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1.
Arch Insect Biochem Physiol ; 108(3): e21845, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34605064

RESUMO

With the wide cultivation of transgenic plants throughout the world and the rising risk of resistance to Bacillus thuringiensis crystal (Cry) toxins, it is essential to design an adaptive resistance management strategy for continued use. Neuropeptide F (NPF) of insects has proven to be valuable for the production of novel-type transgenic plants via its important role in the control of feeding behavior. In this study, the gene encoding NPF was cloned from the diamondback moth, Plutella xylostella, an important agricultural pest. Real-time quantitative reverse transcription-polymerase chain reaction and in situ hybridization showed a relatively high expression of P. xylostella-npf (P. x-npf) in endocrine cells of the midgut of fourth instar larvae, and it was found to participate in P. xylostella feeding behavior and Cry1Ac-induced feeding inhibition. Prokaryotic expression and purification provided structure unfolded P. x-npf from inclusion bodies for diet surface overlay bioassays and the results demonstrated a significant synergistic effect of P. x-npf on Cry1Ac toxicity by increasing intake of noxious food which contains Cry toxins, especially quick death at an early stage of feeding. Our findings provided a potential new way to efficiently control pests by increasing intake of lower dose Cry toxins and a novel hint for the complex Cry toxin mechanism.


Assuntos
Toxinas de Bacillus thuringiensis , Endotoxinas , Proteínas Hemolisinas , Mariposas , Neuropeptídeos , Animais , Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Comportamento Alimentar/fisiologia , Expressão Gênica , Genes de Insetos , Proteínas Hemolisinas/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/metabolismo , Mariposas/fisiologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Controle de Pragas/métodos
2.
Arch Insect Biochem Physiol ; 108(2): e21834, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34288075

RESUMO

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), is a major pest of potato plants worldwide and is notorious for its ability to develop resistance to insecticides. Cry3 toxins synthesized by Bacillus thuringiensis ssp. tenebrionis have been used successfully to manage this pest. Resistance to Cry toxins is a concerning problem for many insect pests; therefore, it is important to determine the mechanisms by which insects acquire resistance to these toxins. Cadherin-like and ABC transporter proteins have been implicated in the mode of action of Cry toxins as mutations in these genes render lepidopterans resistant to them; however, clear consensus does not exist on whether these proteins also play a role in Cry3 toxin activity and/or development of resistance in coleopterans. In the current study, we identified the L. decemlineata orthologues of the cadherin (LdCAD) and the ABCB transporter (LdABCB1) that have been implicated in the mode of action of Cry toxins in other coleopterans. Suppression of LdABCB1 via RNA interference reduced toxin-related larval mortality, whereas partial silencing of LdCAD did not. Our results suggest that the ABCB is involved in the mode of action of Cry3Aa toxins; however, no evidence was found to support the role of cadherin as a receptor of Cry3Aa in L. decemlineata.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Toxinas de Bacillus thuringiensis/farmacologia , Besouros , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Caderinas/genética , Caderinas/metabolismo , Besouros/efeitos dos fármacos , Besouros/metabolismo , Besouros/microbiologia , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/metabolismo , Larva/microbiologia , Controle Biológico de Vetores , Interferência de RNA
3.
Toxins (Basel) ; 13(5)2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065247

RESUMO

Laboratory selection for resistance of field populations is a well-known and useful tool to understand the potential of insect populations to evolve resistance to insecticides. It provides us with estimates of the frequency of resistance alleles and allows us to study the mechanisms by which insects developed resistance to shed light on the mode of action and optimize resistance management strategies. Here, a field population of Mythimna separata was subjected to laboratory selection with either Vip3Aa, Cry1Ab, or Cry1F insecticidal proteins from Bacillus thuringiensis. The population rapidly evolved resistance to Vip3Aa reaching, after eight generations, a level of >3061-fold resistance, compared with the unselected insects. In contrast, the same population did not respond to selection with Cry1Ab or Cry1F. The Vip3Aa resistant population did not show cross resistance to either Cry1Ab or Cry1F. Radiolabeled Vip3Aa was tested for binding to brush border membrane vesicles from larvae from the susceptible and resistant insects. The results did not show any qualitative or quantitative difference between both insect samples. Our data, along with previous results obtained with other Vip3Aa-resistant populations from other insect species, suggest that altered binding to midgut membrane receptors is not the main mechanism of resistance to Vip3Aa.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/farmacologia , Inseticidas/farmacologia , Mariposas/fisiologia , Animais , Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas , Inseticidas/isolamento & purificação , Larva , Ligação Proteica
4.
Insect Biochem Mol Biol ; 135: 103608, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34119653

