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Phyllotreta striolata (Fabricius), commonly known as the striped flea beetle (SFB), is a notorious insect pest that attacks Brassicaceae plants worldwide, leading to tremendous economic losses. RNA interference (RNAi) has been proposed as a promising strategy for sustainable and eco-friendly pest control. In this study, a total of nine housekeeping genes including PsVATPA, PsHSP90, PsEF1A, PsRPL6, PsRPS24, PsActin, PsTUBA, PsRPS18, and PsRPL4 were evaluated under four different conditions (organization, population, sex, and RNAi). PsEF1A and PsVATPA were identified as the best reference genes for RNAi bioassay. Furthermore, a total of 24 target genes were selected to investigate their RNAi effects in SFB adults with double-stranded RNAs (dsRNAs), five of them showed significant mortality (28.00% to 70.00%), namely Psα-COPI, Psβ-COPI, PsRPS18, Psγ-COPI, and PsArf1COPI. We found that gene transcript levels of the two most lethal genes, Psγ-COPI and PsArf1COPI, were significantly decreased after treated with the target dsRNAs either by feeding or injection method. The findings from this study demonstrated that the introduction of dsRNAs via oral feedings or injection induces the RNAi-mediated silencing of target genes and can lead to insect mortality. Overall, the identified target genes can be explored in developing RNAi-based insecticides for SFB control., Competing Interests: Declaration of Competing Interest The authors have declared that no conflict of interest exists., (Copyright © 2023. Published by Elsevier Inc.)

The Asian long-horned beetle (ALB) Anoplophora glabripennis is a serious invasive forest pest in several countries, including the United States. Methods available to manage or eradicate this pest are extremely limited, but RNA interference (RNAi) technology is a potentially effective method to control ALB. In this study, we used sucrose feeding bioassay for oral delivery of double-strand RNA (dsRNA) to ALB larvae. 32 P-labeled dsRNA orally delivered to ALB larvae using the sucrose droplet feeding method was processed to small interfering RNA. Feeding neonate larvae with dsRNA targeting genes coding for the inhibitor of apoptosis (IAP), vacuolar sorting protein SNF7 (SNF7), and snakeskin (SSK) induced knockdown of target genes and mortality. Feeding 2 µg of dsRNA per day for 3 days did not induce a significant decrease in the expression of target genes or mortality. However, feeding 5 or 10 µg of dsRNA per day for 3 days induced a significant decrease in the expression of target genes and 50-90% mortality. Interestingly, feeding 2.5 µg each of dsIAP plus dsSNF7, dsIAP plus dsSSK, or dsSNF7 plus dsSSK per day for 3 days induced a significant decrease in the expression of both target genes and approximately 80% mortality. Our findings demonstrate that orally delivered dsRNA induces target gene knockdown and mortality in ALB neonate larvae and RNAi technology may have the potential for effective ALB control., (© 2020 Wiley Periodicals, Inc.)

The ciliate Paramecium tetraurelia has four arginine kinase genes (AK1, AK2, AK3, and AK4). Of these genes, only AK3 has a signal sequence for farnesylation, a post-translational modification that enables anchoring of the modified enzyme to the ciliary membrane. To confirm this modification, AK3 was synthesized using a cell-free protein synthesis system and the peptide masses were analyzed using peptide mass fingerprinting (PMF). The PMF analysis indicated that the C-terminal peptide of AK3 is farnesylated. Thus, AK3 can be farnesylated under physiologically appropriate conditions. To determine the subcellular localization of P. tetraurelia AK3, Western blot analysis was performed using an AK3 polyclonal antibody for the proteins extracted from intact cells and ciliary fractions. When extraction was performed using Triton X-100, AK3 was detected the ciliary fraction. This result suggested that the ciliary fraction contains AK3. In addition, we investigated the role of P. tetraurelia AKs in ciliary movement using the feeding RNA interference method. The swimming velocity of AK1- and AK3-silenced cells was significantly reduced to half the value of that control cells. In summary, P. tetraurelia AK3 is likely to be located in the ciliary membrane and influences swimming velocity, presumably through the phosphoarginine shuttle system present in cilia., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

RNA interference (RNAi) techniques have emerged as powerful tools in the development of novel management strategies for the control of insect pests, such as Henosepilachna vigintioctopunctata , which is a major solanaceous pest in Asia. Our results showed that levels of HvSnf7 expression were greater in larval midguts than in other tissues. Silencing of HvSnf7 led to greater H. vigintioctopunctata mortality rates and appeared to be time- and partially dose-dependent. Bacterially expressed dsHvSnf7 that was applied to detached plant leaves caused 98, 88, and 60% mortality in 1st and 3rd instars, and adults after 10, 12, and 14 d, respectively; when applied to living plants, bacterially expressed dsHvSnf7 led to mortality in 1st and 3rd instars, with no effect on adults. Bacterially expressed dsHvSnf7 led to improved plant protection against H. vigintioctopunctata . Ultrastructural changes caused by HvSnf7-RNAi in larval midguts showed extensive loss of cellular contents that indicate loss of membrane integrity. This study indicate that HvSnf7 potentially can be used as RNAi target gene for controlling of H. vigintioctopunctata .

Planarians have become widely recognized as one of the major animal models for regeneration studies in invertebrates. To induce RNA interference (RNAi) by feeding in planarians, the widely accepted protocol is one in which animals undergo two or three feedings of food containing double-stranded RNA (dsRNA) plus visible food coloring (e.g., blood) for confirmation of feeding by individual animals. However, one possible problem is that incorporated food coloring is often retained within the gut for several days, which makes it difficult to confirm the success of each round of dsRNA feeding based on the difference of the color density within the gut before and after feeding. As a consequence, the difference of appetite levels among individuals undergoing dsRNA feeding leads to phenotypic variability among them due to insufficient knockdown. In our attempts to overcome this problem, we have developed a novel method for achieving robust confirmation of the success of dsRNA feeding in individuals fed multiple times by means of including a combination of three different colored chalks (pink, yellow and blue) as food coloring. Notably, we found that this method is superior to the conventional method for positively marking individuals that actively consumed the dsRNA-containing food during four times of once-daily feeding. Using these selected animals, we obtained stable and sufficiently strong RNAi-induced phenotypes. We termed this improved multi-colored chalk-spiked method of feeding RNAi "Candi" and propose its benefits for gene function analysis in planarians., (© 2017 Japanese Society of Developmental Biologists.)