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1.
BMC Genomics ; 22(1): 639, 2021 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-34479486

RESUMO

BACKGROUND: Resistance of pest insect species to insecticides, including B. thuringiensis (Bt) pesticidal proteins expressed by transgenic plants, is a threat to global food security. Despite the western corn rootworm, Diabrotica virgifera virgifera, being a major pest of maize and having populations showing increasing levels of resistance to hybrids expressing Bt pesticidal proteins, the cell mechanisms leading to mortality are not fully understood. RESULTS: Twenty unique RNA-seq libraries from the Bt susceptible D. v. virgifera inbred line Ped12, representing all growth stages and a range of different adult and larval exposures, were assembled into a reference transcriptome. Ten-day exposures of Ped12 larvae to transgenic Bt Cry3Bb1 and Gpp34/Tpp35Ab1 maize roots showed significant differential expression of 1055 and 1374 transcripts, respectively, compared to cohorts on non-Bt maize. Among these, 696 were differentially expressed in both Cry3Bb1 and Gpp34/Tpp35Ab1 maize exposures. Differentially-expressed transcripts encoded protein domains putatively involved in detoxification, metabolism, binding, and transport, were, in part, shared among transcripts that changed significantly following exposures to the entomopathogens Heterorhabditis bacteriophora and Metarhizium anisopliae. Differentially expressed transcripts in common between Bt and entomopathogen treatments encode proteins in general stress response pathways, including putative Bt binding receptors from the ATP binding cassette transporter superfamily. Putative caspases, pro- and anti-apoptotic factors, as well as endoplasmic reticulum (ER) stress-response factors were identified among transcripts uniquely up-regulated following exposure to either Bt protein. CONCLUSIONS: Our study suggests that the up-regulation of genes involved in ER stress management and apoptotic progression may be important in determining cell fate following exposure of susceptible D. v. virgifera larvae to Bt maize roots. This study provides novel insights into insect response to Bt intoxication, and a possible framework for future investigations of resistance mechanisms.


Assuntos
Bacillus thuringiensis , Besouros , Praguicidas , Animais , Bacillus thuringiensis/genética , Sobrevivência Celular , Besouros/genética , Endotoxinas/toxicidade , Resistência a Inseticidas , Larva/genética , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Regulação para Cima , Zea mays/genética
2.
Pest Manag Sci ; 77(2): 860-868, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32946636

RESUMO

BACKGROUND: Western corn rootworm (WCR) pyrethroid resistance has been confirmed in the western US Corn Belt. Toxicological and biochemical studies indicated that multiple mechanisms of resistance might be involved in the resistance trait, such as enhanced metabolism and/or kdr target-site mutation(s) in the voltage-gated sodium channels. To characterize the mechanisms of WCR pyrethroid resistance at the molecular level, pairwise comparisons were made between RNA-Seq data collected from pyrethroid-resistant and -susceptible WCR populations. Gene expression levels and sodium channel sequences were evaluated. RESULTS: Seven transcripts exhibited significantly different expression (q ≤ 0.05) when comparing field-collected pyrethroid-resistant (R-Field) and -susceptible (S-Field) WCR populations. Three of the differentially expressed transcripts were P450s overexpressed in R-Field (9.2-26.2-fold). A higher number (99) of differentially expressed transcripts was found when comparing laboratory-derived pyrethroid-resistant (R-Lab) and -susceptible (S-Lab) WCR populations. Eight of the significant transcripts were P450s overexpressed in R-Lab (2.7-39.8-fold). This study did not detect kdr mutations in pyrethroid-resistant WCR populations. Other differentially expressed transcripts that may play a role in WCR pyrethroid resistance are discussed. CONCLUSION: This study revealed that P450-mediated metabolism is likely to be a major mechanism of WCR pyrethroid resistance, which could affect the efficacy of other insecticides sharing similar metabolic pathways. Additionally, results suggested that although laboratory selection of a pyrethroid-resistant WCR population may help to characterize resistance mechanisms, a field-selected population provided rare and perhaps major variants corresponding to the resistance trait.


