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In most holometabolous insects, sex differentiation occurs via a hierarchical cascade of transcription factors, with doublesex (dsx) regulating genes that control sex-specific traits. Although less is known in hemimetabolous insects, early evidence suggests that substantial differences exist from more evolutionarily advanced insects. Here, we identified and characterized dsx in Lygus hesperus (western tarnished plant bug), a hemipteran pest of many agricultural crops in western North America. The full-length transcript for L. hesperus dsx (Lhdsx) and several variants encode proteins with conserved DNA binding and oligomerization domains. Transcript profiling revealed that Lhdsx is ubiquitously expressed, likely undergoes alternative pre-mRNA splicing, and, unlike several model insects, is sex-biased rather than sex-specific. Embryonic RNA interference (RNAi) of Lhdsx only impacted sex development in adult males, which lacked both internal reproductive organs and external genitalia. No discernible impacts on adult female development or reproductivity were observed. RNAi knockdown of Lhdsx in nymphs likewise only affected adult males, which lacked the characteristic dimorphic coloration but had dramatically elevated vitellogenin transcripts. Gene knockout of Lhdsx by CRISPR/Cas9 editing yielded only females in G 0 and strongly biased heterozygous G 1 offspring to females with the few surviving males showing severely impaired genital development. These results indicate that L. hesperus male development requires Lhdsx , whereas female development proceeds via a basal pathway that functions independently of dsx. A fundamental understanding of sex differentiation in L. hesperus could be important for future gene-based management strategies of this important agricultural pest. [Display omitted] • Lygus hesperus has multiple doublesex transcripts that exhibit sex-biased expression. • RNAi knockdown of Lygus hesperus doublesex impedes male sexual development but has no effect on female development. • Following CRISPR/Cas9-mediated doublesex knockout, the sex ratio of the resulting progeny is heavily biased towards females. [ABSTRACT FROM AUTHOR]

[Display omitted] • Lygus hesperus has 7 β-tubulin genes. • β-tubulin2 is predominantly expressed in the testes. • Knockdown or knockout of β-tubulin2 reduces both spermatozoa abundance and quality, which limits fertilization and the subsequent egg hatch. Lygus hesperus Knight is an important insect pest of crops across western North America, with field management heavily reliant on the use of chemical insecticides. Because of the evolution of resistance to these insecticides, effective and environmentally benign pest management strategies are needed. Traditional sterile insect technique (SIT) has been successfully employed to manage or eradicate some insect pests but involves introducing irradiated insects with random mutations into field populations. New genetically-driven SIT techniques are a safer alternative, causing fixed mutations that manipulate individual genes in target pests to produce sterile individuals for release. Here, we identified seven β-tubulin coding genes from L. hesperus and show that Lhβtub2 is critical in male sperm production and fertility. Lhβtub2 is expressed primarily in the male testes and targeting of this gene by RNA interference or gene editing leads to male sterility. [ABSTRACT FROM AUTHOR]

Background: The grain aphid (Sitobion avenae F.) is a major agricultural pest which causes significant yield losses of wheat in China, Europe and North America annually. Transcriptome profiling of the grain aphid alimentary canal after feeding on wheat plants could provide comprehensive gene expression information involved in feeding, ingestion and digestion. Furthermore, selection of aphid-specific RNAi target genes would be essential for utilizing a plant-mediated RNAi strategy to control aphids via a non-toxic mode of action. However, due to the tiny size of the alimentary canal and lack of genomic information on grain aphid as a whole, selection of the RNAi targets is a challenging task that as far as we are aware, has never been documented previously. Results: In this study, we performed de novo transcriptome assembly and gene expression analyses of the alimentary canals of grain aphids before and after feeding on wheat plants using Illumina RNA sequencing. The transcriptome profiling generated 30,427 unigenes with an average length of 664 bp. Furthermore, comparison of the transcriptomes of alimentary canals of pre- and post feeding grain aphids indicated that 5490 unigenes were differentially expressed, among which, diverse genes and/or pathways were identified and annotated. Based on the RPKM values of these unigenes, 16 of them that were significantly up or down-regulated upon feeding were selected for dsRNA artificial feeding assay. Of these, 5 unigenes led to higher mortality and developmental stunting in an artificial feeding assay due to the down-regulation of the target gene expression. Finally, by adding fluorescently labelled dsRNA into the artificial diet, the spread of fluorescence signal in the whole body tissues of grain aphid was observed. Conclusions: Comparison of the transcriptome profiles of the alimentary canals of pre- and post-feeding grain aphids on wheat plants provided comprehensive gene expression information that could facilitate our understanding of the molecular mechanisms underlying feeding, ingestion and digestion. Furthermore, five novel and effective potential RNAi target genes were identified in grain aphid for the first time. This finding would provide a fundamental basis for aphid control in wheat through plant mediated RNAi strategy. [ABSTRACT FROM AUTHOR]

