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BACKGROUND: It is important to understand how non-target insects such as parasitoids may be impacted directly or indirectly by RNA interference with double-stranded RNA (dsRNA) that has emerged as a novel pest control tool. We examined the potential effects of a dsRNA targeting an inhibitor of apoptosis (IAP) of the Asian longhorned beetle Anoplophora glabripennis on its gregarious larval ectoparasitoid Ontsira mellipes, directly on adult wasp's survival via injection of 4 µg of dsIAP per wasp, and indirectly on the detectability and suitability of host larvae injected with 2, 4 or 8 µg of dsIAP per larva. RESULTS: Compared with no injection or injection with a control dsGFP targeting a region of gene coding for a green fluorescence protein (GFP), dsIAP did not affect adult wasp's survival. Ontsira mellipes locates hosts in the wood by sensing their movement. Host larvae did not completely cease movement after the injection of dsIAP and were still detected and parasitized. Clutch size was reduced and only 3.8% of the parasitoid offspring developed into adults on host larvae treated at the highest dose. However, clutch size was not affected and 25.5% of the parasitoid offspring developed into adults on host larvae treated at the lowest dose. The fitness of developed wasps (development time, sex ratio, body size, and fecundity) was not affected when compared to the control treatments. No dsIAP was detected in parasitoid larvae. CONCLUSION: The results show no direct effect of the dsRNA on its parasitoid, but the potential indirect effect of dsRNA-affected host on the parasitoid, which may be minimized through optimizing dsRNA dosage to promote compatible applications of both management options for this invasive forest pest. © 2022 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.
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Escarabajos , Avispas , Animales , Interferencia de ARN , Larva , Fertilidad , ARN Bicatenario , Interacciones Huésped-ParásitosRESUMEN
In insects, the shedding of the old exoskeleton is accomplished through ecdysis which is typically followed by the expansion and tanning of the new cuticle. Four neuropeptides, eclosion hormone (EH), ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP) and bursicon (Bur) are known to control ecdysis. However, the regulation of these neuropeptide genes is still poorly understood. Here, we report that in the red flour beetle (RFB) Tribolium castaneum and the fall armyworm (FAW) Spodoptera frugiperda, knockdown or knockout of the SoxC gene caused eclosion defects. The expansion and tanning of wings were not complete. In both RFB and FAW, the knockdown or knockout of SoxC resulted in a decrease in the expression of EH gene. Electrophoretic mobility shift assays revealed that the SfSoxC protein directly binds to a motif present in the promoter of SfEH. The luciferase reporter assays in Sf9 cells confirmed these results. These data suggest that transcription factor SoxC plays a key role in ecdysteroid induction of genes coding for neuropeptides such as EH involved in the regulation of insect eclosion.
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Developing safe and effective double-stranded RNA (dsRNA) delivery systems remains a major challenge for gene silencing, especially in lepidopteran insects. This study evaluated the protamine sulfate (PS)/lipid/dsRNA nanoparticle (NP) delivery system for RNA interference (RNAi) in cells and larvae of the fall armyworm (FAW), Spodoptera frugiperda, a major worldwide pest. A highly efficient gene delivery formulation was prepared using a cationic biopolymer, PS, and a cationic lipid, Cellfectin (CF), complexed with dsRNA. The NPs were prepared by a two-step self-assembly method. The formation of NPs was revealed by dynamic light scattering and transmission electron microscopy. The formation of CF/dsRNA/PS NPs was spherical in shape and size, ranging from 20 to 100 nm with a positive charge (+23.3 mV). Interestingly, prepared CF/dsRNA/PS NPs could protect dsRNA (95%) from nuclease degradation and thus significantly improve the stability of dsRNA. Formulations prepared by combining EGFP DNA with CF/PS increased transfection efficiency in Sf9 cells compared to PS/EGFP and CF/EGFP NPs. Also, the PS/CF/dsRNA NPs enhanced the endosomal escape for the intracellular delivery of dsRNA. The gene knockdown efficiency was assessed in Sf9 Luciferase (Luc) stable cells after a 72 h incubation with CF/dsRNA/PS, PS/dsRNA, CF/dsRNA, or naked dsRNA. Knockdown of the Luc gene was detected in CF/dsRNA/PS (76%) and PS/dsRNA (42.4%) not CF/dsRNA (19.5%) and naked dsRNA (10.3%) in Sf9 Luc cells. Moreover, CF/dsIAP/PS (25 µg of dsRNA targeting the inhibitor of apoptosis, IAP, gene of FAW) NPs showed knockdown of the IAP gene (39.5%) and mortality (55%) in FAW larvae. These results highlight the potential application of PS/lipid/dsRNA NPs for RNA-mediated control of insect pests.