Assuntos
Besouros , Inseticidas , Piretrinas , Animais , Besouros/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Piretrinas/farmacologia , Zea mays/genética
3.
Toxins (Basel) ; 12(9)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32872277

RESUMO

Insecticidal toxins from Bacillus thuringiensis (Bt) are valuable tools for pest management worldwide, contributing to the management of human disease insect vectors and phytophagous insect pests of agriculture and forestry. Here, we report the effects of dual and triple Bt toxins expressed in transgenic cotton cultivars on the fitness and demographic performance of Helicoverpa zea (Boddie)-a noctuid pest, known as cotton bollworm and corn earworm. Life-history traits were determined for individuals of three field populations from a region where H. zea overwintering is likely. Triple-gene Bt cotton cultivars that express Cry and Vip3Aa toxins killed 100% of the larvae in all populations tested. In contrast, dual-gene Bt cotton that express Cry1Ac+Cry1F and Cry1Ac+Cry2Ab allowed population growth with the intrinsic rate of population growth (rm) 38% lower than on non-Bt cotton. The insects feeding on Bt cotton plants that express Cry1Ac+Cry2Ab, Cry1Ac+Cry1F, or Cry1Ab+Cry2Ae exhibited reduced larval weight, survival rate, and increased development time. Additionally, fitness parameters varied significantly among the insect populations, even on non-Bt cotton plants, likely because of their different genetic background and/or previous Bt toxin exposure. This is the first report of the comparative fitness of H. zea field populations on dual-gene Bt cotton after the recent reports of field resistance to certain Bt toxins. These results document the population growth rates of H. zea from an agricultural landscape with 100% Bt cotton cultivars. Our results will contribute to the development and validation of resistance management recommendations.


Assuntos
Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Gossypium/parasitologia , Proteínas Hemolisinas/metabolismo , Mariposas/metabolismo , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/parasitologia , Animais , Toxinas de Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Endotoxinas/genética , Feminino , Regulação da Expressão Gênica de Plantas , Aptidão Genética , Gossypium/genética , Gossypium/metabolismo , Proteínas Hemolisinas/genética , Masculino , Mariposas/embriologia , Mariposas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Dinâmica Populacional , Fatores de Tempo
4.
Pest Manag Sci ; 76(12): 4240-4247, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32614113

RESUMO

BACKGROUND: Pesticide resistance is a growing issue worldwide, and susceptibility of pest populations should be monitored in migratory intersection regions for successful resistance management. We determined the susceptibility of eight noctuid species from the Florida Panhandle to bifenthrin (pyrethroid) and chlorantraniliprole (diamide). Larvae from field and laboratory populations were exposed to commercial insecticide formulations using the leaf-dip method in concentration-mortality bioassays. RESULTS: The field populations of Helicoverpa zea (Boddie), Spodoptera frugiperda (Smith), S. eridania (Stoll), S. exigua (Hubner) and Chloridea virescens (Fabricius) had reduced susceptibility to bifenthrin compared with the laboratory populations. Resistance ratios to bifenthrin were as high as 10 071-fold in S. exigua and 436-fold in S. frugiperda, while there was no reduced susceptibility in Agrotis ipsilon (Hufnagel). The susceptibility to chlorantraniliprole was similar between the field and laboratory populations studied, except for S. exigua that exhibited 630-fold resistance to the diamide. The probit regression equations indicated that the larval mortality of S. exigua and S. frugiperda populations was <80% with bifenthrin at the concentration equivalent to the label rate. Likewise, the estimated mortality of S. exigua larvae with chlorantraniliprole at the label rate concentration was <80%. CONCLUSIONS: The lepidopteran pest populations tested were variable in susceptibility to bifenthrin by contrast to more consistent susceptibility to chlorantraniliprole. These results help in the choice of effective insecticides for integrated pest management and resistance management in cropping systems colonized by migratory lepidopteran pests from the U.S. Gulf Coast region. © 2020 Society of Chemical Industry.