RESUMO

Bacillus thuringiensis (Bt) bacteria produce Cry toxins that kill insect pests. Insect specificity of Cry toxins relies on their binding to larval gut membrane proteins such as cadherin and ATP-binding cassette (ABC) transporter proteins. Mutations in ABC transporters have been implicated in high levels of resistance to Cry toxins in multiple pests. Spodoptera frugiperda is an insect pest susceptible to Cry1Fa and Cry1Ab toxins while Mythimna separata is tolerant to Cry1Fa and less susceptible to Cry1Ab. Here, we analyzed the potential role of ABCC2 in determining the susceptibility of S. frugiperda to Cry1Fa and Cry1Ab, by expressing SfABCC2 or MsABCC2 in Hi5 insect cell line and by the systematic replacements of extracellular loops (ECLs) between these two proteins. Expression of SfABCC2 in Hi5 conferred susceptibility to both Cry1Fa and Cry1Ab, in contrast to the expression of MsABCC2 that mediated low toxicity to Cry1Ab and no toxicity to Cry1Fa in agreement with their larvicidal toxicities. The SfABCC2 and MsABCC2 amino acid sequences showed differential residues among ECL1, ECL2, ECL4 and ECL6 loops, while ECL3 and ECL5 share the same primary sequence. The exchange of ECLs between SfABCC2 and MsABCC2 demonstrated that ECL4 and ECL2 contribute to Cry1Fa toxicity, where ECL4 plays a major role. The medium region (named M2) of ECL4 was identified as the most important region of SfABCC2 involved in Cry1Fa toxicity as shown by point mutations in this region. These findings will be helpful to understand the mechanisms of action of Bt toxins in S. frugiperda.


Assuntos
Toxinas de Bacillus thuringiensis/farmacologia , Resistência a Inseticidas/genética , Mariposas , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Animais , Proteínas de Bactérias/farmacologia , Linhagem Celular , Endotoxinas/farmacologia , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Spodoptera/efeitos dos fármacos , Spodoptera/genética , Spodoptera/metabolismo
5.
Arch Insect Biochem Physiol ; 107(3): e21794, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33948968

RESUMO

Bombyx mori as a representative in Lepidoptera is an important economic insect in agriculture production. Bacillus thuringiensis (Bt) is a bacterial pathogen in silkworm production. Understanding how silkworm respond to Bt-toxin can provide guidance to cultivate resistant silkworm strains. Cry1Ac is one type of Bt-toxin. In current research, Dazao, a susceptible B. mori strain to Bt-toxin, was treated by Cry1Ac toxin and compared its transcriptome with untreated samples. This analysis detected 1234 differentially expressed genes (DEGs). Gene Ontology, KEGG, and UniProt keyword enrichment analysis showed that DEGs include ATP-binding cassette (ABC) transporter, stress response, cuticle, and protein synthesis, and folding process. Five ABC genes were upregulated after Cry1Ac treatment including ABCA2, ABCA3, and ABCC4. They are also known as the transporters of Bt-toxin in lepidopteran insect. Expression of cuticle proteins was significantly increased at 6 h after Cry1Ac treatment. Sex-specific storage-proteins and heat shock protein were also upregulated in Cry1Ac treated samples. Our data provide an expression profile about the response of Cry1Ac toxin in susceptible B. mori strain.


Assuntos
Toxinas de Bacillus thuringiensis/farmacologia , Bombyx/efeitos dos fármacos , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Transcriptoma/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bombyx/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Insetos/metabolismo
6.
Insect Mol Biol ; 30(4): 436-445, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33955085

RESUMO

Alternative splicing is a common feature in eukaryotes that not only increases the transcript diversity, but also has functional consequences. In insects, alternative splicing has been found associated with resistance to pesticides and Bt toxins. Up to date, the alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) has not been studied. To investigate its alternative splicing pattern and relation to Bt resistance, we carried out single-molecule real-time (SMRT) transcript sequencing and Iso-seq analysis on resistant, eCry3.1Ab-selected and susceptible, unselected, western corn rootworm neonate midguts which fed on seedling maize with and without eCry3.1Ab for 12 and 24 h. We present transcriptome-wide alternative splicing patterns of western corn rootworm midgut in response to feeding on eCry3.1Ab-expressing corn using a comprehensive approach that combines both RNA-seq and SMRT transcript sequencing techniques. The results showed genes in western corn rootworm are highly alternatively spliced, which happens on 67.73% of multi-exon genes. One of the alternative splicing events we identified was a novel peritrophic matrix protein with two alternative splicing isoforms. Analysis of differential exon usage between resistant and susceptible colonies showed that in eCry3.1Ab-resistant western corn rootworm, expression of one isoform was significantly higher than in the susceptible colony, while no significant differences between colonies were observed with the other isoform. Our results provide the first survey of alternative splicing in western corn rootworm and suggest that the observed alternatively spliced isoforms of peritrophic matrix protein may be associated with eCry3.1Ab resistance in western corn rootworm.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Toxinas de Bacillus thuringiensis , Besouros , Endotoxinas , Proteínas Hemolisinas , Animais , Toxinas de Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/farmacologia , Besouros/efeitos dos fármacos , Besouros/genética , Endotoxinas/genética , Endotoxinas/farmacologia , Técnicas Genéticas , Genoma de Inseto/efeitos dos fármacos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/genética , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/microbiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA-Seq , Transcriptoma/efeitos dos fármacos , Zea mays/genética
7.
Appl Environ Microbiol ; 87(13): e0046621, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33893113

