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1.
Annu Rev Entomol ; 67: 387-406, 2022 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-34995087

RESUMO

It is increasingly clear that pest species vary widely in their propensities to develop insecticide resistance. This review uses a comparative approach to analyze the key pest management practices and ecological and biochemical or genetic characteristics of the target that contribute to this variation. We focus on six heliothine species, three of which, Helicoverpa armigera, Heliothis virescens, and Helicoverpa zea, have developed resistances to many pesticide classes. The three others, Helicoverpa punctigera, Helicoverpa assulta, and Helicoverpa gelotopoeon, also significant pests, have developed resistance to very few pesticide classes. We find that host range and movement between alternate hosts are key ecological traits that influence effective selection intensities for resistance. Operational issues are also critical; area-wide, cross-pesticide management practices that account for these ecological factors are key to reducing selection intensity. Without such management, treatment using broad-spectrum chemicals serves to multiply the effects of host plant preference, preadaptive detoxification ability, and high genetic diversity to create a pesticide treadmill for the three high-propensity species.Without rigorous ongoing management, such a treadmill could still develop for newer, more selective chemistries and insecticidal transgenic crops.

2.
Mol Ecol Resour ; 2022 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-35007400

RESUMO

The cotton bollworm Helicoverpa armigera is a cosmopolitan pest and its diverse habitats plausibly contribute to the formation of diverse lineages. Despite the significant threat it poses to economic crops worldwide, its evolutionary history and genetic basis of local adaptation are poorly understood. In this study, we de novo assembled a high-quality chromosome-level reference genome of H. a. armigera (contig N50 = 7.34 Mb), with 99.13% of the HaSCD2 assembly assigned into 31 chromosomes (Z-chromosome + 30 autosomes). We constructed an ultra-dense variation map across 14 cotton bollworm populations and identified a novel lineage in northwestern China. Historical inference showed that effective population size changes coincided with global temperature fluctuation. We identified nine differentiated genes in the three H. armigera lineages (H. a. armigera, H. a. conferta, and the new northwestern Chinese lineage), of which per and clk genes are involved in circadian rhythm. Selective sweep analyses identified a series of GO categories related to climate adaptation, feeding behavior and insecticide tolerance. Our findings reveal fundamental knowledge of the local adaptation of different cotton bollworm lineages and will guide the formulation of cotton bollworm management measures at different scales.

3.
Insect Biochem Mol Biol ; 140: 103696, 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34800643

RESUMO

Insect CYP2 and mitochondrial clan P450s are relatively conserved genes encoding enzymes generally thought to be involved in biosynthesis or metabolism of endobiotics. However, emerging evidence argues they have potential roles in chemical defense as well, but their actual detoxification functions remain largely unknown. Here, we focused on the full complement of 8 CYP2 and 10 mitochondrial P450s in the generalist herbivore, Helicoverpa armigera. Their varied spatiotemporal expression profiles were analyzed and reflected their specific functions. For functional study of the mitochondrial clan P450s, the redox partners, adrenodoxin reductase (AdR) and adrenodoxin (Adx), were identified from genomes of eight insects and an efficient in vitro electron transfer system of mitochondrial P450 was established by co-expression with Adx and AdR of H. armigera. All CYP2 clan P450s and 8 mitochondrial P450s were successfully expressed in Sf9 cells and compared functionally. In vitro metabolism assays showed that two CYP2 clan P450s (CYP305B1 and CYP18A1) and CYP333B3 (mito clan) could epoxidize aldrin to dieldrin, while CYP305B1 and CYP339A1 (mito clan) have limited but significant hydroxylation capacities to esfenvalerate. CYP303A1 of the CYP2 clan exhibits high metabolic efficiency to 2-tridecanone. Screening the xenobiotic metabolism competence of CYP2 and mitochondrial clan P450s not only provides new insights on insect chemical defense but also can give indications on their physiological functions in H. armigera and other insects.