Assuntos
Inseticidas , Piretrinas , Animais , Diamida , Florida , Resistência a Inseticidas , Inseticidas/farmacologia , Larva , Piretrinas/farmacologia , Spodoptera
5.
Pest Manag Sci ; 76(11): 3606-3614, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32400940

RESUMO

BACKGROUND: Most recently, major federal regulatory agencies deregulated an in planta RNA interference (RNAi) trait against a devastating corn pest, the western corn rootworm Diabrotica virgifera virgifera, in the United States and Canada. The impact of double-stranded RNA (dsRNA) plant-incorporated protectants (PIPs) and dietary RNAi to non-target organisms, however, still needs further investigation. In this study, we assessed the potential risks of a Diabrotica virgifera virgifera active dsRNA to a group of predatory biological control agents, including Hippodamia convergens, Harmonia axyridis, Coleomegilla maculata, and Coccinella septempunctata. The overarching hypothesis is that the insecticidal dsRNA targeting Diabrotica virgifera virgifera has no or negligible adverse effect on lady beetles. RESULTS: A 400-bp fragment with the highest sequence similarity between target and tested species was selected as the template for dsRNA synthesis. For the dietary RNAi toxicity assay, newly hatched first instar larvae were administered with v-ATPase A dsRNAs designed from Diabrotica virgifera virgifera and the four lady beetles, respectively. A dsRNA from ß-glucuronidase (GUS), a plant gene, and H2 O were served as the negative controls. The endpoint included both sub-organismal (gene expression), and organismal (survival rate, development time, pupa and adult weight) measurements. The results from dietary RNAi toxicity assay demonstrate significantly impacts of Diabrotica virgifera virgifera-active dsRNAs on lady beetles under the worst-case scenario at both transcriptional and phenotypic level. Interestingly, substantial differences among the four lady beetle species were observed toward the ingested exogenous dsRNAs. CONCLUSION: Such differential response to dietary RNAi may shed light on the mechanisms underlying the mode-of-action of RNAi-based biopesticides. © 2020 Society of Chemical Industry.


Assuntos
Besouros , Animais , Canadá , Besouros/genética , Larva/genética , Controle Biológico de Vetores , Interferência de RNA , RNA de Cadeia Dupla/genética , Zea mays/genética
6.
Pestic Biochem Physiol ; 164: 165-172, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32284123

RESUMO

Western corn rootworm (WCR) pyrethroid resistance has been previously reported in the United States (US) western Corn Belt, and cross-resistance and synergism studies suggested that both target site insensitivity and enhanced metabolism may be conferring WCR resistance to pyrethroids. The present study aimed to investigate the potential mechanisms of WCR pyrethroid resistance and to estimate the heritability of the resistance trait. Biochemical assays using model substrates and spectrophotometry revealed 2-4-fold higher activity of P450s and esterases in pyrethroid-resistant WCR populations, whereas the biological activity of glutathione S-transferase was similar between populations tested. No mutation in the voltage-gated sodium channel was detected in pyrethroid-resistant WCR individuals by sequencing PCR products containing the para-homologous L1014, T929, and M918 amino acid positions that are commonly associated with target site mutations in other pyrethroid-resistant insects. A pilot estimation of pyrethroid resistance heritability obtained during laboratory selection of a WCR population suggested a major genetic component of the resistance trait and predicted a 10-fold increase in WCR bifenthrin resistance within ~7 generations of insecticide lethal exposure. Results support earlier indirect evidence that enhanced metabolism may be contributing to WCR resistance to pyrethroids and illustrates the potential of WCR pyrethroid resistance evolution.


Assuntos
Besouros , Inseticidas , Piretrinas , Animais , Resistência a Inseticidas , Larva , Zea mays
7.
Sci Rep ; 10(1): 5070, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193506