RESUMO

Deciphering the molecular mechanisms underlying insect resistance to Cry toxins produced by Bacillus thuringiensis (Bt) is pivotal for the sustainable utilization of Bt biopesticides and transgenic Bt crops. Previously, we identified that mitogen-activated protein kinase (MAPK)-mediated reduced expression of the PxABCB1 gene is associated with Bt Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.). However, the underlying transcriptional regulation mechanism remains enigmatic. Here, the PxABCB1 promoter in Cry1Ac-susceptible and Cry1Ac-resistant P. xylostella strains was cloned and analyzed and found to contain a putative Jun binding site (JBS). A dual-luciferase reporter assay and yeast one-hybrid assay demonstrated that the transcription factor PxJun repressed PxABCB1 expression by interacting with this JBS. The expression levels of PxJun were increased in the midguts of all resistant strains compared to the susceptible strain. Silencing of PxJun expression significantly elevated PxABCB1 expression and Cry1Ac susceptibility in the resistant NIL-R strain, and silencing of PxMAP4K4 expression decreased PxJun expression and also increased PxABCB1 expression. These results indicate that MAPK-activated PxJun suppresses PxABCB1 expression to confer Cry1Ac resistance in P. xylostella, deepening our understanding of the transcriptional regulation of midgut Cry receptor genes and the molecular basis of insect resistance to Bt Cry toxins. IMPORTANCE The transcriptional regulation mechanisms underlying reduced expression of Bt toxin receptor genes in Bt-resistant insects remain elusive. This study unveils that a transcription factor PxJun activated by the MAPK signaling pathway represses PxABCB1 expression and confers Cry1Ac resistance in P. xylostella. Our results provide new insights into the transcriptional regulation mechanisms of midgut Cry receptor genes and deepen our understanding of the molecular basis of insect resistance to Bt Cry toxins. To our knowledge, this study identified the first transcription factor that can be involved in the transcriptional regulation mechanisms of midgut Cry receptor genes in Bt-resistant insects.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Mariposas , Proteínas Proto-Oncogênicas c-jun/genética , Animais , Larva/genética , Larva/metabolismo , Mariposas/genética , Mariposas/metabolismo
8.
Sci Rep ; 11(1): 6523, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33753776

RESUMO

Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RNA interference (RNAi) mechanism in insect cells. Despite high commercial interest in insecticidal dsRNA, information on resistance to dsRNA is scarce, particularly for dsRNA products with non-transgenic delivery (ex. foliar/topical application) nearing regulatory review. We report the development of the CEAS 300 population of Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) with > 11,100-fold resistance to a dsRNA targeting the V-ATPase subunit A gene after nine episodes of selection using non-transgenic delivery by foliar coating. Resistance was associated with lack of target gene down-regulation in CEAS 300 larvae and cross-resistance to another dsRNA target (COPI ß; Coatomer subunit beta). In contrast, CEAS 300 larvae showed very low (~ 4-fold) reduced susceptibility to the Cry3Aa insecticidal protein from Bacillus thuringiensis. Resistance to dsRNA in CEAS 300 is transmitted as an autosomal recessive trait and is polygenic. These data represent the first documented case of resistance in an insect pest with high pesticide resistance potential using dsRNA delivered through non-transgenic techniques. Information on the genetics of resistance and availability of dsRNA-resistant L. decemlineata guide the design of resistance management tools and allow research to identify resistance alleles and estimate resistance risks.


