Bryony C. Bonning

On the classification and nomenclature of baculoviruses: A proposal for revision

Summary.Recent evidence from genome sequence analyses demands a substantial revision of the taxonomy and classification of the family Baculoviridae. Comparisons of 29 baculovirus genomes indicated that baculovirus phylogeny followed the classification of the hosts more closely than morphological traits that have previously been used for classification of this virus family. On this basis, dipteran- and hymenopteran-specific nucleopolyhedroviruses (NPV) should be separated from lepidopteran-specific NPVs and accommodated into different genera. We propose a new classification and nomenclature for the genera within the baculovirus family. According to this proposal the updated classification should include four genera: Alphabaculovirus (lepidopteran-specific NPV), Betabaculovirus (lepidopteran-specific Granuloviruses), Gammabaculovirus (hymenopteran-specific NPV) and Deltabaculovirus (dipteran-specific NPV).

Toxins for Transgenic Resistance to Hemipteran Pests

The sap sucking insects (Hemiptera), which include aphids, whiteflies, plant bugs and stink bugs, have emerged as major agricultural pests. The Hemiptera cause direct damage by feeding on crops, and in some cases indirect damage by transmission of plant viruses. Current management relies almost exclusively on application of classical chemical insecticides. While the development of transgenic crops expressing toxins derived from the bacterium Bacillus thuringiensis (Bt) has provided effective plant protection against some insect pests, Bt toxins exhibit little toxicity against sap sucking insects. Indeed, the pest status of some Hemiptera on Bt-transgenic plants has increased in the absence of pesticide application. The increased pest status of numerous hemipteran species, combined with increased prevalence of resistance to chemical insecticides, provides impetus for the development of biologically based, alternative management strategies. Here, we provide an overview of approaches toward transgenic resistance to hemipteran pests.

Next Generation Sequencing Technologies for Insect Virus Discovery

Insects are commonly infected with multiple viruses including those that cause sublethal, asymptomatic, and latent infections. Traditional methods for virus isolation typically lack the sensitivity required for detection of such viruses that are present at low abundance. In this respect, next generation sequencing technologies have revolutionized methods for the discovery and identification of new viruses from insects. Here we review both traditional and modern methods for virus discovery, and outline analysis of transcriptome and small RNA data for identification of viral sequences. We will introduce methods for de novo assembly of viral sequences, identification of potential viral sequences from BLAST data, and bioinformatics for generating full-length or near full-length viral genome sequences. We will also discuss implications of the ubiquity of viruses in insects and in insect cell lines. All of the methods described in this article can also apply to the discovery of viruses in other organisms.

Autographa californica multiple nucleopolyhedrovirus ODV-E56 is a per os infectivity factor, but is not essential for binding and fusion of occlusion-derived virus to the host midgut

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion-derived virus (ODV) envelope protein ODV-E56 is essential for oral infection of larvae of Heliothis virescens. Bioassays with recombinant clones of AcMNPV lacking a functional odv-e56 gene showed that ODV-E56 was required for infectivity of both polyhedra and to a lesser extent, purified ODV. However, binding and fusion assays showed that ODV lacking ODV-E56 bound and fused to midgut cells at levels similar to ODV of wild-type virus. Fluorescence microscopy of midguts from larvae inoculated with ODV-E56-positive and -negative viruses that express GFP indicated that ODV-E56 was required for infection of the midgut epithelium. Purified ODV-E56 bound to several proteins in midgut-derived brush border membrane vesicles, but failed to rescue infectivity of ODV-E56-negative viruses in trans. These results indicate that ODV-E56 is a per os infectivity factor (pif-5) required for primary midgut infection at a point before or after virion binding and fusion.

Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests

Although transgenic crops expressing Bacillus thuringiensis (Bt) toxins have been used successfully for management of lepidopteran and coleopteran pest species, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. To overcome this limitation, we demonstrate that addition of a short peptide sequence selected for binding to the gut of the targeted pest species serves to increase toxicity against said pest. Insertion of a 12-aa pea aphid gut-binding peptide by adding to or replacing amino acids in one of three loops of the Bt cytolytic toxin, Cyt2Aa, resulted in enhanced binding and toxicity against both the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. This strategy may allow for transgenic plant-mediated suppression of other hemipteran pests, which include some of the most important pests of global agriculture.

Interaction of the Bacillus thuringiensis delta endotoxins Cry1Ac and Cry3Aa with the gut of the pea aphid, Acyrthosiphon pisum (Harris)

Hemipteran pests including aphids are not particularly susceptible to the effects of insecticidal Cry toxins derived from the bacterium Bacillus thuringiensis. We examined the physiological basis for the relatively low toxicity of Cry1Ac and Cry3Aa against the pea aphid, Acyrthosiphon pisum (Harris). Cry1Ac was efficiently hydrolyzed by aphid stomach membrane associated cysteine proteases (CP) producing a 60kDa mature toxin, whereas Cry3Aa was incompletely processed and partially degraded. Cry1Ac bound to the aphid gut epithelium but showed low aphid toxicity in bioassays. Feeding of aphids on Cry1Ac in the presence or absence of GalNAc, suggested that Cry1Ac gut binding was glycan mediated. In vitro binding of biotinylated-Cry1Ac to gut BBMVs and competition assays using unlabeled Cry1Ac and GalNAc confirmed binding specificity as well as glycan mediation of Cry1Ac binding. Although Cry3Aa binding to the aphid gut membrane was not detected, Cry3Aa bound 25 and 37kDa proteins in aphid gut BBMV in ligand blot analysis and competition assays confirmed the binding specificity of Cry3Aa. This, combined with low toxicity in feeding assays, suggests that Cry3Aa does bind the gut epithelium to some extent. This is the first systematic examination of the physiological basis for the low efficacy of Cry toxins against aphids, and analysis of Cry toxin-aphid gut interaction.