RESUMO

The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is the main insect pest of peppers (Capsicum spp.) throughout the southern U.S. and a potential target for novel control methods that may require gene expression analyses. Careful selection of adequate reference genes to normalize RT-qPCR data is an important prerequisite for gene expression studies since the expression stability of reference genes can be affected by the experimental conditions leading to biased or erroneous results. The lack of studies on validation of reference genes for RT-qPCR analysis in A. eugenii limits the investigation of gene expression, therefore it is needed a systematic selection of suitable reference genes for data normalization. In the present study, three programs (BestKeeper, geNorm and NormFinder) were used to analyze the expression stability of candidate reference genes (ß-ACT, ArgK, EF1-α, GAPDH, RPL12, RPS23, α-TUB, 18S and 28S) in A. eugenii under different experimental conditions. Our results revealed that the most stably expressed reference genes in A. eugenii varied according to the experimental condition evaluated: developmental stages (EF1-α, 18S and RPL12), sex (RPS23 and RPL12), low temperature (GAPDH and α-TUB), high temperature (α-TUB and RPS23), all temperatures (α-TUB and GAPDH), starvation (RPL12 and α-TUB), and dsRNA exposure (α-TUB and RPL12). Our study provides for the first time valuable information on appropriate reference genes that can be used in the analysis of gene expression by RT-qPCR in biological experiments involving A. eugenii.


Assuntos
Besouros/genética , Besouros/fisiologia , Bases de Dados Genéticas/normas , Perfilação da Expressão Gênica/métodos , Expressão Gênica/genética , Genes de Insetos/genética , Estudos de Associação Genética/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Animais , Capsicum/parasitologia , Besouros/crescimento & desenvolvimento , Inanição/genética , Temperatura
8.
Insect Biochem Mol Biol ; 118: 103285, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31760137

RESUMO

Insecticides are a key tool in the management of many insect pests of agriculture, including soybean aphids. The selection imposed by insecticide use has often lead to the evolution of resistance by the target pest through enhanced detoxification mechanisms. We hypothesised that exposure of insecticide-susceptible aphids to sublethal doses of insecticides would result in the up-regulation of genes involved in detoxification of insecticides, revealing the genes upon which selection might act in the field. We used the soybean aphid biotype 1 reference genome, version 6.0 as a reference to analyze RNA-Seq data. We identified multiple genes with potential detoxification roles that were up-regulated 12 h after sublethal exposure to esfenvalerate or thiamethoxam. However, these genes were part of a dramatic burst of differential gene expression in which thousands of genes were up- or down-regulated, rather than a defined response to insecticides. Interestingly, the transcriptional burst observed at 12 h s declined dramatically by 24-hrs post-exposure, suggesting a general stress response that may become fine-tuned over time.


Assuntos
Afídeos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Genes de Insetos/efeitos dos fármacos , Inseticidas/metabolismo , Nitrilas/metabolismo , Piretrinas/metabolismo , Tiametoxam/metabolismo , Animais , Afídeos/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
9.
Pest Manag Sci ; 76(4): 1500-1512, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31677217

RESUMO

BACKGROUND: RNA interference (RNAi) triggered by maize plants expressing RNA hairpins against specific western corn rootworm (WCR) transcripts have proven to be effective at controlling this pest. To provide robust crop protection, mRNA transcripts targeted by double-stranded RNA must be sensitive to knockdown and encode essential proteins. RESULTS: Using WCR adult feeding assays, we identified Sec23 as a highly lethal RNAi target. Sec23 encodes a coatomer protein, a component of the coat protein (COPII) complex that mediates ER-Golgi transport. The lethality detected in WCR adults was also observed in early instar larvae, the life stage causing most of the crop damage, suggesting that WCR adults can serve as an alternative to larvae for dsRNA screening. Surprisingly, over 85% transcript inhibition resulted in less than 40% protein knockdown, suggesting that complete protein knockdown is not necessary for Sec23 RNAi-mediated mortality. The efficacy of Sec23 dsRNA for rootworm control was confirmed in planta; T0 maize events carrying rootworm Sec23 hairpin transgenes showed high levels of root protection in greenhouse assays. A reduction in larval survival and weight were observed in the offspring of WCR females exposed to Sec23 dsRNA LC25 in diet bioassays. CONCLUSION: We describe Sec23 as RNAi target for in planta rootworm control. High mortality in exposed adult and larvae and moderate sublethal effects in the offspring of females exposed to Sec23 dsRNA LC25 , suggest the potential for field application of this RNAi trait and the need to factor in responses to sublethal exposure into insect resistance management programs. © 2019 Society of Chemical Industry.