Abstract: The fungal pathogen, Ophiostomo novo-ulmi, has been responsible for the rapid decline of American elm (Ulmus americana) across North America and remains a serious threat to surviving elm populations. The production of pectinolytic polygalacturonase enzymes has been implicated as a virulence factor for many fungal pathogens, including O. novo-ulmi. Previous work has shown that the targeted disruption of the endopolygalacturonase gene locus epg1 of O. novo-ulmi reduced, but did not eliminate pectinase activity. In the present study, we evaluated the use of RNA interference (RNAi) as a method of suppressing expression of the epg1 locus in O. novo-ulmi and compared its efficiency to the gene disruption method. While there was a reduction in epg1-specific mRNA transcripts and in the amount of polygalacturonase enzyme secreted for both methods of gene regulation, neither method completely suppressed the expression of pectinase activity. There was, however, a significantly greater reduction in both transcript levels and secreted enzyme observed for some of the RNAi transformants. As the first demonstration of RNAi in O. novo-ulmi, this method of gene regulation shows promise in future studies of gene expression and pathogenicity. [Copyright &y& Elsevier]

• RNA interference (RNAi) in MPB can be activated with exogenous dsRNAs. • When silenced, essential MPB genes cause beetle mortality. • Oral ingestion and dermal absorption of dsRNAs creates management potential. • The hsp gene is expressed at higher levels in females, regardless of dsRNA exposure. • Differential gene expression is a factor when considering RNAi for pest management. The mountain pine beetle (MPB), Dendroctonus ponderosae , is an eruptive endemic forest pest that is undergoing substantial range expansion in response to recent climatic changes, breaching geographic barriers, exploiting novel hosts, and affecting millions of hectares of conifer forests in western North America. Current management approaches have been unable to keep pace with MPB population outbreaks, and novel and aggressive management responses are required as MPB's range expansion progresses. Gene silencing through RNA interference (RNAi) is an emerging pest management approach that is being developed for agricultural pests, and has also been shown to be effective against some xylophagous forest pests, including the southern pine beetle (SPB), D. frontalis. When essential genes are targeted, RNAi can cause rapid insect mortality; here we focus on evaluating its effectiveness in MPB. We identified reference genes for quantitative real-time PCR (qPCR) and validated RNAi responses in MPB by analyzing gene expression and beetle survival. Using an adult bioassay that combined oral ingestion and dermal absorption of dsRNAs targeting three genes (hsp, iap, and shi), we measure gene expression and demonstrate silencing, as well as insect mortality, following dsRNA exposure. All three genes were silenced and all treatment beetles died within 7 d. This validates reference genes for expression analyses and demonstrates that MPB, similar to the congeneric SPB, has a highly sensitive RNAi response. Additionally, we document sex-specific differences in gene expression for one of the three target genes, hsp ; any differences in gene expression and subsequent mortality based on sex must be considered as this technology progresses as a pest management tool. RNAi causes rapid insect mortality when essential genes are targeted, is highly specific to the target pest, and has no environmental contamination risks, making it an attractive approach for further development in forest pest suppression. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is a naturally occurring process inhibiting gene expression, and recent advances in our understanding of the mechanism have allowed its development as a tool against insect pests. A major challenge for deployment in the field is the development of convenient and efficient methods for production of double stranded RNA (dsRNA). We assessed the potential for deploying bacterially produced dsRNA as a bio-pesticide against an invasive forest pest, the emerald ash borer (EAB). EAB feeds on the cambial tissue of ash trees (Fraxinus spp.), causing rapid death. EAB has killed millions of trees in North America since its discovery in 2002, prompting the need for innovative management strategies. In our study, bacterial expression and synthesis of dsRNA were performed with E. coli strain HT115 using the L4440 expression vector. EAB-specific dsRNAs (shi and hsp) over-expressed in E. coli were toxic to neonate EAB after oral administration, successfully triggering gene silencing and subsequent mortality; however, a non-specific dsRNA control was not included. Our results suggest that ingestion of transformed E. coli expressing dsRNAs can induce an RNAi response in EAB. To our knowledge, this is the first example of an effective RNAi response induced by feeding dsRNA-expressing bacteria in a forest pest. [ABSTRACT FROM AUTHOR]