Assuntos
Besouros/efeitos dos fármacos , Resistência a Medicamentos/genética , Inseticidas/farmacologia , RNA de Cadeia Dupla/farmacologia , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/farmacologia , Besouros/genética , Besouros/patogenicidade , Colorado , Endotoxinas/genética , Endotoxinas/farmacologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Proteínas de Insetos/genética , Larva/genética , Larva/crescimento & desenvolvimento , Interferência de RNA , RNA de Cadeia Dupla/genética , Solanum tuberosum/crescimento & desenvolvimento , Solanum tuberosum/parasitologia
9.
PLoS One ; 16(2): e0246696, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33591990

RESUMO

Maize with enhanced ß-carotene production was engineered to counteract pervasive vitamin A deficiency in developing countries. Second-generation biofortified crops are being developed with additional traits that confer pest resistance. These include crops that can produce Bacillus thuringiensis Berliner (Bt) insecticidal proteins. Currently, it is unknown whether ß-carotene can confer fitness benefits through to insect pests, specifically through altering Ostrinia nubilalis foraging behaviour or development in the presence of Bt insecticidal toxin. Therefore the effects of dietary ß-carotene plus Bt insecticidal protein on feeding behaviour, mortality, and physiology in early and late instars of O. nubilalis larvae were investigated. The results of two-choice experiments showed that irrespective of ß-carotene presence, at day five 68%-90% of neonates and 69%-77% of fifth-instar larvae avoided diets with Cry1A protein. Over 65% of neonate larvae preferred to feed on diets with ß-carotene alone compared to 39% of fifth-instar larvae. Higher mortality (65%-97%) in neonates fed diets supplemented with ß-carotene alone and in combination with Bt protein was found, whereas <36% mortality was observed when fed diets without supplemented ß-carotene or Bt protein. Diets with both ß-carotene and Bt protein extended 25 days the larval developmental duration from neonate to fifth instar (compared to Bt diets) but did not impair larval or pupal weight. Juvenile hormone and 20-hydroxyecdysone regulate insect development and their levels were at least 3-fold higher in larvae fed diets with ß-carotene for 3 days. Overall, these results suggest that the effects of ß-carotene and Bt protein on O. nubilalis is dependent on larval developmental stage. This study is one of the first that provides insight on how the interaction of novel traits may modulate crop susceptibility to insect pests. This understanding will in turn inform the development of crop protection strategies with greater efficacy.


Assuntos
Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Mariposas/fisiologia , beta Caroteno/farmacologia , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Endotoxinas/metabolismo , Comportamento Alimentar/fisiologia , Proteínas Hemolisinas/metabolismo , Resistência a Inseticidas/efeitos dos fármacos , Inseticidas/farmacologia , Larva/fisiologia , Lepidópteros/metabolismo , Mariposas/metabolismo , Controle Biológico de Vetores/métodos , Plantas Geneticamente Modificadas/metabolismo , Pupa/metabolismo , Zea mays/genética , Zea mays/metabolismo , beta Caroteno/metabolismo
10.
Pest Manag Sci ; 77(5): 2385-2394, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33415809

RESUMO

BACKGROUND: Management of the corn pest, western corn rootworm (WCR), Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), relies heavily on the planting of transgenic corn expressing toxins produced by the bacterium Bacillus thuringiensis (Bt). This has resulted in the evolution of resistance to all of the four commercially available Bt toxins targeting coleopteran insects. In this study, we evaluated the susceptibility of a Cry34/35Ab1-resistant WCR colony in seedling and diet toxicity assays after removal from selection for six and nine generations. In addition, female fecundity, egg fertility, adult lifespan, larval development, and adult emergence were evaluated in two Cry34/35Ab1-resistant and two susceptible WCR colonies to assess fitness costs. RESULTS: Susceptibility to Cry34/35Ab1 was restored in a colony removed from selection after six and nine generations based on diet toxicity assays and comparisons of relative survival, head capsule width, and dry weight in plant assays. Thus, pronounced fitness costs associated with resistance to Cry34/35Ab1 were documented by susceptibility being restored within six generations. In separate studies evaluating specific fitness costs, larval fitness when reared on isoline corn did not differ between resistant and susceptible colonies. However, beetles from susceptible colonies lived longer than resistant beetles which resulted in females from susceptible colonies producing significantly more eggs than resistant colonies, with no differences in egg fertility. CONCLUSIONS: The presence of a fitness cost that may contribute to the restoration of susceptibility to Bt has not been documented in other Cry3-resistant WCR populations and could have significant impact on the deployment of resistance management practices. Published 2021. This article is a U.S. Government work and is in the public domain in the USA.