Assuntos
Zea mays , Animais , Besouros , Feminino , Larva , Controle Biológico de Vetores , Plantas Geneticamente Modificadas , Interferência de RNA , RNA de Cadeia Dupla
10.
Pest Manag Sci ; 76(3): 936-943, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31461216

RESUMO

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 ß-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.


Assuntos
Gorgulhos , Animais , Microinjeções , Interferência de RNA , RNA de Cadeia Dupla , Sri Lanka
11.
Sci Rep ; 9(1): 10703, 2019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31341190

RESUMO

Quantitative reverse transcription PCR (RT-qPCR) is one of the most efficient, reliable and widely used techniques to quantify gene expression. In this study, we evaluated the performance of six southern corn rootworm, Diabrotica undecimpunctata howardi (Barber), housekeeping genes (HKG), ß-actin (Actin), ß-tubulin (Tubulin), elongation factor 1 alpha (EF1α), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 40 S ribosomal protein S9 (RpS9) and ubiquitin-conjugating protein (Ubi), under different experimental conditions such as developmental stage, exposure of neonate and adults to dsRNA, exposure of adults to different temperatures, different 3rd instar larva tissues, and neonate starvation. The HKGs were analyzed with four algorithms, including geNorm, NormFinder, BestKeeper, and delta-CT. Although the six HKGs showed a relatively stable expression pattern among different treatments, some variability was observed. Among the six genes, EF1α exhibited the lowest Ct values for all treatments while Ubi exhibited the highest. Among life stages and across treatments, Ubi exhibited the least stable expression pattern. GAPDH, Actin, and EF1α were among the most stable HKGs in the majority of the treatments. This research provides HKG for accurate normalization of RT-qPCR data in the southern corn rootworm. Furthermore, this information can contribute to future genomic and functional genomic research in Diabrotica species.


Assuntos
Besouros/genética , Genes de Insetos , Técnicas de Genotipagem/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Animais , Técnicas de Genotipagem/métodos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
12.
Pest Manag Sci ; 75(10): 2652-2662, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30729648

RESUMO

BACKGROUND: One concern with the adoption of RNAi-based genetically engineered (GE) crops is the potential harm to valued non-target organisms. Species of Coccinellidae (Coleoptera) are important natural enemies and might be exposed to the insecticidal dsRNA produced by the plant. To assess their susceptibility to dietary RNAi, we fed Adalia bipunctata and Coccinella septempunctata with a dsRNA designed to target the vATPase A of the western corn rootworm, Diabrotica virgifera virgifera (Dvv dsRNA). Specific dsRNAs designed to target the vATPase A of the two ladybird beetle species served as positive controls. RESULTS: Our results revealed that both species were sensitive to dietary RNAi when ingesting their own dsRNAs, with C. septempunctata being more sensitive than A. bipunctata. Dvv dsRNA also adversely affected the two ladybird beetles as indicated by a significantly (but marginally) prolonged developmental time for A. bipunctata and a significantly reduced survival rate for C. septempunctata. These results, however, were obtained at Dvv dsRNA concentrations that were orders of magnitude higher than expected to occur in the field. Gene expression analyses confirmed the bioactivity of the dsRNA treatments and the results from the feeding bioassays. These results are consistent with the bioinformatics analyses, which revealed a higher number of 21-nucleotide-long matches, a requirement for effective RNAi, of the Dvv dsRNA with the vATPase A of C. septempunctata (34 matches) than with that of A. bipunctata (six matches). CONCLUSION: Feeding bioassays revealed that two ladybird species are responsive to dietary RNAi. The two species, however, differed in their sensitivity. © 2019 Society of Chemical Industry.