• L. hesperus increase movement velocity and duration if exposed to noxious odors. • The L. hesperus transcriptome contains sequences for Orco and TRPA1. • RNAi knockdown of TRPA1, but not Orco, reduces the noxious odor response. Lygus hesperus is a key pest of many economically important crops across western North America. Central to many aspects of the lives of these insects is chemical signalling, with identified roles in host plant selection, aggregation and passive mate guarding. The development of novel monitoring and control approaches for this insect will rely on a sound understanding of how these cues are perceived and processed, and their impact on behavior. Towards this end, we investigated allyl isothiocyanate, cinnamaldehyde and citronellal, compounds that are noxious repellents to other insects. We found that L. hesperus avoided areas containing the three compounds and that exposure induced increases in movement velocity and duration in both nymphs and adults. This suggests these compounds may work as repellents. To better understand the underlying physiology of this response, RNA interference by dsRNA injection was used to inhibit the expression of two chemosensory-associated proteins, the odorant receptor co-receptor (Orco) and the transient receptor potential A (TRPA1) channel. While knockdown of Orco did not change the reaction of adult females to citronellal, TRPA1 silencing effectively eliminated the induced increase to movement, suggesting a chemoperceptory role in citronellal detection. [ABSTRACT FROM AUTHOR]

Background: The western corn rootworm, Diabrotica virgifera virgifera, is a pervasive pest of maize in North America and Europe, which has adapted to current pest management strategies. In advance of an assembled and annotated D. v. virgifera genome, we developed transcriptomic resources to use in identifying candidate genes likely to be involved in the evolution of resistance, starting with members of the ATP-binding cassette (ABC) transporter family. Results: In this study, 65 putative D. v. virgifera ABC (DvvABC) transporters were identified within a combined transcriptome assembly generated from embryonic, larval, adult male, and adult female RNA-sequence libraries. Phylogenetic analysis placed the deduced amino-acid sequences of the DvvABC transporters into eight subfamilies (A to H). To supplement our sequence data with functional analysis, we identified orthologs of Tribolium castaneum ABC genes which had previously been shown to exhibit overt RNA interference (RNAi) phenotypes. We identified eight such D. v. virgifera genes, and found that they were functionally similar to their T. castaneum counterparts. Interestingly, depletion of DvvABCB_39715 and DvvABCG_3712 transcripts in adult females produced detrimental reproductive and developmental phenotypes, demonstrating the potential of these genes as targets for RNAi-mediated insect control tactics. Conclusions: By combining sequence data from four libraries covering three distinct life stages, we have produced a relatively comprehensive de novo transcriptome assembly for D. v. virgifera. Moreover, we have identified 65 members of the ABC transporter family and provided the first insights into the developmental and physiological roles of ABC transporters in this pest species. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV), a mosquito-borne alphavirus of increasing public health significance, has caused large epidemics in Africa and the Indian Ocean basin; now it is spreading throughout the Americas. The primary vectors of CHIKV are Aedes (Ae.) aegypti and, after the introduction of a mutation in the E1 envelope protein gene, the highly anthropophilic and geographically widespread Ae. albopictus mosquito. We review here research efforts to characterize the viral genetic basis of mosquito-vector interactions, the use of RNA interference and other strategies for the control of CHIKV in mosquitoes, and the potentiation of CHIKV infection by mosquito saliva. Over the past decade, CHIKV has emerged on a truly global scale. Since 2013, CHIKV transmission has been reported throughout the Caribbean region, in North America, and in Central and South American countries, including Brazil, Columbia, Costa Rica, El Salvador, French Guiana, Guatemala, Guyana, Nicaragua, Panama, Suriname, and Venezuela. Closing the gaps in our knowledge of driving factors behind the rapid geographic expansion of CHIKV should be considered a research priority. The abundance of multiple primate species in many of these countries, together with species of mosquito that have never been exposed to CHIKV, may provide opportunities for this highly adaptable virus to establish sylvatic cycles that to date have not been seen outside of Africa. The short-term and long-term ecological consequences of such transmission cycles, including the impact on wildlife and people living in these areas, are completely unknown.