RNA Interference in the Tobacco Hornworm, Manduca sexta, Using Plastid-Encoded Long Double-Stranded RNA

RNA interference (RNAi) is a promising method for controlling pest insects by silencing the expression of vital insect genes to interfere with development and physiology; however, certain insect Orders are resistant to this process. In this study, we set out to test the ability of in planta-expressed dsRNA synthesized within the plastids to silence gene expression in an insect recalcitrant to RNAi, the lepidopteran species, Manduca sexta (tobacco hornworm). Using the Manduca vacuolar-type H+ ATPase subunit A (v-ATPaseA) gene as the target, we first evaluated RNAi efficiency of two dsRNA products of different lengths by directly feeding the in vitro-synthesized dsRNAs to M. sexta larvae. We found that a long dsRNA of 2222 bp was the most effective in inducing lethality and silencing the v-ATPaseA gene, when delivered orally in a water droplet. We further transformed the plastid genome of the M. sexta host plant, Nicotiana tabacum, to produce this long dsRNA in its plastids and performed bioassays with M. sexta larvae on the transplastomic plants. In the tested insects, the plastid-derived dsRNA had no effect on larval survival and no statistically significant effect on expression of the v-ATPaseA gene was observed. Comparison of the absolute quantities of the dsRNA present in transplastomic leaf tissue for v-ATPaseA and a control gene, GFP, of a shorter size, revealed a lower concentration for the long dsRNA product compared to the short control product. We suggest that stability and length of the dsRNA may have influenced the quantities produced in the plastids, resulting in inefficient RNAi in the tested insects. Our results imply that many factors dictate the effectiveness of in planta RNAi, including a likely trade-off effect as increasing the dsRNA product length may be countered by a reduction in the amount of dsRNA produced and accumulated in the plastids

The Effect of Diet on Midgut and Resulting Changes in Infectiousness of AcMNPV Baculovirus in the Cabbage Looper, Trichoplusia ni

Insecticide resistance has been reported in many important agricultural pests, and alternative management methods are required. Baculoviruses qualify as an effective, yet environmentally benign, biocontrol agent but their efficacy against generalist herbivores may be influenced by diet. However, few studies have investigated the tritrophic interactions of plant, pest, and pathogen from both a gene expression and physiological perspective. Here we use microscopy and transcriptomics to examine how diet affects the structure of peritrophic matrix (PM) in Trichoplusia ni larvae and consequently their susceptibility to the baculovirus, AcMNPV. Larvae raised on potato leaves had lower transcript levels for chitinase and chitin deacetylase genes, and possessed a thicker and more multi-layered PM than those raised on cabbage or artificial diet, which could contribute to their significantly lower susceptibility to the baculovirus. The consequences of these changes underline the importance of considering dietary influences on pathogen susceptibility in pest management strategies

The ABCB Multidrug Resistance Proteins Do Not Contribute to Ivermectin Detoxification in the Colorado Potato Beetle, Leptinotarsa decemlineata (Say)

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a significant agricultural pest that has developed resistance to many insecticides that are used to control it. Investigating the mechanisms of insecticide detoxification in this pest is important for ensuring its continued control, since they may be contributors to such resistance. Multidrug resistance (MDR) genes that code for the ABCB transmembrane efflux transporters are one potential source of insecticide detoxification activity that have not been thoroughly examined in L. decemlineata. In this study, we annotated the ABCB genes found in the L. decemlineata genome and then characterized the expression profiles across midgut, nerve, and Malpighian tubule tissues of the three full transporters identified. To investigate if these genes are involved in defense against the macrocyclic lactone insecticide ivermectin in this insect, each gene was silenced using RNA interference or MDR protein activity was inhibited using a chemical inhibitor, verapamil, before challenging the insects with a dose of ivermectin. Survival of the insects did not significantly change due to gene silencing or protein inhibition, suggesting that MDR transporters do not significantly contribute to defense against ivermectin in L. decemlineata

Plastid Transformation of Micro-Tom Tomato with a Hemipteran Double-Stranded RNA Results in RNA Interference in Multiple Insect Species