Assuntos
Besouros/efeitos dos fármacos , RNA de Cadeia Dupla/toxicidade , Animais , Besouros/crescimento & desenvolvimento , Besouros/fisiologia , Dieta , Controle de Insetos , Mariposas , Interferência de RNA/fisiologia , Especificidade da Espécie
13.
J Econ Entomol ; 112(3): 1354-1364, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-30753514

RESUMO

The southern corn rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae), was exposed over multiple generations to vacuolar (v)ATPase-A double-stranded (ds)RNA, first as adults and later, as neonate larvae. During adult selection, high mortality and lower fecundity were observed in the RNAi-selected cages after beetles were exposed to sublethal dsRNA concentrations that varied between LC40 and LC75. During larval selection, a delay in adult emergence and effects on population growth parameters were observed after neonates were exposed to sublethal dsRNA concentrations that varied between LC50 and LC70. Some of the parameters measured for adult emergence such as time to reach maximum linear adult emergence, time elapsed before attaining linear emergence, termination point of the linear emergence, and total days of linear emergence increase, were significantly different between RNAi-selected and control colonies for at least one generation. Significant differences were also observed in population growth parameters such as growth rate, net reproductive rate, doubling time, and generation time. After seven generations of selection, there was no indication that resistance evolved. The sublethal effects caused by exposures of southern corn rootworm to dsRNAs can affect important life history traits and fitness especially through delays in adult emergence and reduction in population growth. Although changes in susceptibility did not occur, the observation of sublethal effects suggests important responses to potential selection pressure. Assuming resistance involves a recessive trait, random mating between susceptible and resistant individuals is an important factor that allows sustainable use of transgenic plants, and delays in adult emergence observed in our studies could potentially compromise this assumption.


Assuntos
Besouros , ATPases Vacuolares Próton-Translocadoras , Animais , Endotoxinas , Larva , Plantas Geneticamente Modificadas , Crescimento Demográfico , RNA de Cadeia Dupla , Zea mays
14.
PLoS One ; 13(12): e0208647, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30521608

RESUMO

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is an important pest of maize in the Americas and has recently been introduced into Africa. Fall armyworm populations have developed resistance to control strategies that depend on insecticides and transgenic plants expressing Bacillus thuringiensis toxins. The study of various resistance mechanisms at the molecular level and the development novel control strategies have been hampered by a lack of functional genomic tools such as gene editing in this pest. In the current study, we explored the possibility of using the CRISPR/Cas9 system to modify the genome of FAW. We first identified and characterized the abdominal-A (Sfabd-A) gene of FAW. Sfabd-A single guide RNA (sgRNA) and Cas9 protein were then injected into 244 embryos of FAW. Sixty-two embryos injected with Sfabd-A sgRNA hatched. Of these hatched embryos, twelve developed into larvae that displayed typical aba-A mutant phenotypes such as fused segments. Of the twelve mutant larvae, three and five eventually developed into female and male moths, respectively. Most mutant moths were sterile, and one female produced a few unviable eggs when it was outcrossed to a wild-type male. Genotyping of 20 unhatched Sfabd-A sgRNA-injected embryos and 42 moths that developed from Sfabd-A sgRNA-injected embryos showed that 100% of the unhatched embryos and 50% of the moths contained indel mutations at the Sfabd-A genomic locus near the guide RNA target site. These results suggest that the CRISPR/Cas9 system is highly efficient in editing FAW genome. Importantly, this gene editing technology can be used to validate gene function to facilitate an understanding of the resistance mechanism and lead to the development of novel pest management approaches.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Silenciamento de Genes , Proteínas de Insetos/genética , Spodoptera/genética , Animais , Sequência de Bases , Feminino , Proteínas de Insetos/metabolismo , Masculino , Mutagênese Sítio-Dirigida , Controle Biológico de Vetores , Fenótipo , Filogenia , Spodoptera/anatomia & histologia , Spodoptera/metabolismo
15.
Pestic Biochem Physiol ; 150: 66-70, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30195389

RESUMO

RNA interference (RNAi) has proven effective for controlling pest insects such as western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Previous studies have shown that WCR adults display a robust RNAi response to orally-administered double-stranded RNA (dsRNA). However, it is unclear how quickly the response occurs after ingestion or how long RNAi effect lasts after WCR stop ingesting diet containing dsRNA. In the current study, WCR adult females were provided with diet treated with dsRNAs of Laccase 2 and Argonaute 2, two nonessential genes, for 8 days. RNAi response in WCR females commenced as early as 10 h after exposure to dsRNA and lasted up to 40 days after exposure to dsRNA ended. Our results show that dsRNA-mediated RNAi response in WCR females is rapid and long-lasting. These findings suggest that even a short-term ingestion of transgenic plants expressing dsRNA by WCR may have a sustained impact on this insect.