Assuntos
Bacillus thuringiensis , Besouros , Animais , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Besouros/genética , Endotoxinas/genética , Endotoxinas/farmacologia , Feminino , Resistência a Inseticidas/genética , Larva/genética , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Zea mays/genética
11.
Pest Manag Sci ; 77(2): 949-955, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32985759

RESUMO

BACKGROUND: Transgenic crops producing Cry and Vip3 insecticidal proteins from the bacterium Bacillus thuringiensis provide effective control of the fall armyworm, Spodoptera frugiperda J. E. Smith. However, cases of practical S. frugiperda resistance to transgenic corn producing Cry1F, Cry1Ab and Cry1A.105 proteins have been reported in the Western hemisphere. Importantly, S. frugiperda resistance to Cry1F corn in Puerto Rico was previously associated with lower susceptibility to synthetic pesticides. When characterized, resistance to transgenic corn in S. frugiperda involved alterations in an ABC transporter subfamily C2 (SfABCC2) gene. The main goal of this work was to test the role of mutations in SfABCC2 that result in resistance to Cry1F in susceptibility to synthetic and semisynthetic small molecule pesticides. RESULTS: Marginal but significantly reduced susceptibility to bifenthrin and increased susceptibility to spinetoram was detected in a Cry1F-resitant S. frugiperda strain from Puerto Rico carrying a frameshift mutation in the SfABCC2 gene. Gene editing by CRISPR/Cas9 created a SfABCC2 knockout in a laboratory reference S. frugiperda strain. When compared to the parental reference, the knockout strain displayed 25-fold resistance to Cry1F but no alteration in susceptibility to small molecule pesticides. CONCLUSION: These results support that resistance to Cry1F due to mutations in the SfABCC2 gene do not affect susceptibility to the tested small molecule pesticides.


Assuntos
Bacillus thuringiensis , Praguicidas , Transportadores de Cassetes de Ligação de ATP , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/genética , Endotoxinas/farmacologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Mutação , Plantas Geneticamente Modificadas , Spodoptera/genética , Zea mays/genética
12.
Curr Pharm Biotechnol ; 22(1): 176-181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-31893989

RESUMO

OBJECTIVE: To detect the pyrogen in CAR-T cells product employing the HL60-IL-6 assay. METHODS: The HL60 cells were incubated with CAR-T cells injection or endotoxin standard for 48 hours. After then, the secreted cytokine interleukin-6 (IL-6) from HL60 cells was determined by ELISA. According to the four-parameter logistic curve fitted by Optical Density (OD) value corresponding to IL-6 and endotoxin standard concentration, the endotoxin equivalents of pyrogen content in the CAR-T cells products can be measured. Then, the method was validated, including the limit of detection (LOD), limit of quantitation, the recovery rate and the comparison of the determined results by HL60-IL-6 assay with that by the conventional pyrogen test, the Rabbit Pyrogen Test (RPT). RESULTS: The HL60-IL-6 assay applied to pyrogen test in CAR-T cells products has been established and validated, The LOD was 0.03 EU/mL while the LOQ was 0.07 EU/mL, the recovery rates were 121.4% and 94.5% respectively. The results determined by HL60-IL-6 assay were consistent with that by the RPT. CONCLUSION: The HL60-IL-6 assay can be employed in CAR-T cell products in vitro pyrogen test.


Assuntos
Interleucina-6/metabolismo , Pirogênios/análise , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Endotoxinas/farmacologia , Ensaio de Imunoadsorção Enzimática , Células HL-60 , Humanos , Limite de Detecção , Pirogênios/farmacologia , Coelhos , Linfócitos T/imunologia , Linfócitos T/metabolismo
13.
Insect Sci ; 28(3): 574-589, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32478944

RESUMO

The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a major target pest of Bt crops (e.g., corn, cotton, and soybean) in North and South America. This pest has recently invaded Africa and Asia including China and the invasion has placed a great threat to the food security in many countries of these two continents. Due to the extensive use of Bt crops, practical resistance of S. frugiperda to Cry1F corn (TC 1507) with field control problems has widely occurred in Puerto Rico, Brazil, Argentina, and the mainland United States. Analyzing data generated from decade-long studies showed that several factors might have contributed to the wide development of the resistance. These factors include (1) limited modes of action of Bt proteins used in Bt crops; (2) cross-resistance among Cry1 proteins; (3) use of nonhigh dose Bt crop traits; (4) that the resistance is complete on Bt corn plants; (5) abundant in initial Cry1F resistance alleles; and (6) lack of fitness costs/recessive fitness costs of the resistance. The long-term use of Bt crop technology in the Americas suggests that Bt corn can be an effective tool for controlling S. frugiperda in China. IRM programs for Bt corn in China should be as simple as possible to be easily adopted by small-scale growers. The following aspects may be considered in its Bt corn IRM programs: (1) use of only "high dose" traits for both S. frugiperda and stalk borers; (2) developing and implementing a combined resistance monitoring program; (3) use "gene pyramiding" as a primary IRM strategy; and (4) if possible, Bt corn may not be planted in the areas where S. frugiperda overwinters. Lessons and experience gained from the global long-term use of Bt crops should have values in improving IRM programs in the Americas, as well as for a sustainable use of Bt corn technology in China.