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Nanopartículas , ARN Bicatenario , Animales , Larva , Lípidos/farmacología , Protaminas/genética , Protaminas/metabolismo , Protaminas/farmacología , Interferencia de ARN , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , SpodopteraRESUMEN
SignificanceJuvenile hormone (JH), a sesquiterpenoid, regulates many aspects of insect development, including maintenance of the larval stage by preventing metamorphosis. In contrast, ecdysteroids promote metamorphosis by inducing the E93 transcription factor, which triggers apoptosis of larval cells and remodeling of the larval midgut. We discovered that JH suppresses precocious larval midgut-remodeling by inducing an epigenetic modifier, histone deacetylase 3 (HDAC3). JH-induced HDAC3 deacetylates the histone H4 localized at the promoters of proapoptotic genes, resulting in the suppression of these genes. This eventually prevents programmed cell death of midgut cells and midgut-remodeling during larval stages. These studies identified a previously unknown mechanism of JH action in blocking premature remodeling of the midgut during larval feeding stages.
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Aedes/fisiología , Apoptosis , Sistema Digestivo/metabolismo , Histona Desacetilasas/metabolismo , Hormonas Juveniles/metabolismo , Animales , Apoptosis/genética , Sistema Digestivo/anatomía & histología , Ecdisona/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Histona Desacetilasas/genética , Histonas/metabolismo , Larva , Pupa/metabolismoRESUMEN
Developing strategies to optimize double-stranded RNA (dsRNA) delivery remains a significant challenge in improving RNA interference (RNAi) in insects. Nanoformulations may provide an avenue for the safe and effective delivery of dsRNA. We investigated nanoparticle-mediated gene silencing using biodegradable polymers, poly-l-lysine (PLL), and polyphenol (-)-epigallocatechin gallate (EGCG) for dsRNA delivery into Spodoptera frugiperda (Sf9) cells. Negatively charged cores were formed by EGCG and dsRNA complexes, and PLL was used to encapsulate the cores. The nanoparticles were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM), and energy-dispersive spectrometry (EDS) analysis. The stability of the nanoparticles was assessed by incubating them in nuclease-containing Sf9 cell conditioned media. The effectiveness of the nanoparticles was investigated in Sf9 cells stably expressing the luciferase gene. The results revealed that the nanoparticles formed were small and spherical. The PLL/EGCG/dsRNA nanoparticles exhibited better stability compared to that of PLL/dsRNA or naked dsRNA. Nanoparticles prepared with dsRNA targeting the luciferase gene induced an efficient knockdown (66.7%) of the target gene. In Sf9 cells, nanoparticles prepared with Cy3- or CyPHer-5E-labeled dsRNA showed higher cellular uptake and endosomal escape, respectively, than the naked dsRNA. The improvement in uptake and cytosolic delivery may have helped to increase the knockdown efficiency. In Sf9 cells, the nanoparticles prepared with dsRNA targeting the inhibitor of apoptosis gene induced apoptosis by knocking down its expression. In conclusion, we demonstrate that PLL/EGCG/dsRNA nanoparticles are stable, highly efficient, and effective in dsRNA delivery and knockdown of the target gene.
