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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.

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, 2nd instar nymphs, 4th 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.

The bagrada bug, Bagrada hilaris (Burmeister), is an emerging agricultural pest in the Americas, threatening agricultural production in the southwestern United States, Mexico and Chile, as well as in the Old World (including Africa, South Asia and, more recently, Mediterranean areas of Europe). Substantive transcriptomic sequence resources for this damaging species would be beneficial towards understanding its capacity for developing insecticide resistance, identifying viruses that may be present throughout its population and identifying genes differentially expressed across life stages that could be exploited for biomolecular pesticide formulations. This study establishes B . hilaris transcriptomic resources for eggs, 2 nd and 4 th larval instars, as well as male and female adults. Three gene families involved in xenobiotic detoxification—glutathione S-transferases, carboxylesterases and cytochrome P450 monooxygenases—were phylogenetically characterized. These data were also qualitatively compared with previously published results for two closely related pentatomid species—the brown marmorated stink bug, Halyomorpha halys (Stål), and the harlequin bug, Murgantia histrionica (Hahn)—to elucidate shared enzymatic components of terpene-based sex pheromone biosynthetic pathways. Lastly, the sequence data were screened for potential RNAi- and virus-related content and for genes implicated in insect growth and development.

The PRAA4-1 T strain of Chromobacterium subtsugae was the first insecticidal bacterium to be registered by the U.S. Environmental Protection Agency for use in crop protection applications since approval for Bacillus thuringiensis was granted in 1961. C. subtsugae , a Gram-negative betaproteobacterium, exhibits oral toxicity against a broad range of important insects, including dipteran, coleopteran, lepidopteran, and at least some hemipteran and tetranychidan pests. Chromobacterium sphagni is a closely related bacterium exhibiting a distinctly narrower activity spectrum than that of C. subtsugae : it is toxic to lepidopteran, but not dipteran or coleopteran pest insects. The molecular mode of activity for either species is not well characterized at present, and it remains unclear whether these bacterial species affect insects similarly, notwithstanding their close evolutionary relatedness. In this study, synchronized third-instar larvae of the destructive lepidopteran forest pest, Lymantria dispar dispar (European spongy moth), were separately fed with cultures of C. subtsugae strain PRAA4-1 T or C. sphagni strain 14B-1 T and sampled after 24 h post infection. Gene expression levels in healthy reference versus treated insects were independently compared at the whole-insect and midgut-only tissue levels to characterize host-specific transcriptional responses to intoxication. Treatment induced up-regulation of such antimicrobial peptides as attacin and cecropin, of two cytochrome P450-encoding genes, and of gelsolin, a molecule involved in actin organization. Some differentially expressed genes were novel or uncharacterized, hence future work with lepidopteran species will be necessary to understand insect physiological responses to Chromobacterium infection.

The gypsy moth, Lymantria dispar L., is one of the most destructive forest pests in the world. While the subspecies established in North America is the European gypsy moth ( L. dispar dispar ), whose females are flightless, the two Asian subspecies, L. dispar asiatica and L. dispar japonica , have flight-capable females, enhancing their invasiveness and warranting precautionary measures to prevent their permanent establishment in North America. Various molecular tools have been developed to help distinguish European from Asian subspecies, several of which are based on the mitochondrial barcode region. In an effort to identify additional informative markers, we undertook the sequencing and analysis of the mitogenomes of 10 geographic variants of L. dispar , including two or more variants of each subspecies, plus the closely related L. umbrosa as outgroup. Several regions of the gypsy moth mitogenomes displayed nucleotide substitutions with potential usefulness for the identification of subspecies and/or geographic origins. Interestingly, the mitogenome of one geographic variant displayed significant divergence relative to the remaining variants, raising questions about its taxonomic status. Phylogenetic analyses placed this population from northern Iran as basal to the L. dispar clades. The present findings will help improve diagnostic tests aimed at limiting risks of AGM invasions.

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.

