Insect pest control by RNA interference (RNAi)-mediated gene expression knockdown can be undermined by many factors, including small sequence differences between double-stranded RNA (dsRNA) and the target gene. It can also be compromised by effects that are independent of the dsRNA sequence on non-target organisms (known as sequence-non-specific effects). This study investigated the species-specificity of RNAi in plant sap-feeding hemipteran pests. We first demonstrated sequence-non-specific suppression of aphid feeding by dsRNA at dietary concentrations ≥0.5 µg µL⁻¹. Then we quantified the expression of NUC (nuclease) genes in insects administered homologous dsRNA (with perfect sequence identity to the target species) or heterologous dsRNA (generated against a related gene of non-identical sequence in a different insect species). For the aphids Acyrthosiphon pisum and Myzus persicae, significantly reduced NUC expression was obtained with the homologous but not heterologous dsRNA at 0.2 µg µL⁻¹, despite high dsNUC sequence identity. Follow-up experiments demonstrated significantly reduced expression of NUC genes in the whitefly Bemisia tabaci and mealybug Planococcus maritimus administered homologous dsNUCs, but not heterologous aphid dsNUCs. Our demonstration of inefficient expression knockdown by heterologous dsRNA in these insects suggests that maximal dsRNA sequence identity is required for RNAi targeting of related pest species, and that heterologous dsRNAs at appropriate concentrations may not be a major risk to non-target sap-feeding hemipterans.

Summary: Spodoptera frugiperda (fall armyworm) is a notorious pest that threatens maize production worldwide. Current control measures involve the use of chemical insecticides and transgenic maize expressing Bacillus thuringiensis (Bt) toxins. Although additional transgenes have confirmed insecticidal activity, limited research has been conducted in maize, at least partially due to the technical difficulty of maize transformation. Here, we describe implementation of a sugarcane mosaic virus (SCMV) vector for rapidly testing the efficacy of both endogenous maize genes and heterologous genes from other organisms for the control of S. frugiperda in maize. Four categories of proteins were tested using the SCMV vector: (i) maize defence signalling proteins: peptide elicitors (Pep1 and Pep3) and jasmonate acid conjugating enzymes (JAR1a and JAR1b); (ii) maize defensive proteins: the previously identified ribosome‐inactivating protein (RIP2) and maize proteinase inhibitor (MPI), and two proteins with predicted but unconfirmed anti‐insect activities, an antimicrobial peptide (AMP) and a lectin (JAC1); (iii) lectins from other plant species: Allium cepa agglutinin (ACA) and Galanthus nivalis agglutinin (GNA); and (iv) scorpion and spider toxins: peptides from Urodacus yaschenkoi (UyCT3 and UyCT5) and Hadronyche versuta (Hvt). In most cases, S. frugiperda larval growth was reduced by transient SCMV‐mediated overexpression of genes encoding these proteins. Additionally, experiments with a subset of the SCMV‐expressed genes showed effectiveness against two aphid species, Rhopalosiphum maidis (corn leaf aphid) and Myzus persicae (green peach aphid). Together, these results demonstrate that SCMV vectors are a rapid screening method for testing the efficacy and insecticidal activity of candidate genes in maize.

As cyanobacterial harmful algal bloom (cHAB) events increase in scale, severity, frequency, and duration around the world, rapid and accurate monitoring and characterization tools have become critically essential for regulatory and management decision-making. The composition of cHAB-forming cyanobacteria community can change significantly over time and space and be altered by sample preservation and transportation, making in situ monitoring necessary to obtain real-time and localized information. Sandwich hybridization assay (SHA) utilizes capture oligonucleotide probes for sensitive detection of target-specific nucleic acid sequences. As an amplification-free molecular biology technology, SHA can be adapted for in-situ, real-time or near real-time detection and qualitatively or semi-quantitatively monitoring of cHAB-forming cyanobacteria, owing to its characteristics such as being rapid, portable, inexpensive, and amenable to automation, high sensitivity, specificity and robustness, and multiplexing (i.e., detecting multiple targets simultaneously). Despite its successful application in the monitoring of marine and freshwater phytoplankton, there is still room for improvement. The ability to identify a cHAB community rapidly would decrease delays in cyanotoxin analyses, reduce costs, and increase sample throughput, allowing for timely actions to improve environmental and human health and the understanding of short- and long-term bloom dynamics. Real-time detection and quantitation of HAB-forming cyanobacteria is essential for improving environmental and public health and reducing associated costs. We review and propose to apply SHA for in situ cHABs monitoring.