Plant-mediated RNA interference (RNAi) holds great promise for insect pest control, as plants can be transformed to produce double-stranded RNA (dsRNA) to selectively down-regulate insect genes essential for survival. For optimum potency, dsRNA can be produced in plant plastids, enabling the accumulation of unprocessed dsRNAs. However, the relative effectiveness of this strategy in inducing an RNAi response in insects using different feeding mechanisms is understudied. To investigate this, we first tested an in vitro-synthesized 189 bp dsRNA matching a highly conserved region of the v-ATPaseA gene from cotton mealybug (Phenacoccus solenopsis) on three insect species from two different orders that use leaf-chewing, lacerate-and-flush, or sap-sucking mechanisms to feed, and showed that the dsRNA significantly down-regulated the target gene. We then developed transplastomic Micro-tom tomato plants to produce the dsRNA in plant plastids and showed that the dsRNA is produced in leaf, flower, green fruit, red fruit, and roots, with the highest dsRNA levels found in the leaf. The plastid-produced dsRNA induced a significant gene down-regulation in insects using leaf-chewing and lacerate-and-flush feeding mechanisms, while sap-sucking insects were unaffected. Our results suggest that plastid-produced dsRNA can be used to control leaf-chewing and lacerate-and-flush feeding insects, but may not be useful for sap-sucking insects

Plastid Transformation of Micro-Tom Tomato with a Hemipteran Double-Stranded RNA Results in RNA Interference in Multiple Insect Species

Plant-mediated RNA interference (RNAi) holds great promise for insect pest control, as plants can be transformed to produce double-stranded RNA (dsRNA) to selectively down-regulate insect genes essential for survival. For optimum potency, dsRNA can be produced in plant plastids, enabling the accumulation of unprocessed dsRNAs. However, the relative effectiveness of this strategy in inducing an RNAi response in insects using different feeding mechanisms is understudied. To investigate this, we first tested an in vitro-synthesized 189 bp dsRNA matching a highly conserved region of the v-ATPaseA gene from cotton mealybug (Phenacoccus solenopsis) on three insect species from two different orders that use leaf-chewing, lacerate-and-flush, or sap-sucking mechanisms to feed, and showed that the dsRNA significantly down-regulated the target gene. We then developed transplastomic Micro-tom tomato plants to produce the dsRNA in plant plastids and showed that the dsRNA is produced in leaf, flower, green fruit, red fruit, and roots, with the highest dsRNA levels found in the leaf. The plastid-produced dsRNA induced a significant gene down-regulation in insects using leaf-chewing and lacerate-and-flush feeding mechanisms, while sap-sucking insects were unaffected. Our results suggest that plastid-produced dsRNA can be used to control leaf-chewing and lacerate-and-flush feeding insects, but may not be useful for sap-sucking insects

Transcriptomic analysis of the Malpighian tubules of Trichoplusia ni: Clues to mechanisms for switching from ion secretion to ion reabsorption in the distal ileac plexus

Excretion of metabolic wastes and toxins in insect Malpighian tubules (MTs) is coupled to secretion of ions and fluid. Larval lepidopterans demonstrate a complex and regionalized MT morphology, and recent studies of larvae of the cabbage looper, Trichoplusia ni, have revealed several unusual aspects of ion transport in the MTs. Firstly, cations are reabsorbed via secondary cells (SCs) in T. ni, whereas in most insects SCs secrete ions. Secondly, SCs are coupled to neighbouring principal cells (PCs) via gap junctions to enable such ion reabsorption. Thirdly, PCs in the SC-containing distal ileac plexus (DIP) region of the tubule reverse from cation secretion to reabsorption in response to dietary ion loading. Lastly, antidiuresis is observed in response to a kinin neuropeptide, which targets both PCs and SCs, whereas in most insects kinins are diuretics that act exclusively via SCs. Recent studies have generated a basic model of ion transport in the DIP of the larval T. ni. RNAseq was used to elucidate previously uncharacterised aspects of ion transport and endocrine regulation in the DIP, with the aim of painting a composite picture of ion transport and identifying putative regulatory mechanisms of ion transport reversal in this tissue. Results indicated an overall expression of 9103 transcripts in the DIP, 993 and 382 of which were differentially expressed in the DIP of larvae fed high-K+ and high-Na+ diets respectively. Differentially expressed transcripts include ion-motive ATPases, ion channels and co-transporters, aquaporins, nutrient and xenobiotic transporters, cell adhesion and junction components, and endocrine receptors. Notably, several transcripts for voltage-gated ion channels and cell volume regulation-associated products were detected in the DIP and differentially expressed in larvae fed ion-rich diet. The study provides insights into the transport of solutes (sugars, amino acids, xenobiotics, phosphate and inorganic ions) by the DIP of lepidopterans. Our data suggest that this region of the MT in lepidopterans (as previously reported) transports cations, fluid, and xenobiotics/toxic metals. Besides this, the DIP expresses genes coding for the machinery involved in Na+- and H+-dependent reabsorption of solutes, chloride transport, and base recovery. Additionally, many of the transcripts expressed by the DIP a capacity of this region to respond to, process, and sometimes produce, neuropeptides, steroid hormones and neurotransmitters. Lastly, the DIP appears to possess an arsenal of septate junction components, differential expression of which may indicate junctional restructuring in the DIP of ion-loaded larvae