The NJAY (New Jersey aster yellows) strain of 'Candidatus Phytoplasma asteris' is a significant plant pathogen responsible for causing severe lettuce yellows in the U.S. state of New Jersey. A draft genome sequence was prepared for this organism. A total of 177,847 reads were assembled into 75 contigs > 518 bp with a total base value of 652,092 and an overall [G+C] content of 27.1%. A total of 733 protein coding genes were identified. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession MAPF00000000. This draft genome was used for genome- and gene-based comparative phylogenetic analyses with other phytoplasmas, including the closely related 'Ca. Phytoplasma asteris' strain, aster yellows witches'- broom (AY-WB). NJAY and AY-WB exhibit approximately 0.5% dissimilarity at the nucleotide level among their shared genomic segments. Evidence indicated that NJAY harbors four plasmids homologous to those known to encode pathogenicity determinants in AY-WB, as well as a chromosome-encoded mobile unit. Apparent NJAY orthologs to the important AY-WB virulence factors, SAP11 and SAP54, were identified. A number of secreted proteins, both membrane-bound and soluble, were encoded, with many bearing similarity to known AY-WB effector molecules and others representing possible secreted proteins that may be novel to the NJAY lineage.

▪ Abstract  During the past decade, research has yielded new knowledge about the plant and insect host ranges, geographical distribution, and phylogenetic relationships of phytoplasmas, and a taxonomic system has emerged in which distinct phytoplasmas are named as separate “Candidatus phytoplasma species.” In large part, this progress has resulted from the development and use of molecular methods to detect, identify, and classify phytoplasmas. While these advances continue, research has recently begun on the phytoplasma genome, how phytoplasmas cause disease, the role of mixed phytoplasmal infections in plant diseases, and molecular/genetic phenomena that underlie symptom development in plants. These and other recent advances are laying the foundation for future progress in understanding the mechanisms of phytoplasma pathogenicity, organization of the phytoplasma genome, evolution of new phytoplasma strains and emergence of new diseases, bases of insect transmissibility and specificity of transmission, and plant gene expression in response to phytoplasmal infection, as well as the design of novel approaches to achieve effective control of phytoplasmal diseases.

The European gypsy moth, Lymantria dispar dispar (LDD), is an invasive insect and a threat to urban trees, forests and forest-related industries in North America. For use as a comparator with a previously published genome based on the LD652 pupal ovary-derived cell line, as well as whole-insect genome sequences obtained from the Asian gypsy moth subspecies L. dispar asiatica and L. dispar japonica, the whole-insect LDD genome was sequenced, assembled and annotated. The resulting assembly was 998 Mb in size, with a contig N50 of 662 Kb and a GC content of 38.8%. Long interspersed nuclear elements constitute 25.4% of the whole-insect genome, and a total of 11,901 genes predicted by automated gene finding encoded proteins exhibiting homology with reference sequences in the NCBI NR and/or UniProtKB databases at the most stringent similarity cutoff level (i.e., the gold tier). These results will be especially useful in developing a better understanding of the biology and population genetics of L. dispar and the genetic features underlying Lepidoptera in general.

RNA interference (RNAi) technology uses dsRNAs to silence specific targeted genes by downregulating their expression. It has become a potent tool for functional and regulatory studies of insect genes and has potential to be applied for insect control. Though it has been challenging to generate effective RNAi in lepidopteran insects, in the current study this technology was applied to develop specific RNAi-based molecular tools that could be used to negatively impact the invasive lepidopteran forest pest, gypsy moth (GM). GM midgut-specific genes were selected for dsRNA design from larval transcriptome profiles. Two methods were used to produce specific dsRNAs, bacterial expression and in vitro synthesis, which were then fed per os to GM larvae. Depletion of uncharacterized gene targets known as locus 365 and locus 28365, or their stacked combination, depleted target transcripts in a sequence specific manner and resulted in 60% reduction in body mass. Treated GM females that were able to moult to the adult stage displayed an approximately two-fold reduction in egg masses. These have potential to be developed as molecular biopesticides for GM.