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Materiales Biocompatibles/química , Catequina/química , Nanopartículas/química , Polilisina/química , ARN Bicatenario/química , Spodoptera/genética , Animales , Materiales Biocompatibles/síntesis química , Catequina/síntesis química , Silenciador del Gen , Ensayo de Materiales , Tamaño de la Partícula , Polilisina/síntesis química , ARN Bicatenario/genética , Spodoptera/citologíaRESUMEN
Juvenile hormones (JH) and ecdysone coordinately regulate metamorphosis in Aedes aegypti. We studied the function of an epigenetic regulator and multifunctional transactivator, CREB binding protein (CBP) in A. aegypti. RNAi-mediated knockdown of CBP in Ae. aegypti larvae resulted in suppression of JH primary response gene, Krüppel-homolog 1 (Kr-h1), and induction of primary ecdysone response gene, E93, resulting in multiple effects including early metamorphosis, larval-pupal intermediate formation, mortality and inhibition of compound eye development. RNA sequencing identified hundreds of genes, including JH and ecdysone response genes regulated by CBP. In the presence of JH, CBP upregulates Kr-h1 by acetylating core histones at the Kr-h1 promoter and facilitating the recruitment of JH receptor and other proteins. CBP suppresses metamorphosis regulators, EcR-A, USP-A, BR-C, and E93 through the upregulation of Kr-h1 and E75A. CBP regulates the expression of core eye specification genes including those involved in TGF-ß and EGFR signaling. These studies demonstrate that CBP is an essential player in JH and 20E action and regulates metamorphosis and compound eye development in Ae. aegypti.
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Aedes/metabolismo , Proteína de Unión a CREB/metabolismo , Ojo/crecimiento & desarrollo , Metamorfosis Biológica/fisiología , Organogénesis/fisiología , Aedes/genética , Animales , Proteína de Unión a CREB/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Ecdisona/genética , Ecdisona/metabolismo , Ecdisona/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Hormonas Juveniles/metabolismo , Hormonas Juveniles/farmacología , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Larva , Organogénesis/efectos de los fármacos , Organogénesis/genética , Regiones Promotoras Genéticas , Pupa/crecimiento & desarrollo , Transducción de Señal , Factores de Transcripción/metabolismo , Fiebre Amarilla/genéticaRESUMEN
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.
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Escarabajos/genética , Interferencia de ARN , ARN Bicatenario , Administración Oral , Animales , Escarabajos/crecimiento & desarrollo , Técnicas de Silenciamiento del Gen , Proteínas Inhibidoras de la Apoptosis/genética , Control de Insectos , Proteínas de Insectos/genética , Larva/genética , Larva/crecimiento & desarrolloRESUMEN
Mosquito-borne diseases are a major threat to human health and are responsible for millions of deaths globally each year. Vector control is one of the most important approaches used in reducing the incidence of these diseases. However, increasing mosquito resistance to chemical insecticides presents challenges to this approach. Therefore, new strategies are necessary to develop the next generation vector control methods. Because of the target specificity of dsRNA, RNAi-based control measures are an attractive alternative to current insecticides used to control disease vectors. In this study, Chitosan (CS) was cross-linked to sodium tripolyphosphate (TPP) to produce nano-sized polyelectrolyte complexes with dsRNA. CS-TPP-dsRNA nanoparticles were prepared by ionic gelation method. The encapsulation efficiency, protection of dsRNA from nucleases, cellular uptake, in vivo biodistribution, larval mortality and gene knockdown efficiency of CS-TPP-dsRNA nanoparticles were determined. The results showed that at a 5:1 weight ratio of CS-TPP to dsRNA, nanoparticles of less than 200 nm mean diameter and a positive surface charge were formed. Confocal microscopy revealed the distribution of the fed CS-TPP-dsRNA nanoparticles in midgut, fat body and epidermis of yellow fever mosquito, Aedes aegypti larvae. Bioassays showed significant mortality of larvae fed on CS-TPP-dsRNA nanoparticles. These assays also showed knockdown of a target gene in CS-TPP-dsRNA nanoparticle fed larvae. These data suggest that CS-TPP nanoparticles may be used for delivery of dsRNA to mosquito larvae.