Assuntos
Toxinas de Bacillus thuringiensis , Endotoxinas , Proteínas Hemolisinas , Resistência a Inseticidas , Controle de Pragas/métodos , Spodoptera/efeitos dos fármacos , Zea mays/genética , América , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/farmacologia , China , Produtos Agrícolas/genética , Endotoxinas/genética , Endotoxinas/farmacologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Espécies Introduzidas , Plantas Geneticamente Modificadas/genética , Spodoptera/fisiologia
14.
Insect Sci ; 28(3): 627-638, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32558234

RESUMO

The fall armyworm (FAW), Spodoptera frugiperda, is a major pest native to the Americas that has recently invaded the Old World. Point mutations in the target-site proteins acetylcholinesterase-1 (ace-1), voltage-gated sodium channel (VGSC) and ryanodine receptor (RyR) have been identified in S. frugiperda as major resistance mechanisms to organophosphate, pyrethroid and diamide insecticides respectively. Mutations in the adenosine triphosphate-binding cassette transporter C2 gene (ABCC2) have also been identified to confer resistance to Cry1F protein. In this study, we applied a whole-genome sequencing (WGS) approach to identify point mutations in the target-site genes in 150 FAW individuals collected from China, Malawi, Uganda and Brazil. This approach revealed three amino acid substitutions (A201S, G227A and F290V) of S. frugiperda ace-1, which are known to be associated with organophosphate resistance. The Brazilian population had all three ace-1 point mutations and the 227A allele (mean frequency = 0.54) was the most common. Populations from China, Malawi and Uganda harbored two of the three ace-1 point mutations (A201S and F290V) with the 290V allele (0.47-0.58) as the dominant allele. Point mutations in VGSC (T929I, L932F and L1014F) and RyR (I4790M and G4946E) were not detected in any of the 150 individuals. A novel 12-bp insertion mutation in exon 15 of the ABCC2 gene was identified in some of the Brazilian individuals but absent in the invasive populations. Our results not only demonstrate robustness of the WGS-based genomic approach for detection of resistance mutations, but also provide insights for improvement of resistance management tactics in S. frugiperda.


Assuntos
Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Spodoptera , Acetilcolinesterase/genética , Animais , Diamida/farmacologia , Genes de Insetos , Genoma de Inseto , Organofosfatos/farmacologia , Mutação Puntual/genética , Mutação Puntual/fisiologia , Piretrinas/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Spodoptera/efeitos dos fármacos , Spodoptera/genética , Canais de Sódio Disparados por Voltagem/genética , Sequenciamento Completo do Genoma
15.
Insect Sci ; 28(4): 1121-1138, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32458593

RESUMO

The use of genetically modified varieties tolerant to herbicides (HT varieties) and resistant to insects (Bt varieties) in combination with application of a broad-spectrum herbicide such as glyphosate could be an effective option for the simultaneous control of weeds and pests in maize. Nevertheless, the possible impact of these tools on nontarget arthropods still needs to be evaluated. In a field study in central Spain, potential changes in populations of canopy-dwelling arthropods in Bt maize under different weed management options, including glyphosate application, were investigated. Canopy-dwelling arthropods were sampled by visual inspection and yellow sticky traps. The Bt variety had no effect on any group of studied arthropods, except for the expected case of corn borers-the target pests of Bt maize. Regarding the effects of herbicide regimes, the only observed difference was a lower abundance of Cicadellidae and Mymaridae on yellow sticky traps in plots not treated with pre-emergence herbicides. This effect was especially pronounced in a treatment involving two glyphosate applications. The decrease in Cicadellidae and Mymaridae populations was associated with a higher density of weeds in plots, which may have hindered colonization of the crop by leafhoppers. These differences, however, were only significant in the last year of the study. The low likelihood of the use of glyphosate- and herbicide-tolerant varieties for weed control triggering important effects on the nontarget arthropod fauna of the maize canopy is discussed.


Assuntos
Artrópodes/efeitos dos fármacos , Toxinas de Bacillus thuringiensis , Endotoxinas , Proteínas Hemolisinas , Herbicidas/farmacologia , Zea mays , Animais , Toxinas de Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/farmacologia , Biodiversidade , Produtos Agrícolas/genética , Endotoxinas/genética , Endotoxinas/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Hemípteros/efeitos dos fármacos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Himenópteros/efeitos dos fármacos , Controle de Pragas , Plantas Daninhas/efeitos dos fármacos , Plantas Geneticamente Modificadas , Dinâmica Populacional , Controle de Plantas Daninhas , Zea mays/genética
16.
Insect Sci ; 28(4): 1147-1158, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32662592

