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

Abstract

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 ...