus Diabrotica were estimated using flow cytometry along with that of Acalymma vittatum as an outgroup. Genome sizes ranged between 1.56 and 1.64 gigabase pairs and between 2.26 and 2.59 Gb, respectively, for the Diabrotica subgroups fucata and virgifera; the Acalymma vittatum genome size was around 1.65 Gb. This result indicated that a substantial increase in genome size occurred in the ancestor of the virgifera group. Further analysis of the fucata group and the virgifera group genome sequencing reads indicated that the genome size difference between the Diabrotica subgroups could be attributed to a higher content of transposable elements, mostly miniature inverted-transposable elements and gypsy-like long terminal repeat retroelements.

Increased survival of western corn rootworm on transgenic corn within three generations of on-plant greenhouse selection.

To delay evolution of insect resistance to transgenic crops producing Bacillus thuringiensis (Bt) toxins, nearby "refuges" of host plants not producing Bt toxins are required in many regions. Such refuges are expected to be most effective in slowing resistance when the toxin concentration in Bt crops is high enough to kill all or nearly all insects heterozygous for resistance. However, Bt corn, Zea mays, introduced recently does not meet this "high-dose" criterion for control of western corn rootworm (WCR), Diabrotica virgifera virgifera. A greenhouse method of rearing WCR on transgenic corn expressing the Cry3Bb1 protein was used in which approximately 25% of previously unexposed larvae survived relative to isoline survival (compared to 1-4% in the field). After three generations of full larval rearing on Bt corn (Constant-exposure colony), WCR larval survival was equivalent on Bt corn and isoline corn in greenhouse trials, and the LC(50) was 22-fold greater for the Constant-exposure colony than for the Control colony in diet bioassays with Cry3Bb1 protein on artificial diet. After six generations of greenhouse selection, the ratio of larval recovery on Bt corn to isoline corn in the field was 11.7-fold greater for the Constant-exposure colony than the Control colony. Removal from selection for six generations did not decrease survival on Bt corn in the greenhouse. The results suggest that rapid response to selection is possible in the absence of mating with unexposed beetles, emphasizing the importance of effective refuges for resistance management.

Molecular characterization of western corn rootworm pyrethroid resistance.

BACKGROUND: Western corn rootworm (WCR) pyrethroid resistance has been confirmed in the western US Corn Belt. Toxicological and biochemical studies indicated that multiple mechanisms of resistance might be involved in the resistance trait, such as enhanced metabolism and/or kdr target-site mutation(s) in the voltage-gated sodium channels. To characterize the mechanisms of WCR pyrethroid resistance at the molecular level, pairwise comparisons were made between RNA-Seq data collected from pyrethroid-resistant and -susceptible WCR populations. Gene expression levels and sodium channel sequences were evaluated. RESULTS: Seven transcripts exhibited significantly different expression (q ≤ 0.05) when comparing field-collected pyrethroid-resistant (R-Field) and -susceptible (S-Field) WCR populations. Three of the differentially expressed transcripts were P450s overexpressed in R-Field (9.2-26.2-fold). A higher number (99) of differentially expressed transcripts was found when comparing laboratory-derived pyrethroid-resistant (R-Lab) and -susceptible (S-Lab) WCR populations. Eight of the significant transcripts were P450s overexpressed in R-Lab (2.7-39.8-fold). This study did not detect kdr mutations in pyrethroid-resistant WCR populations. Other differentially expressed transcripts that may play a role in WCR pyrethroid resistance are discussed. CONCLUSION: This study revealed that P450-mediated metabolism is likely to be a major mechanism of WCR pyrethroid resistance, which could affect the efficacy of other insecticides sharing similar metabolic pathways. Additionally, results suggested that although laboratory selection of a pyrethroid-resistant WCR population may help to characterize resistance mechanisms, a field-selected population provided rare and perhaps major variants corresponding to the resistance trait.

Transcriptional responses of soybean aphids to sublethal insecticide exposure.

Insecticides are a key tool in the management of many insect pests of agriculture, including soybean aphids. The selection imposed by insecticide use has often lead to the evolution of resistance by the target pest through enhanced detoxification mechanisms. We hypothesised that exposure of insecticide-susceptible aphids to sublethal doses of insecticides would result in the up-regulation of genes involved in detoxification of insecticides, revealing the genes upon which selection might act in the field. We used the soybean aphid biotype 1 reference genome, version 6.0 as a reference to analyze RNA-Seq data. We identified multiple genes with potential detoxification roles that were up-regulated 12 h after sublethal exposure to esfenvalerate or thiamethoxam. However, these genes were part of a dramatic burst of differential gene expression in which thousands of genes were up- or down-regulated, rather than a defined response to insecticides. Interestingly, the transcriptional burst observed at 12 h s declined dramatically by 24-hrs post-exposure, suggesting a general stress response that may become fine-tuned over time.

Comparative performance of single nucleotide polymorphism and microsatellite markers for population genetic analysis.

Microsatellite loci are standard genetic markers for population genetic analysis, whereas single nucleotide polymorphisms (SNPs) are more recent tools that require assessment of neutrality and appropriate use in population genetics. Twelve SNP markers were used to describe the genetic structure of Diabrotica virgifera virgifera (LeConte; Coleoptera: Chrysomelidae) in the United States of America and revealed a high mean observed heterozygosity (0.40 +/- 0.059) and low global F(ST) (0.029). Pairwise F(ST) estimates ranged from 0.007 to 0.045, and all but 2 populations showed significant levels of genetic differentiation (P < or = 0.008). Population parameters and conclusions based on SNP markers were analogous to that obtained by use of microsatellite markers from the identical population samples. SNP-based F(ST) estimates were 3-fold higher than corresponding estimates from microsatellites, wherein lower microsatellite F(ST) estimates likely resulted from an overestimate of migration rates between subpopulations due to convergence of allele size (homoplasy). No significant difference was observed in the proportion of SNP or microsatellite markers loci that were nonneutral within populations. SNP markers provided estimates of population genetic parameters consistent with those from microsatellite data, and their low back mutation rates may result in reduced propensity for error in estimation of population parameters.

Mitochondrial DNA variation and range expansion in western bean cutworm (Lepidoptera: Noctuidae): no evidence for a recent population bottleneck.

The western bean cutworm, Striacosta albicosta (Smith) (Lepidoptera: Noctuidae), is a pest of both corn and dry bean crops. At the beginning of the 21st century, the species began to extend its range out of the Great Plains, eastward through the Corn Belt. This rapid range expansion is remarkable because the species distribution had been stable for at least the previous half century, despite the apparent abundance of suitable habitat (i.e., cornfields) immediately to the east. We hypothesized that if the western bean cutworm had to overcome a stable barrier to movement before starting the current range expansion, it probably experienced a genetic bottleneck in doing so. To test this hypothesis, variation in the mitochondrial NADH dehydrogenase one (ND1) gene was studied in populations from Wyoming, Nebraska, and Iowa. No differences in overall genetic diversity or haplotype frequencies indicative of a bottleneck were observed between the recently founded populations in Iowa and the established populations in Wyoming and Nebraska. This result suggests that the sudden loss of an ecological exclusion mechanism, allowing the species to move east in appreciable numbers, is more likely to have triggered the range expansion than the surmounting of an extrinsic barrier to movement. The nature of this mechanism is unknown but might be related to recent changes in corn farming practices and technology.