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Aedes , Quitosano , Resistencia a Medicamentos/efectos de los fármacos , Control de Mosquitos , Nanopartículas/química , Polifosfatos , Interferencia de ARN , ARN Bicatenario , Aedes/genética , Aedes/metabolismo , Animales , Quitosano/química , Quitosano/farmacología , Larva/genética , Larva/metabolismo , Polifosfatos/química , Polifosfatos/farmacología , ARN Bicatenario/química , ARN Bicatenario/genética , ARN Bicatenario/farmacología , Fiebre AmarillaRESUMEN
Asian Longhorned Beetle (ALB) Anoplophora glabripennis is a serious invasive forest pest in several countries including the United States, Canada, and Europe. RNA interference (RNAi) technology is being developed as a novel method for pest management. Here, we identified the ALB core RNAi genes including those coding for Dicer, Argonaute, and double-stranded RNA-binding proteins (dsRBP) as well as for proteins involved in dsRNA transport and the systemic RNAi. We also compared expression of six potential reference genes that could be used to normalize gene expression and selected gapdh and rpl32 as the most reliable genes among different tissues and stages of ALB. Injection of double-stranded RNA (dsRNA) targeting gene coding for inhibitor of apoptosis (IAP) into larvae and adults resulted in a significant knockdown of this gene and caused the death of 90% of the larvae and 100% of adults. No mortality of both larvae and adults injected with dsRNA targeting gene coding for green fluorescence protein (GFP, as a negative control) was observed. These data suggest that functional RNAi machinery exists in ALB and a potential RNAi-based method could be developed for controlling this insect.
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Escarabajos/genética , Genes de Insecto , Interferencia de ARN , Animales , Escarabajos/clasificación , Biología Computacional/métodos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Filogenia , Reproducibilidad de los ResultadosRESUMEN
This study reported the synthesis of Vicenin-2 gold nanoparticles (VN-AuNPs) and evaluated their effect on the glucose utilization efficiency of 3T3-L1 adipocytes. The VN-AuNPs were characterized by microscopic, DLS and spectral analysis. The bio-reducing efficiency of Vicenin-2 (VN) was computed and confirmed by HPLC analysis. The stability of VN-AuNPs in various physiological media was explored. The cytotoxicity and glucose uptake assays were performed in 3T3-L1 adipocytes. The docking of VN with PTP1B and AMPK was also performed. The color change and UV absorption at 537 nm preliminarily confirmed the VN reduced gold nanoparticles. The VN-AuNPs appeared as spherical particles (57 nm) and face centered cubic crystals under TEM and XRD analysis, respectively. Its zeta potential was found to be -6.53 mV. The FT-IR spectra of VN and its AuNPs confirmed its stability. The computed reducing potential of VN was similar to the extent of VN utilized during the synthesis of VN-AuNPs. The VN-AuNPs showed a remarkable stability in different physiological media. At 100 µM concentration, VN-AuNPs displayed 78.21% cell viability. A concentration dependent increase in glucose uptake was noted in 3T3-L1 adipocytes when incubated with VN-AuNPs. The docking data revealed a strong interaction of VN with the binding pockets of PTP1B and AMPK. This demonstrates that the fabricated VN-AuNPs might enhance the intracellular VN availability mediated cellular glucose utilization and this would serve as a novel nanodrug for the management of diabetes.
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Proteínas Quinasas Activadas por AMP/metabolismo , Apigenina/farmacología , Glucosa/metabolismo , Glucósidos/farmacología , Oro/farmacología , Nanopartículas del Metal/química , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Adipocitos , Animales , Apigenina/química , Apigenina/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Biología Computacional , Glucósidos/química , Glucósidos/toxicidad , Oro/química , Oro/toxicidad , Nanopartículas del Metal/toxicidad , Ratones , Simulación del Acoplamiento MolecularRESUMEN
Enzymatic browning by polyphenoloxidase (PPO) affects food quality and taste in fruits and vegetables. Thus, the study was designed to reduce browning in apple juice by coumarin. The ethanolic extract of cinnamon was prepared and its coumarin content was quantitated by HPLC, using authentic coumarin (AC) as standard. The effect of cinnamon extract (CE) and AC on enzymatic browning, its time dependent effects, and the specific activity of PPO and peroxidase (POD) were studied in apple juice. The docking of coumarin with PPO and POD was also performed to elucidate its antibrowning mechanism. The CE (73%) and AC (82%) showed better reduction in browning, maintained its antibrowning effect at all time points, and significantly (p < 0.05) reduced the specific activity of PPO and POD when compared with controls. Coumarin showed strong interaction with binding pockets of PPO and POD, suggesting its potential use as inhibitor to enzyme mediated browning in apple juice.