Proteases as Insecticidal Agents

Proteases from a variety of sources (viruses, bacteria, fungi, plants, and insects) have toxicity towards insects. Some of these insecticidal proteases evolved as venom components, herbivore resistance factors, or microbial pathogenicity factors, while other proteases play roles in insect development or digestion, but exert an insecticidal effect when over-expressed from genetically engineered plants or microbial pathogens. Many of these proteases are cysteine proteases, although insect-toxic metalloproteases and serine proteases have also been examined. The sites of protease toxic activity range from the insect midgut to the hemocoel (body cavity) to the cuticle. This review discusses these insecticidal proteases along with their evaluation and use as potential pesticides.

A novel protein that binds juvenile hormone esterase in fat body tissue and pericardial cells of the tobacco hornworm Manduca sexta L.

Juvenile hormone esterase degrades juvenile hormone, which acts in conjunction with ecdysteroids to control gene expression in insects. Circulating juvenile hormone esterase is removed from insect blood by pericardial cells and degraded in lysosomes. In experiments designed to characterize proteins involved in the degradation of juvenile hormone esterase, a pericardial cell cDNA phage display library derived from the tobacco hornworm mothManduca sexta L. was constructed and screened for proteins that bind juvenile hormone esterase. A 732-base pair cDNA encoding a novel 29-kDa protein (P29) was isolated. Western and Northern analyses indicated that P29 is present in both pericardial cell and fat body tissues and is expressed in each larval instar. In immunoprecipitation experiments, P29 bound injected recombinant juvenile hormone esterase taken up by pericardial cells and nativeM. sexta juvenile hormone esterase in fat body tissue, where the enzyme is synthesized. Binding assays showed that P29 bound juvenile hormone esterase more strongly than it did a mutant form of the enzyme with mutations that perturb lysosomal targeting. Based on these data, we propose that P29 functions in pericardial cells to facilitate lysosomal degradation of juvenile hormone esterase.

The nucleopolyhedroviruses of Rachiplusia ou and Anagrapha falcifera are isolates of the same virus.

The 7.8 kb EcoRI-G fragment of Rachiplusia ou multicapsid nucleopolyhedrovirus (RoMNPV), containing the polyhedrin gene, was cloned and sequenced. The sequence of the fragment was 92.3% identical to the sequence of the corresponding region in the Autographa californica (Ac)MNPV genome. A comparison of the EcoRI-G sequence with other MNPV sequences revealed that RoMNPV was most closely related to AcMNPV. However, the predicted amino acid sequence of RoMNPV polyhedrin shared more sequence identity with the polyhedrin of Orygia pseudotsugata MNPV. In addition, the RoMNPV sequence was almost completely identical (99.9%) to a previously published 6.3 kb sequence of Anagrapha falcifera MNPV (AfMNPV). The EcoRI and HindIII restriction fragment profiles of RoMNPV and AfMNPV also were nearly identical, with an additional EcoRI band detected in RoMNPV DNA. Bioassays of these viruses with three different hosts (the European corn borer, Ostrinia nubilalis Hubner, the corn earworm, Helicoverpa zea Boddie, and the tobacco budworm, Heliothis virescens Fabricius) failed to detect any differences in the biological activities of RoMNPV and AfMNPV. These results indicate that RoMNPV and AfMNPV are different isolates of the same virus. The taxonomic relationship of Ro/AfMNPV and AcMNPV is discussed.

Deep Sequencing of the Transcriptomes of Soybean Aphid and Associated Endosymbionts

Background The soybean aphid has significantly impacted soybean production in the U.S. Transcriptomic analyses were conducted for further insight into leads for potential novel management strategies. Methodology/Principal Findings Transcriptomic data were generated from whole aphids and from 2,000 aphid guts using an Illumina GAII sequencer. The sequence data were assembled de novo using the Velvet assembler. In addition to providing a general overview, we demonstrate (i) the use of the Multiple-k/Multiple-C method for de novo assembly of short read sequences, followed by BLAST annotation of contigs for increased transcript identification: From 400,000 contigs analyzed, 16,257 non-redundant BLAST hits were identified; (ii) analysis of species distributions of top non-redundant hits: 80% of BLAST hits (minimum e-value of 1.0-E3) were to the pea aphid or other aphid species, representing about half of the pea aphid genes; (iii) comparison of relative depth of sequence coverage to relative transcript abundance for genes with high (membrane alanyl aminopeptidase N) or low transcript abundance; (iv) analysis of the Buchnera transcriptome: Transcripts from 57.6% of the genes from Buchnera aphidicola were identified; (v) identification of Arsenophonus and Wolbachia as potential secondary endosymbionts; (vi) alignment of full length sequences from RNA-seq data for the putative salivary gland protein C002, the silencing of which has potential for aphid management, and the putative Bacillus thuringiensis Cry toxin receptors, aminopeptidase N and alkaline phosphatase. Conclusions/Significance This study provides the most comprehensive data set to date for soybean aphid gene expression: This work also illustrates the utility of short-read transcriptome sequencing and the Multiple-k/Multiple-C method followed by BLAST annotation for rapid identification of target genes for organisms for which reference genome sequences are not available, and extends the utility to include the transcriptomes of endosymbionts.