4.
Toxins (Basel) ; 13(9)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34564662

RESUMO

Transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal proteins have been extensively planted for insect pest control, but the evolution of Bt resistance in target pests threatens the sustainability of this approach. Mutations of cadherin in the midgut brush border membrane was associated with Cry1Ac resistance in several lepidoptera species, including the Asian corn borer, Ostrinia furnacalis, a major pest of maize in Asian-Western Pacific countries. However, the causality of O. furnacalis cadherin (OfCad) with Cry1Ac resistance remains to be clarified. In this study, in vitro and in vivo approaches were employed to examine the involvement of OfCad in mediating Cry1Ac toxicity. Sf9 cells transfected with OfCad showed significant immunofluorescent binding with Cry1Ac toxin and exhibited a concentration-dependent mortality effect when exposed to Cry1Ac. The OfCad knockout strain OfCad-KO, bearing homozygous 15.4 kb deletion of the OfCad gene generated by CRISPR/Cas9 mutagenesis, exhibited moderate-level resistance to Cry1Ac (14-fold) and low-level resistance to Cry1Aa (4.6-fold), but no significant changes in susceptibility to Cry1Ab and Cry1Fa, compared with the original NJ-S strain. The Cry1Ac resistance phenotype was inherited as autosomal, recessive mode, and significantly linked with the OfCad knockout in the OfCad-KO strain. These results demonstrate that the OfCad protein is a functional receptor for Cry1Ac, and disruption of OfCad confers a moderate Cry1Ac resistance in O. furnacalis. This study provides new insights into the mode of action of the Cry1Ac toxin and useful information for designing resistance monitoring and management strategies for O. furnacalis.

5.
J Binocul Vis Ocul Motil ; 71(4): 167-174, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34449280

RESUMO

PURPOSE: This study reports clinical opinions and preferences on the non-surgical management of intermittent exotropia (IXT) among practitioners in China. METHODS: An online survey was developed and distributed through professional bodies. The study was conducted from July 25th to August 3rd, 2019. A total of 300 ophthalmologists and 188 optometrists responded. RESULTS: Of 488 participants, 257 (53%) considered fusion defects as the main cause of IXT, and 299 (61%) took IXT as a progressive disorder. Two hundred and seventy-one (56%) participants considered orthoptic exercises as the most effective non-surgical intervention for IXT. Likewise, 245 (50%) participants reported that orthoptic exercises were their most frequent non-surgical option, followed by observation (178, 37%). There are discrepancies between ophthalmologists and optometrists. A greater proportion of ophthalmologists (201, 67%) shared the view that IXT worsens over time (98, 52%) (p = .001). Additionally, ophthalmologists (121, 40%) tended to prefer observation compared with optometrists (57, 30%) (p = .021). CONCLUSIONS: This study shows that there is no general consensus on the non-surgical management of IXT in China. Given the lack of robust evidence, the findings from this study highlight the need for future randomized clinical trials to validate the effectiveness of non-surgical interventions, orthoptic exercises in particular, and to establish treatment guidelines accordingly.


Assuntos
Exotropia , Oftalmologistas , China , Doença Crônica , Exotropia/terapia , Humanos , Inquéritos e Questionários
6.
PLoS Genet ; 17(7): e1009680, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34252082

RESUMO

The evolution of insecticide resistance represents a global constraint to agricultural production. Because of the extreme genetic diversity found in insects and the large numbers of genes involved in insecticide detoxification, better tools are needed to quickly identify and validate the involvement of putative resistance genes for improved monitoring, management, and countering of field-evolved insecticide resistance. The avermectins, emamectin benzoate (EB) and abamectin are relatively new pesticides with reduced environmental risk that target a wide number of insect pests, including the beet armyworm, Spodoptera exigua, an important global pest of many crops. Unfortunately, field resistance to avermectins recently evolved in the beet armyworm, threatening the sustainable use of this class of insecticides. Here, we report a high-quality chromosome-level assembly of the beet armyworm genome and use bulked segregant analysis (BSA) to identify the locus of avermectin resistance, which mapped on 15-16 Mbp of chromosome 17. Knockout of the CYP9A186 gene that maps within this region by CRISPR/Cas9 gene editing fully restored EB susceptibility, implicating this gene in avermectin resistance. Heterologous expression and in vitro functional assays further confirm that a natural substitution (F116V) found in the substrate recognition site 1 (SRS1) of the CYP9A186 protein results in enhanced metabolism of EB and abamectin. Hence, the combined approach of coupling gene editing with BSA allows for the rapid identification of metabolic resistance genes responsible for insecticide resistance, which is critical for effective monitoring and adaptive management of insecticide resistance.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Resistência a Inseticidas/genética , Spodoptera/genética , Animais , Mapeamento Cromossômico/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Genoma/genética , Inseticidas/farmacologia , Ivermectina/análogos & derivados , Ivermectina/farmacologia , Larva/genética , Spodoptera/metabolismo
7.
Insect Biochem Mol Biol ; 135: 103597, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34089822