Assuntos
Besouros/genética , Interferência de RNA , RNA de Cadeia Dupla/administração & dosagem , Animais , Proteínas Argonauta/genética , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Proteínas de Insetos/metabolismo , Lacase/genética , Controle Biológico de Vetores , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
PLoS One ; 13(8): e0201849, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30092086

RESUMO

The cellular uptake of dsRNA after dietary exposure is critical for RNAi efficiency; however, the mechanism of its uptake in many insects remains to be understood. In this study, we evaluated the roles of the endocytic pathway genes Clathrin heavy chain (Chc), Clathrin adaptor protein AP50, ADP ribosylation factor-like 1 (Arf72A), Vacuolar H+ ATPase 16 kDa subunit (Vha16), and small GTPase Rab7 and putative sid-1-like genes (silA and silC) in RNAi response in western corn rootworm (WCR) using a two-stage dsRNA exposure bioassay. Silencing of Chc, Vha16, and AP50 led to a significant decrease in the effects of laccase2 dsRNA reporter, indicating that these genes are involved in RNAi response. However, the knockdown of either Arf72A or Rab7 did not suppress the response to laccase2 dsRNA. The silencing of the silC gene did not lead to a significant reduction in mortality or increase in the expression of V-ATPase A reporter. While the silencing of the silA gene significantly decreased insect mortality, significant changes in V-ATPase A expression were not detected. These results suggest that clathrin-dependent endocytosis is a biological mechanism that plays an important role during RNAi response in WCR adults. The fact that no definitive support for the roles of silA or silC in RNAi response was obtained support the idea that RNAi response varies greatly in different insect species, demanding additional studies focused on elucidating their involvement in this mechanism.


Assuntos
Clatrina/metabolismo , Besouros/metabolismo , Endocitose/fisiologia , Proteínas de Insetos/metabolismo , Interferência de RNA/fisiologia , Animais , Besouros/genética , Endocitose/genética , Técnicas de Silenciamento de Genes , Proteínas de Insetos/genética , Controle Biológico de Vetores , RNA de Cadeia Dupla/metabolismo , Transcriptoma , Zea mays
17.
Pestic Biochem Physiol ; 148: 103-110, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29891360

RESUMO

The use of transgenic crops that induce silencing of essential genes using double-stranded RNA (dsRNA) through RNA interference (RNAi) in western corn rootworm, Diabrotica virgifera virgifera, is likely to be an important component of new technologies for the control of this important corn pest. Previous studies have demonstrated that the dsRNA response in D. v. virgifera depends on the presence of RNAi pathway genes including Dicer-2 and Argonaute 2, and that downregulation of these genes limits the lethality of environmental dsRNA. A potential resistance mechanism to lethal dsRNA may involve loss of function of RNAi pathway genes. Howver, the potential for resistance to evolve may depend on whether these pathway genes have essential functions such that the loss of function of core proteins in the RNAi pathway will have fitness costs in D. v. virgifera. Fitness costs associated with potential resistance mechanisms have a central role in determining how resistance can evolve to RNAi technologies in western corn rootworm. We evaluated the effect of dsRNA and microRNA pathway gene knockdown on the development of D. v. virgifera larvae through short-term and long-term exposures to dsRNA for Dicer and Argonaute genes. Downregulation of Argonaute 2, Dicer-2, Dicer-1 did not significantly affect larval survivorship or development through short and long-term exposure to dsRNA. However, downregulation of Argonaute 1 reduced larval survivorship and delayed development. The implications of these results as they relate to D. v. virgifera resistance to lethal dsRNA are discussed.


