Background: Polydnaviruses, double-stranded DNA viruses with segmented genomes, have evolved as obligate endosymbionts of parasitoid wasps. Virus particles are replication deficient and produced by female wasps from proviral sequences integrated into the wasp genome. These particles are co-injected with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and, thereby, survival of proviral DNA. Here we characterize and compare the encapsidated viral genome sequences of bracoviruses in the family Polydnaviridae associated with Glyptapanteles gypsy moth parasitoids, along with near complete proviral sequences from which both viral genomes are derived. Results: The encapsidated Glyptapanteles indiensis and Glyptapanteles flavicoxis bracoviral genomes, each composed of 29 different size segments, total approximately 517 and 594 kbp, respectively. They are generated from a minimum of seven distinct loci in the wasp genome. Annotation of these sequences revealed numerous novel features for polydnaviruses, including insect-like sugar transporter genes and transposable elements. Evolutionary analyses suggest that positive selection is widespread among bracoviral genes. Conclusions: The structure and organization of G. indiensis and G. flavicoxis bracovirus proviral segments as multiple loci containing one to many viral segments, flanked and separated by wasp gene-encoding DNA, is confirmed. Rapid evolution of bracovirus genes supports the hypothesis of bracovirus genes in an 'arms race' between bracovirus and caterpillar. Phylogenetic analyses of the bracoviral genes encoding sugar transporters provides the first robust evidence of a wasp origin for some polydnavirus genes. We hypothesize transposable elements, such as those described here, could facilitate transfer of genes between proviral segments and host DNA.

Double-stranded RNA (dsRNA)-mediated gene silencing, also known as RNA interference (RNAi), has been a breakthrough technology for functional genomic studies and represents a potential tool for the management of insect pests. Since the inception of RNAi numerous studies documented successful introduction of exogenously synthesized dsRNA or siRNA into an organism triggering highly efficient gene silencing through the degradation of endogenous RNA homologous to the presented siRNA. Managing hemipteran insect pests, especially Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug (BMSB), is critical to food productivity. BMSB was recently introduced into North America where it is both an invasive agricultural pest of high value specialty, row, and staple crops, as well as an indoor nuisance pest. RNAi technology may serve as a viable tool to manage this voracious pest, but delivery of dsRNA to piercing-sucking insects has posed a tremendous challenge. Effective and practical use of RNAi as molecular biopesticides for biocontrol of insects like BMSB in the environment requires that dsRNAs be delivered in vivo through ingestion. Therefore, the key challenge for molecular biologists in developing insect-specific molecular biopesticides is to find effective and reliable methods for practical delivery of stable dsRNAs such as through oral ingestion. Here demonstrated is a reliable delivery system of effective insect-specific dsRNAs through oral feeding through a new delivery system to induce a significant decrease in expression of targeted genes such as JHAMT and Vg. This state-of-the-art delivery method overcomes environmental delivery challenges so that RNAi is induced through insect-specific dsRNAs orally delivered to hemipteran and other insect pests.

Transcriptomic profiles of the serious lepidopteran insect pest Lymantria dispar (gypsy moth) were characterized in the larval midgut in response to infection by Bacillus thuringiensis kurstaki, a biopesticide commonly used for its control. RNA-Seq approaches were used to define a set of 49,613 assembled transcript sequences, of which 838, 1,248 and 3,305 were respectively partitioned into high-, mid- and low-quality tiers on the basis of homology information. Digital gene expression profiles suggested genes differentially expressed at 24 hours post infection, and qRT-PCR analyses were performed for verification. The differentially expressed genes primarily associated with digestive function, including α-amylase, lipase and carboxypeptidase; immune response, including C-type lectin 4; developmental genes such as arylphorin; as well as a variety of binding proteins: cellular retinoic acid binding protein (lipid-binding), insulin-related peptide binding protein (protein-binding) and ovary C/EBPg transcription factor (nucleic acid-binding). This is the first study conducted to specifically investigate gypsy moth response to a bacterial infection challenge using large-scale sequencing technologies, and the results highlight important genes that could be involved in biopesticide resistance development or could serve as targets for biologically-based control mechanisms of this insect pest.

Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug, is an invasive agricultural and nuisance pest rapidly expanding its incidence in North America. This voracious pest poses a significant threat to rural and urban agriculture, especially to specialty crops such as apples, grapes and ornamentals, as well as staple crops including soybean and corn. The object of this study was to generate transcript sequence resources for H. halys. RNA-seq libraries derived from distinct developmental stages and sexes were sequenced and assembled into 248,569 putatively unique transcripts (PUTs). PUTs were segmented into three disjoint tiers of varying reliability, with 4,794 classified as gold tier (highest quality), 16,878 as silver, and 14,357 as bronze. The gold-tier PUTs associated with 2,580 distinct non-redundant protein sequences from the NCBI NR database—1,785 of these (69%) mapped to annotated UniProtKB database proteins, from which 1,273 unique Pfam families and 459 unique Molecular Function GO terms were encountered. Of the silver tier's 6,527 PUTs associated with unique proteins, 4,193 mapped to UniProtKB (64%), from which 1,941 and 640 unique Pfam and Molecular Function GO terms were extracted. H. halys PUTs related to important life processes like immunity, endocrinology, reproduction, development, behavior, neurotransmission, neurotoxicity, olfaction, and small RNA pathways were validated through quantitative Real-Time PCR (qRT-PCR) for differential expression during distinct life stages (eggs, 2^nd instar nymphs, 4^th instar nymphs, female adults, male adults). PUTs similar to hypothetical proteins identified in symbiont microbes, including Pantoea and Nosema species, were more abundantly expressed in adults versus nymphs. These comprehensive H. halys transcriptomic resources can be utilized to aid development of novel control methodologies to disrupt life processes; to conduct reverse genetic screens to determine host gene function; and to design environmentally unobtrusive means to control host populations or target specific H. halys life stages, such as molecular biopesticides.

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

One of the more unusual groups of insect pathogens consists of members of the family Polydnaviridae, insect DNA viruses that live in mutual symbioses with their associated parasitoid wasp (Hymentoptera) carriers until they are injected into specific lepidopteran hosts. Once inside this secondary host, polydnaviruses cause a wide variety of negative effects that ultimately ensure the survival of the parasitoid larvae. Because of their unusual life strategy and genetic features, it had been difficult to fully characterise polydnaviruses in terms of evolutionary history, replication cycle and functions in the host that might normally be well characterised for more conventional viruses. Recently, our understanding of polydnavirus evolutionary origins, gene content, genome organisation and functions in parasitism has greatly increased. Key findings are summarised in this review with emphasis on evolution of polydnavirus genes and genomes, their functional roles in insect pathology and their potential applications in insect biological control and biotechnology.