RESUMO

Lepidopteran P450s of the CYP6B and CYP9A subfamilies are thought to play important roles in host plant adaptation and insecticide resistance. An increasing number of paralogs and orthologs with high levels of sequence identity have been found in these subfamilies by mining recent genome projects. However, the biochemical function of most of them remains unknown. A better understanding of the evolution of P450 genes and of the catalytic competence of the enzymes they encode is needed to facilitate studies of host plant use and insecticide resistance. Here, we focused on the full complement of CYP6B (4 genes) and CYP9A (7 genes) in the generalist herbivore, Helicoverpa armigera. These P450s were heterologously expressed in Sf9 cells and compared functionally. In vitro assays showed that all CYP6B and CYP9A P450s can metabolize esfenvalerate efficiently, except for the evolutionarily divergent CYP6B43. A new 2'-hydroxy-metabolite of esfenvalerate was identified and found to be the main metabolite produced by CYP9A12. All tested P450s showed only low induction responses to esfenvalerate. To put these results from H. armigera P450s in perspective, 158 complete CYP6B and 100 complete CYP9A genes from 34 ditrysian species were manually curated. The CYP9A subfamily was more widespread than the CYP6B subfamily and the latter showed dramatic gains and losses, with ten species lacking CYP6B genes. Two adjacent CYP6B loci were found on chromosome 21, with different fates during the evolution of Lepidoptera. The diversity and functional redundancy of CYP6B and CYP9A genes challenge resistance management and pest control strategies as many P450s are available to insects to cope with chemical stresses they encounter.


Assuntos
Sistema Enzimático do Citocromo P-450 , Resistência a Inseticidas/genética , Mariposas , Piretrinas , Animais , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Genes de Insetos , Genoma de Inseto , Controle de Insetos/métodos , Proteínas de Insetos/efeitos dos fármacos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/metabolismo , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/metabolismo , Filogenia , Piretrinas/metabolismo , Piretrinas/farmacologia
8.
Pest Manag Sci ; 77(11): 4874-4883, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34176224

RESUMO

BACKGROUND: Although decoding the molecular mechanisms underlying insecticide resistance has often proven difficult, recent progress has revealed that specific mutations in the ryanodine receptor (RyR) of the diamondback moth, Plutella xylostella, can confer resistance to diamide insecticides. The extent to which specific RyR mutations contribute to the diamide resistance phenotype, the associated genetic traits and fitness costs remain limited. RESULTS: Three field-evolved PxRyR mutations (G4946E, I4790 M, and I4790 K) were respectively introgressed into a common susceptible background strain (IPP-S) of P. xylostella with marker-assisted backcrossing. The mutations alone can result in moderate to high levels of resistance to five commercial diamides (flubendiamide, chlorantraniliprole, cyantraniliprole, tetraniliprole, and cyclaniliprole), and the resistance intensity mediated by the three mutations was hierarchical in order of I4790 K (1199- to >2778-fold) > G4946E (39- to 739-fold) > I4790 M (16- to 57-fold). Flubendiamide resistance was autosomal and incompletely recessive, and was significantly linked with the introgressed mutations in the three constructed strains. In addition, the resistance levels to flubendiamide of hybrid progeny from any two resistant strains fell in between the status of their parents. Furthermore, by comparing the net replacement rate, the fitness of 4946E, 4790 M and 4790 K strains were 0.77, 0.93 and 0.92 relative to the IPP-S strain, respectively. CONCLUSION: Three independent PxRyR mutations confer varying degrees of resistance to diamides in P. xylostella. Among the three mutations, I4790 K confers highest levels of resistance (> 1000-fold) to all five commercial diamides. The findings can guide resistance management practices for diamides in P. xylostella and other arthropods.


Assuntos
Inseticidas , Mariposas , Animais , Diamida/farmacologia , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Mariposas/genética , Mutação Puntual , Pirazóis , Piridinas , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Tetrazóis , ortoaminobenzoatos/farmacologia
9.
Sci Rep ; 11(1): 10377, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001946

RESUMO

Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefits. Understanding the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. Previous work shows that resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2 in lab- and field-selected populations of the pink bollworm (Pectinophora gossypiella), one of the world's most destructive pests of cotton. Here we used CRISPR/Cas9 gene editing to test the hypothesis that mutations in the pink bollworm gene encoding ABCA2 (PgABCA2) can cause resistance to Cry2Ab. Consistent with this hypothesis, introduction of disruptive mutations in PgABCA2 in a susceptible strain of pink bollworm increased the frequency of resistance to Cry2Ab and facilitated creation of a Cry2Ab-resistant strain. All Cry2Ab-resistant individuals tested in this study had disruptive mutations in PgABCA2. Overall, we found 17 different disruptive mutations in PgABCA2 gDNA and 26 in PgABCA2 cDNA, including novel mutations corresponding precisely to single-guide (sgRNA) sites used for CRISPR/Cas9. Together with previous results, these findings provide the first case of practical resistance to Cry2Ab where evidence identifies a specific gene in which disruptive mutations can cause resistance and are associated with resistance in field-selected populations.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Toxinas de Bacillus thuringiensis/genética , Gossypium/parasitologia , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Animais , Bacillus thuringiensis/genética , Sistemas CRISPR-Cas/genética , Humanos , Larva/efeitos dos fármacos , Larva/genética , Larva/patogenicidade , Lepidópteros/efeitos dos fármacos , Lepidópteros/genética , Lepidópteros/patogenicidade , Mariposas/genética , Mariposas/patogenicidade , Mutação/genética
10.
Front Microbiol ; 12: 670402, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054780