As a popular tool for gene function characterization and gene therapy, RNA interference (RNAi)-based gene silencing has been increasingly explored for potential applications to control invasive species. At least two major hurdles exist when applying this approach to invasive plants: (1) the design and screening of species- and gene-specific biomacromolecules (i.e., gene-silencing agents or GSAs) made of DNA, RNA, or peptides that can suppress the expression of target genes efficiently, and (2) the delivery vehicle needed to penetrate plant cell walls and other physical barriers (e.g., leaf cuticles). In this study, we investigated the cell-penetrating peptide (CPP)-mediated delivery of multiple types of GSAs (e.g., double-stranded RNA (dsRNA), artificial microRNA (amiRNA), and antisense oligonucleotide (ASO)) to knock down a putative phytoene desaturase (PDS) gene in the invasive common reed (Phragmites australis spp. australis). Both microscopic and quantitative gene expression evidence demonstrated the CPP-mediated internalization of GSA cargos and transient suppression of PDS expression in both treated and systemic leaves up to 7 days post foliar application. Although various GSA combinations and application rates and frequencies were tested, we observed limitations, including low gene-silencing efficiency and a lack of physiological trait alteration, likely owing to low CPP payload capacity and the incomplete characterization of the PDS-coding genes (e.g., the recent discovery of two PDS paralogs) in P. australis. Our work lays a foundation to support further research toward the development of convenient, cost-effective, field-deployable, and environmentally benign gene-silencing technologies for invasive P. australis management.

Previously, sugarcane mosaic virus (SCMV) was developed as a vector for transient expression of heterologous genes in Zea mays (maize). Here, we show that SCMV can also be applied for virus‐induced gene silencing (VIGS) of endogenous maize genes. Comparison of sense and antisense VIGS constructs targeting maize phytoene desaturase (PDS) showed that antisense constructs resulted in a greater reduction in gene expression. In a time course of gene expression after infection with VIGS constructs targeting PDS, lesion mimic 22 (Les22), and Iodent japonica 1 (Ij1), efficient expression silencing was observed 2, 3, and 4 weeks after infection with SCMV. However, at Week 5, expression of Les22 and Ij1 was no longer significantly reduced compared with control plants. The defense signaling molecule jasmonate‐isoleucine (JA‐Ile) can be inactivated by 12C‐hydroxylation and hydrolysis, and knockout of these genes leads to herbivore resistance. JA‐Ile hydroxylases and hydrolases have been investigated in Arabidopsis, rice, and Nicotiana attenuata. To determine whether the maize homologs of these genes function in plant defense, we silenced expression of ZmCYP94B1 (predicted JA‐Ile hydroxylase) and ZmJIH1 (predicted JA‐Ile hydrolase) by VIGS with SCMV, which resulted in elevated expression of two defense‐related genes, Maize Proteinase Inhibitor (MPI) and Ribosome Inactivating Protein 2 (RIP2). Although ZmCYP94B1 and ZmJIH1 gene expression silencing increased resistance to Spodoptera frugiperda (fall armyworm), Schistocerca americana (American birdwing grasshopper), and Rhopalosiphum maidis (corn leaf aphid), there was no additive effect from silencing the expression of both genes. Further work will be required to determine the more precise functions of these enzymes in regulating maize defenses.