RNA interference (RNAi) is a promising technology for the development of next‐generation insect pest control products. Though RNAi is efficient and systemic in coleopteran insects, it is inefficient and variable in lepidopteron insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW),
RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW),
- Award ID(s):
- 1821936
- NSF-PAR ID:
- 10144881
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Archives of Insect Biochemistry and Physiology
- Volume:
- 104
- Issue:
- 4
- ISSN:
- 0739-4462
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract Spodoptera frugiperda by conjugating double‐stranded RNA (dsRNA) with biodegradable chitosan (Chi). dsRNA conjugated with chitosan was protected from degradation by endonucleases present in Sf9 cell‐conditioned medium, hemolymph, and midgut lumen contents collected from the FAW larvae. Chi–dsRNA complexes showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing chitosan formulated dsRNA in Sf9 cells and the tissues induced a significant knockdown of endogenous genes. Chi–dsIAP fed to FAW larvae induced knockdown ofiap gene, growth retardation, and mortality. Processing of dsRNA into small interfering RNA was detected with chitosan‐conjugated32P‐UTP‐labeled ds green fluorescent protein in Sf9 cells and FAW larval tissues. Overall, these data suggest that dsRNA conjugated with chitosan helps dsRNA escape from the endosomes and improves RNAi efficiency in FAW cells and tissues. -
more » « less
Abstract BACKGROUND Calmodulin (CaM) is an essential protein in cellular activity and plays important roles in many processes in insect development. RNA interference (RNAi) has been hypothesized to be a promising method for pest control. CaM is a good candidate for RNAi target. However, the sequence and function of CaM in
Nilaparvata lugens are unknown. Furthermore, the double‐stranded RNA (dsRNA) target to CaM gene in pest control is still unavailable.RESULTS In the present study, two alternatively spliced variants of
CaM transcripts, designatedNlCaM1 andNlCaM2 , were cloned fromN. lugens NlCaM includingNlCaM1 andNlCaM2 mRNA was detectable in all developmental stages and tissues ofN. lugens NlCaM expression by RNAi with different dsRNAs led to an inability to molt properly, increased mortality, which ranged from 49.7 to 92.5%, impacted development of the ovaries and led to female infertility. There were no significant reductions in the transcript levels of vitellogenin and its receptor or in the total vitellogenin protein level relative to the control group. However, a significant reduction in vitellogenin protein was detected in ovaries injected with dsNlCaM. In addition, a specific dsRNA ofNlCaM for control ofN. lugens CONCLUSION NlCaM plays important roles mainly in nymph development and uptake of vitellogenin by ovaries in vitellogenesis inN. lugens . dsRNA derived from the less conserved 3′‐UTR ofNlCaM shows great potential for RNAi‐basedN. lugens -
ABSTRACT The endosomal sorting complex required for transport (ESCRT) machinery is necessary for budding of many enveloped viruses. Recently, it was demonstrated that Vps4, the key regulator for recycling of the ESCRT-III complex, is required for efficient infection by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, ESCRT assembly, regulation, and function are complex, and little is known regarding the details of participation of specific ESCRT complexes in AcMNPV infection. In this study, the core components of ESCRT-I (Tsg101 and Vps28) and ESCRT-III (Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60) were cloned from Spodoptera frugiperda . Using a viral complementation system and RNA interference (RNAi) assays, we found that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. In cells knocking down or overexpressing dominant negative (DN) forms of the components of ESCRT-I and ESCRT-III complexes, entering virions were partially trapped within the cytosol. To examine only egress, cells were transfected with the double-stranded RNA (dsRNA) targeting an individual ESCRT-I or ESCRT-III gene and viral bacmid DNA or viral bacmid DNA that expressed DN forms of ESCRT-I and ESCRT-III components. We found that ESCRT-III components (but not ESCRT-I components) are required for efficient nuclear egress of progeny nucleocapsids. In addition, we found that several baculovirus core or conserved proteins (Ac11, Ac76, Ac78, GP41, Ac93, Ac103, Ac142, and Ac146) interact with Vps4 and components of ESCRT-III. We propose that these viral proteins may form an “egress complex” that is involved in recruiting ESCRT-III components to a virus egress domain on the nuclear membrane. IMPORTANCE The ESCRT system is hijacked by many enveloped viruses to mediate budding and release. Recently, it was found that Vps4, the key regulator of the cellular ESCRT machinery, is necessary for efficient entry and egress of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, little is known about the roles of specific ESCRT complexes in AcMNPV infection. In this study, we demonstrated that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. The components of ESCRT-III (but not ESCRT-I) are also necessary for efficient nuclear egress of progeny nucleocapsids. Several baculovirus core or conserved proteins were found to interact with Vps4 and components of ESCRT-III, and these interactions may suggest the formation of an “egress complex” involved in the nuclear release or transport of viral nucleocapsids.more » « less
-
more » « less
Abstract Apoptosis has been widely studied from mammals to insects. Inhibitor of apoptosis (IAP) protein is a negative regulator of apoptosis. Recent studies suggest that
iap genes could be excellent targets for RNA interference (RNAi)‐mediated control of insect pests. However, not much is known aboutiap genes in one of the well‐known insect model species,Tribolium castaneum . The orthologues of fiveiap genes were identified inT. castaneum by searching its genome at NCBI (https://www.ncbi.nlm.nih.gov/ ) and UniProt (https://www.uniprot.org/ ) databases usingDrosophila melanogaster andAedes aegypti IAP protein sequences as queries. RNAi assays were performed inT. castaneum cell line (TcA) and larvae. The knockdown ofiap1 gene induced a distinct apoptotic phenotype in TcA cells and induced 91% mortality inT. castaneum larvae. Whereas, knockdown ofiap5 resulted in a decrease in cell proliferation in TcA cells and developmental defects inT. castaneum larvae which led to 100% mortality. Knockdown of the other threeiap genes identified did not cause a significant effect on cells or insects. These data increase our understanding ofiap genes in insects and provide opportunities for developingiap1 andiap5 as targets for RNAi‐based insect pest control. -
more » « less
Abstract The E93 transcription factor is a member of helix‐turn‐helix transcription factor family containing a Pip‐squeak motif. This ecdysone primary response gene was identified as a regulator of cell death in
Drosophila melanogaster where it is involved in ecdysone‐induced autophagy and caspase activity that mediate degeneration of larval tissues during metamorphosis from larva to pupa. However, its function in adult insects is not well studied. To study E93 function in the red flour beetle,Tribolium castaneum , double‐stranded RNA (dsRNA) targeting E93 (dsE93) was injected into newly emerged adults. Knockdown of E93 caused a decrease in the synthesis of vitellogenin (Vg), oocyte development, and egg‐laying. Sequencing of RNA isolated from adults injected with dsE93 and controldsmalE (dsRNA targetingEscherichia coli malE gene) followed by differential gene expression analysis showed upregulation of genes involved in the metabolism of reserved nutrients.E93 knockdown induced changes in gene expression resulted in a decrease in Vg synthesis in the fat body and oocyte maturation in ovaries. Mating experiments showed that females injected with dsE93 did not lay eggs. Knockdown ofE93 caused a reduction in the number and size of lipid droplets in the fat body when compared with that in control beetles injected withdsmalE . These data suggest that during the first 2–3 days after the emergence of adult females, E93 suppresses genes coding for enzymes that metabolize reserved nutrients until initiation of vitellogenesis and oogenesis.
