Improving RNAi in the Brown Marmorated Stink Bug: Identification of target genes and reference genes for RT-qPCR

Mogilicherla, Kanakachari; Howell, Jeffrey L.; Palli, Subba Reddy

doi:10.1038/s41598-018-22035-z

Cited by 59 publications

(67 citation statements)

References 42 publications

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“…29,30 In addition, we found that Snf7 dsRNA was the most lethal among the dsRNAs tested. This result is in agreement with a number of studies that have shown high mortality in several insect species exposed to Snf7 dsRNA, including Diabrotica virgifera virgifera, 36 Diabrotica undecimpunctata howardi, 37 Halyomorpha halys 38 and Amrasca biguttula biguttula. 39 Interestingly, ingestion of V-ATPase A dsRNA induced the highest level of gene knockdown at day 10 (75.6%), although no significant mortality of the weevils was observed.…”

Section: Discussionsupporting

confidence: 92%

Delivery of gene‐specific dsRNA by microinjection and feeding induces RNAi response in Sri Lanka weevil, Myllocerus undecimpustulatus undatus Marshall

Pinheiro

Taylor

et al. 2019

Pest Management Science

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BACKGROUND RNA interference (RNAi) is widely used in entomological research for functional analysis of genes and is being considered as a new tool for insect pest management. Sri Lanka weevil (SLW) is a highly polyphagous pest of agronomically important plants, but currently only a few control methods are available for this insect. RESULTS In the present study, we evaluated the stability of candidate reference genes β‐ACT, α‐TUB, EF1‐α, RPL12 and GAPDH, and identified EF1‐α as the most reliable for gene expression normalization. Four target genes involved in different cellular processes, including Prosα2, RPS13, Snf7 and V‐ATPase A were selected to evaluate whether RNAi response in SLW adults can be triggered by microinjection and oral feeding of their double‐stranded RNAs (dsRNAs). Three days after injection of the dsRNAs for the target genes, their transcript levels were significantly reduced (up to 91.4%) when compared to the control. Additionally, weevils fed with the target dsRNAs showed significant decreases in gene transcript levels and significant mortality was observed in insects treated with Prosα2 and Snf7 dsRNAs (78.6 to 92.7%). CONCLUSION Our data demonstrate that microinjection and feeding of dsRNA produce a strong RNAi response in SLW, indicating that RNAi‐based strategies could be explored to develop a selective and environmentally safe control method against SLW. © 2019 Society of Chemical Industry

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Section: Discussionsupporting

confidence: 92%

Delivery of gene‐specific dsRNA by microinjection and feeding induces RNAi response in Sri Lanka weevil, Myllocerus undecimpustulatus undatus Marshall

Pinheiro

Taylor

et al. 2019

Pest Management Science

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“…Compared to the control larvae, the mean expression level of diap1, v-ATPase A and v-ATPase E in the f-RNAi treated larvae reduced to 50.2, 3.6, 3.4%, respectively. The knockdown effect of diap1 f-RNAi seems to be lower than that of v-ATPase genes, although, the reduced rate of diap1 mRNA level is nearly equivalent to that in previous diap1 f-RNAi studies 14,20,22,25 showing the obvious phenotype. Considering these facts, we determined that our knockdown effect of diap1 is reasonable.…”

Section: Resultssupporting

confidence: 50%

“…For these reasons, diap1 is one of the candidate target gene for RNAi pesticides and has been investigated in various pests. So far, RNAi of the diap1 gene by injecting the dsRNA into the hemocoel of pest has been shown to induce over 70% mortality in T. castaneum (Coleoptera) 14 , Anoplophora glabripennis (Coleoptera) 20 , Musca domestica (Diptera) 21 , Delia radicum (Diptera) 21 , Heliothis virescens (Lepidoptera) 22 , Lygus lineolaris (Hemiptera) 23 and Apolygus lucorum (Hemiptera) 24 . Diap1 silencing by f-RNAi has also been reported to induce 30– 78% of mortality in Agrilus planipennis 24 , T. castaneum 14 , and Diaphorina citri 25 .…”