RESUMO

A seed blend refuge has been implemented in the U.S. Corn Belt for Bt maize resistance management. The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a target pest of Bt maize in the Americas. The larvae of this pest are mobile, which may affect the efficacy of seed blend refuges. In this study, field and greenhouse trials were conducted to determine the performance of Bt-susceptible (aabb) and -heterozygous dual-gene-resistant (AaBb) genotypes of S. frugiperda in seed blends of non-Bt and pyramided Bt maize. Three field trials evaluated larval survival, larval growth, and plant injury with aabb in seed blends of Bt maize expressing Cry1A.105/Cry2Ab2/Vip3A with 0-30% non-Bt seeds. Greenhouse tests investigated the performance of aabb and AaBb in seed blends of Cry1A.105/Cry2Ab2 with 0-30% non-Bt seeds. In pure non-Bt maize plots, after 9-13 d of neonates being released on the plants, 0.39 and 0.65 larvae/plant survived with leaf injury ratings of 4.7 and 5.9 (Davis's 1-9 scale) in the field and greenhouse, respectively. In contrast, live larvae and plant injury were virtually not observed on Bt plants across all planting patterns. Larval occurrence and plant injury by aabb on non-Bt plants were similar between seed blends and pure non-Bt plantings, suggesting that the blended refuges could provide an equivalent susceptible population as structured refuge under the test conditions. In the greenhouse, the two insect genotypes in seed blends performed similarly, indicating that the seed blends did not provide more favorable conditions for AaBb over aabb. The information generated from this study should be useful in managing S. frugiperda and evaluating if send blends could be suitable refuge options for Bt resistance management in the regions where the insect is a primary target pest.


Assuntos
Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Controle Biológico de Vetores , Spodoptera/crescimento & desenvolvimento , Zea mays/genética , Animais , Toxinas de Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Produtos Agrícolas/genética , Endotoxinas/genética , Genótipo , Proteínas Hemolisinas/genética , Resistência a Inseticidas/genética , Larva/crescimento & desenvolvimento , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Spodoptera/efeitos dos fármacos , Spodoptera/genética
17.
J Invertebr Pathol ; 178: 107507, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33249063

RESUMO

Transgenic plants expressing insecticidal proteins from the Bacillus thuringiensis (Bt) have provided an effective way to control target pests. However, the toxicity of Bt proteins against yellow peach moth (YPM), Conogethes punctiferalis (Guenée), one of the most serious maize pests in China, has not received much study. Therefore, we performed diet-overlay bioassays to evaluate the insecticidal activities of Cry1Ab, Cry1Ac, Cry1Fa, Cry1Ah, Cry1Ie, Cry2Aa, and Vip3Aa19, as well as the interaction between Cry1-Class, Cry2Aa, and Vip3Aa19 against YPM. Results showed that the LC50 values ranged from 1.08 to 178.12 ng/cm2 (protein/diet). Among these proteins, Cry1Ab and Cry1Ac had lower LC50 values and LC90 values. In YPM bioassays, the combinations of Cry2Aa with Cry1Ac, Cry1Ie, and Cry1Ab showed antagonism while a mixture of Cry2Aa with Cry1Fa and Cry1Ah exhibited synergism. When Vip3Aa19 was combined with Cry proteins, all combinations interacted positively, with variation in synergistic factors (SF). Three ratios 1:1, 1:2, and 2:1 of Cry1Ah and Vip3Aa19 protein combination showed SF values of 5.20, 5.63, and 8.98, respectively. These findings can be applied in the establishment of new pyramided transgenic crops with suitable candidates as well as in resistance management strategies.


Assuntos
Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Mariposas , Animais , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/farmacologia , Bioensaio , Produtos Agrícolas , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Mariposas/microbiologia , Controle de Pragas/métodos , Plantas Geneticamente Modificadas , Zea mays
18.
Pest Manag Sci ; 77(3): 1169-1177, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33236463

RESUMO

BACKGROUND: Transgenic crops producing insecticidal proteins derived from Bacillus thuringiensis (Bt) are used globally to kill key insect pests and provide numerous benefits, including improved pest management, increased profits, reduced insecticide use, and increased biological control. Unfortunately, such benefits are rapidly being lost by the evolution of Bt resistance by pests. RESULTS: The main strategy to delay resistance relies on the use of non-Bt refuge plants to produce sufficient susceptible insects that mate with rare resistant insects emerging from Bt crops, essentially diluting and/or removing resistance alleles from pest populations. A key assumption for the success of this refuge strategy is that inheritance of resistance is recessive. In China, dominant resistance to Cry1Ac Bt cotton by the cotton bollworm Helicoverpa armigera is increasing and is associated with a mutation in the tetraspanin HaTSPAN1 gene, conferring more than 125-fold resistance. Here, we used amplicon sequencing to test the hypotheses that the HaTSPAN1 mutation either arose from a single event and spread or that the mutation evolved independently several times throughout northern China. From three laboratory strains and 28 field populations sampled from northern China, we identified six resistant and 50 susceptible haplotypes. Phylogenetic analysis indicates that the HaTSPAN1 mutation arose from at least four independent origins and spread to their current distributions. CONCLUSION: The results provide valuable information about the evolutionary origins of dominant resistance to Cry1Ac Bt cotton in northern China and offer rationale for the rapid increase in field-evolved resistance in these areas, where the implementation of additional practical resistance management is needed. © 2020 Society of Chemical Industry.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , China , Endotoxinas/genética , Endotoxinas/farmacologia , Gossypium/genética , Proteínas Hemolisinas/genética , Resistência a Inseticidas/genética , Mariposas/genética , Filogenia , Plantas Geneticamente Modificadas , Mutação Puntual , Tetraspaninas
19.
Pest Manag Sci ; 77(4): 1963-1970, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33314557