Assuntos
Proteínas Argonauta/genética , Besouros/genética , Técnicas de Silenciamento de Genes , Genes de Insetos , RNA Helicases/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , Ribonuclease III/genética , Animais , Besouros/crescimento & desenvolvimento , Besouros/fisiologia , Produtos Agrícolas/genética , Produtos Agrícolas/parasitologia , Regulação para Baixo , Larva/genética , Larva/crescimento & desenvolvimento , MicroRNAs/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/parasitologia
18.
J Econ Entomol ; 111(1): 337-347, 2018 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-29186445

RESUMO

The risks associated with soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), in the North Central soybean systems has fostered the adoption of prophylactic chemical control practices, such as planting seeds treated with neonicotinoid insecticides, especially thiamethoxam. Consequently, there is a concern that increased selection pressure imposed on the arthropod-pest complex by this insecticide may lead to resistance. Therefore, in vivo bioassays were conducted to determine the susceptibility of soybean aphid to thiamethoxam among North Central U.S. POPULATIONS: Concentration-mortality data were collected using contact glass-vial and detached-leaf systemic bioassays. The results of these experiments indicate that both bioassays were reliable to detect shifts in susceptibility between different soybean aphid clones. The LC50s of field populations of soybean aphid were significantly different when mortality was recorded in contact and systemic exposure assays. Nevertheless, the magnitude of the resistance ratios was consistent in both methods. In addition, a significant increase in the LC50 and EC50 values was observed among field populations tested in detached-leaf systemic bioassays. These results represent the first extensive efforts to identify the variability in susceptibility of soybean aphid to thiamethoxam in the North Central United States Therefore, our results provide a baseline for future assessment and contribute to a better understanding of the applicability of in vivo bioassays for susceptibility monitoring and resistance detection of soybean aphid to thiamethoxam.


Assuntos
Afídeos , Controle de Insetos , Inseticidas , Neonicotinoides , Nitrocompostos , Oxazinas , Tiazóis , Animais , Geografia , Meio-Oeste dos Estados Unidos , Soja/crescimento & desenvolvimento , Tiametoxam
19.
Insect Sci ; 25(1): 45-56, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27520841

RESUMO

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is highly sensitive to orally delivered double-stranded RNA (dsRNA). RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA-dependent RNA polymerase (RdRP) activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi-mediated knockdown of Dv v-ATPase C mRNA throughout the WCR gut and other tissues using high-sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v-ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.


Assuntos
Besouros , Interferência de RNA , Animais , Larva
20.
PLoS One ; 12(12): e0190208, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29267401

RESUMO

RNA interference (RNAi) based approaches can potentially be used to control insect pests. These approaches may depend on the usage of microRNA (miRNA) or double stranded RNA (dsRNA) mediated gene knockdown, which likely involves proteins that regulate these pathways, such as Argonaute 1 (Ago1), Argonaute 2 (Ago2), Dicer 1 (Dcr1), Dicer 2 (Dcr2), and Drosha in insects. We previously performed functional characterization of Ago2 and Dcr2 of western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) and observed that knockdown of Ago2 and Dcr2 ameliorated the lethal effect induced by the dsRNA-mediated knockdown of an essential gene in WCR, thereby confirming the involvement of Ago2 and Dcr2 in the dsRNA pathway. In the current study, we identified and characterized additional members of the Argonaute and Dicer gene families, namely Ago1, Ago3, Aubergine, and Dcr1, in a previously developed WCR transcriptome. We also identified a Drosha homolog in the same transcriptome. We evaluated the impacts on WCR adult fitness associated with the dsRNA-mediated knockdown of Ago1, Ago2, Dcr1, Dcr2, and Drosha genes. Among these putative RNAi pathway genes, only the knockdown of Ago1 incurred significant fitness costs such as reduced survival and oviposition rate, as well as decreased egg viability. The present study, to our knowledge, represents the first report showing that Ago1 is critical to the survival of insect adults. Our findings suggest that Ago1 plays an essential role in broader life stages of an insect than previously thought. Importantly, since fitness costs were not observed, downregulation or loss of function of RNAi pathway genes such as Ago2 or Dcr2 may confer resistance to pest control measures that rely on the normal functions of these genes. However, the precise roles of these genes under field conditions (i.e., in the presence of possible viral pathogens) requires further investigation.


Assuntos
Besouros/genética , Técnicas de Silenciamento de Genes , Interferência de RNA , Zea mays/parasitologia , Animais , Besouros/fisiologia , Interações Hospedeiro-Parasita , Filogenia
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