Section: Introductionmentioning

confidence: 99%

Oral RNAi of diap1 in a pest results in rapid reduction of crop damage

Chikami

Kawaguchi

Suzuki

et al. 2019

Preprint

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25Selecting an appropriate target gene is critical to the success of feeding RNA interference 26 (f-RNAi)-based pest control. Gene targets have been chosen based on their ability to 27 induce lethality. However, lethality induction by f-RNAi is slow-acting and crop damage 28 can progress during this time. Here, we show that f-RNAi of death-associated inhibitor 29 of apoptosis protein 1 (diap1), but not two conventional targets vacuolar ATPase subunit 30 A and E, induces acute feeding cessation in the solanaceous pest, Henosepilachna 31 vigintioctopunctata during 24-48 hours. We also found that the feeding cessation by 32 diap1 f-RNAi has species-specificity and occurs with only 1.6 ng dsRNA. Our results 33 suggest that diap1 is an appropriate target in the context of rapid reduction of crop damage. 34 We propose that acute feeding disorder should be assessed as a novel criterion for 35 selecting appropriate target genes for RNAi-based pest control in addition to the 36 conventional criterion based on lethality. 37 38 dsRNA 5 on crops. Regardless of application method, the selection of an appropriate target 46 gene is essential to achieve effective pest control 6 . The most target genes reported so far 47 are housekeeping genes such as the v-ATPase genes and metabolic genes such as the 48 chitin synthase genes (representative targets listed in Baum et al. 2 ).49Conventionally, the criterion for selecting target genes is a combination of induction of 50 lethality or growth-inhibition by gene silencing with RNAi 6 . For example, silencing of v-51 ATPase subunit A (v-ATPase A) or v-ATPase subunit E (v-ATPase E) by oral delivery of 52 the dsRNA causes increased mortality in various pests of the order Coleoptera 7,8,9 , 53 Lepidoptera 7,10 , Diptera 7 , Hemiptera 11,12 and Orthoptera 13 . Such induction of lethality or 54 growth-inhibition is extremely effective in terms of pest reduction and eradication. 55However neither lethality nor growth-inhibition are phenotype that are quick to induce, 56 and it takes a certain amount of time before maximal effect of treatment is achieved. 57During the long-time span between treatment and phenotype, insect pests can continue to 58 damage crops. For example, it takes more than a week for the RNAi effect of v-ATPase 59 A or v-ATPase E to occur. Silencing of v-ATPase E requires 9 days to induce 100% 60 mortality through injected and 25 days to induce 100% mortality through oral RNAi in 61 Tribolium castaneum 14 . Therefore, in order to achieve effective RNAi-based control of 62 4 herbivorous pests, it would be desirable to evaluate and search for target genes from the 63 viewpoint of rapid termination of crop damage in addition to the induction of lethality 64 and growth-inhibition. 65In this regard, we found that the gene silencing of death-associated inhibitor of 66 apoptosis protein 1 (diap1) gene by oral-feeding RNAi (f-RNAi) causes acute feeding 67 disorder. diap1 is an insect homolog of the iap genes and is known as a suppressor of 68 apoptosis in the fruit ...

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“…For a role in crop protection, oral uptake of dsRNA and subsequent efficient uptake in the gut have to be evaluated . Supplying dsRNA in artificial diet resulted in the knockdown of targeted genes in different species of Hemiptera including the small brown planthopper, Laodelphax striatellus (Fallén), the brown planthopper, Nilaparvata lugens (Stål), the corn planthopper, Peregrinus maidis (Ashmead), the white‐backed planthopper, Sogatella furcifera (Horvath), the kissing bug, Rhodnius prolixus (Stål), the pea aphid, Acyrthosiphon pisum (Harris), the wheat aphid, Sitobion avenae (Fabricius), the potato/tomato psyllid, Bactericera cockerelli (Sulc), the Asian citrus psyllid, Diaphorina citri (Kuwayama), the brown marmorated stink bug, Halyomorpha halys (Stål) and the tobacco whitefly, Bemisia tabaci (Gennadius) . However, oral delivery in some hemipteran pests, such as the tarnished plant bug Lygus lineolaris (Palisot de Beauvois) and the pea aphid A. pisum , is confounded by high ribonuclease activity in the saliva which degrades the dsRNA .…”

Section: Introductionmentioning

confidence: 99%

Liposome encapsulation and EDTA formulation of dsRNA targeting essential genes increase oral RNAi‐caused mortality in the Neotropical stink bug Euschistus heros

Castellanos

Smagghe

Sharma

et al. 2018

Pest Management Science

104

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Improving RNAi in the Brown Marmorated Stink Bug: Identification of target genes and reference genes for RT-qPCR

Cited by 59 publications

References 42 publications

Delivery of gene‐specific dsRNA by microinjection and feeding induces RNAi response in Sri Lanka weevil, Myllocerus undecimpustulatus undatus Marshall

Delivery of gene‐specific dsRNA by microinjection and feeding induces RNAi response in Sri Lanka weevil, Myllocerus undecimpustulatus undatus Marshall

Oral RNAi of diap1 in a pest results in rapid reduction of crop damage

Liposome encapsulation and EDTA formulation of dsRNA targeting essential genes increase oral RNAi‐caused mortality in the Neotropical stink bug Euschistus heros

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