RESUMO

BACKGROUND: Pyramided Bacillus thuringiensis (Bt) crops could delay insect resistance development by expressing multiple distinct Bt proteins to manage the same insect pest. The efficacy of pyramiding strategy for resistance management could be jeopardized by positive cross-resistance, which is defined as insects showing resistance to one Bt protein also exhibiting resistance to other Bt proteins. The fall armyworm, Spodoptera frugiperda, is a destructive agricultural pest and target of Vip3Aa. In this study, we evaluate the cross-resistance of Vip3Aa resistance in S. frugiperda to Bt cotton and corn plants, as well as purified Bt proteins. RESULTS: Diet bioassay showed that Vip3Aa-resistant (RR), -heterozygous (RS), and -susceptible (SS) insects of S. frugiperda performed similarly against Cry2Ab2 purified protein. The data also indicated that genotypes RR and RS were more susceptible to Cry1F and Cry2Ae purified proteins relative to SS. The diet bioassays suggested that resistance to Vip3Aa does not confer any positive cross-resistance to Cry1F, Cry2Ae or Cry2Ab2 in S. frugiperda. The plant bioassay indicated that the S. frugiperda resistance to Vip3Aa conferred no cross-resistance to corn and no cross-crop resistance to cotton plants expressing Cry1 and/or Cry2 proteins. CONCLUSION: These findings indicate that a lack of positive cross-resistance to Cry1 and Cry2 proteins favors pyramiding strategy for managing S. frugiperda resistance to Vip3Aa protein. © 2020 Society of Chemical Industry.


Assuntos
Bacillus thuringiensis , Proteínas de Bactérias , Animais , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Endotoxinas/farmacologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Larva/genética , Plantas Geneticamente Modificadas/genética , Spodoptera/genética , Zea mays/genética
20.
Pest Manag Sci ; 77(4): 2142-2150, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33336541

RESUMO

BACKGROUND: Extensive planting of transgenetic Bacillus thuringiensis crops has driven the evolution of pest resistance to Cry1Ac. Adjustment of cropping structures has promoted further outbreak of Helicoverpa armigera in China. To control this pest, a combination of pyramiding RNA interference (RNAi) and Cry2Ab is considered a promising strategy for countering cross-resistance and enhancing the toxicity of Cry2Ab to cotton bollworm. We explored the possibility of using calcineurin (CAN) as a target RNAi gene, because it is involved in cotton bollworm responses to the toxicity of Cry2Ab. RESULTS: Cry2Ab treatment led to a significant increase in HaCAN mRNA level and HaCAN activity. Suppression of HaCAN activity due to RNAi-mediated knockdown of HaCAN increased the susceptibility of midgut cells to Cry2Ab. The increase in HaCAN activity shown by heterologous expression of HaCAN reduced the cytotoxicity of Cry2Ab to Sf9 cells. Moreover, ingestion of HaCAN-specific inhibitor FK506 increased the toxicity of Cry2Ab in larvae. Interestingly, HaCAN does not function as a Cry2Ab direct binding protein that participates in Cry2Ab toxicity. CONCLUSIONS: The results in this study provide evidence that suppression of HaCAN not only affected the development of the cotton bollworm, but also enhanced the toxicity of Cry2Ab to the pest. HaCAN is therefore an important candidate gene in cotton bollworm that can be targeted for pest control when the pest infests RNAi+Cry2Ab crops. Meanwhile, the mechanism of action of HaCAN in Cry2Ab toxicity suggested that protein dephosphorylation was involved. © 2020 Society of Chemical Industry.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Calcineurina/genética , China , Endotoxinas/genética , Endotoxinas/farmacologia , Gossypium , Proteínas Hemolisinas/genética , Resistência a Inseticidas , Larva/genética , Mariposas/genética , Plantas Geneticamente Modificadas
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