RESUMO

Evolution of resistance by pests has diminished the efficacy of transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt). In China, where transgenic cotton producing Bt toxin Cry1Ac has been planted since 1997, field control failures have not been reported but the frequency of resistance to Cry1Ac has increased in the cotton bollworm, Helicoverpa armigera. This provides incentive to switch to multi-toxin Bt cotton, which is grown in many other countries. Previous work created four laboratory strains of H. armigera with >100-fold resistance to Cry1Ac, with the genetic basis of resistance known in all but the LF256 strain. Here, we analyzed the genetic basis of resistance in Cry1Ac in LF256 and evaluated cross-resistance of all four strains to three toxins produced by widely planted multi-toxin Bt cotton: Cry1Fa, Cry2Ab, and Vip3Aa. DNA sequencing revealed that LF256 lacked the mutations in three genes (HaTSPAN1, HaABCC2, and HaABCC3) that confer resistance to Cry1Ac in two other strains of H. armigera we analyzed. Together with previous results, the data reported here show that each of the four strains examined has a different genetic basis of resistance to Cry1Ac. Significant positive cross-resistance occurred to Cry1Fa in three of the four strains tested but not to Cry2Ab or Vip3Aa in any strain. Thus, Cry2Ab and Vip3Aa are likely to be especially valuable for increasing the efficacy and durability of Bt cotton against H. armigera populations that have some resistance to Cry1Ac.

11.
Insect Sci ; 2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-33998150

RESUMO

Spinosyns, including spinosad and spinetoram, act on the insect central nervous system, gradually paralyzing or destroying the target insect. Spinosad resistance is associated with loss-of-function mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit in a number of agricultural pests. Using gene editing, nAChR α6 has been verified as a target for spinosyns in five insect species. Recently, a point mutation (G275E) in exon 9 of nAChR α6 was identified in spinosad-resistant strains of Thrips palmi and Tuta absoluta. To date, no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests. In this study, the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) gene-editing technology. Reverse transcriptase-polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain. The results of bioassays showed that the 275E strain was highly resistant to spinosad (230-fold) and spinetoram (792-fold) compared to the unedited background strain, directly confirming that the G275E mutation of the nAChR α6 subunit confers high levels of spinosyn resistance in S. exigua. Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive. This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChR α6 of S. exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene-editing technology to confirm the role played by candidate target site mutations in insecticide resistance.

12.
J Glob Antimicrob Resist ; 24: 370-372, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33581344

RESUMO

OBJECTIVES: The global spread of carbapenem-resistant Enterobacterales is a leading public-health threat. Lack of effective treatment has resulted in use of colistin as a last-resort therapeutic option for multidrug-resistant (MDR) bacterial infections. Here we report the complete genome sequence of a MDR Escherichia coli strain carrying a plasmid-mediated colistin resistance gene mcr-1 recovered from a Chinese paediatric patient. METHODS: Whole-genome sequencing of E. coli strain 1506 was performed using both Oxford Nanopore MinION and Illumina NovaSeq 6000 platforms. De novo hybrid assembly of short Illumina reads and long MinION reads was performed using Unicycler. In silico multilocus sequence typing (MLST), antimicrobial resistance genes (ARGs) and plasmid replicons were identified from the genome sequence. Core genome multilocus sequence typing (cgMLST) analysis between E. coli 1506 and all of the ST48 E. coli strains retrieved from the NCBI GenBank database was performed using BacWGSTdb 2.0 server. RESULTS: The complete genome sequence of E. coli 1506 consists of six contigs comprising 4 849 058 bp, including one chromosome and five plasmids, and was assigned to ST48. Fourteen ARGs were identified, including mcr-1 located on a 33 309-bp IncX4 plasmid. The closest relative of E. coli 1506 was another isolate originating from livestock in Australia, which differed by 614 cgMLST alleles. CONCLUSION: Our study reports the genome sequence of a MDR E. coli carrying mcr-1 isolated from a Chinese paediatric patient. These data may help to understand the antimicrobial resistance mechanisms and genomic features and highlight the growing threat of antimicrobial resistance in children.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Criança , China , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genoma Bacteriano , Genômica , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus
13.
J Cancer ; 12(3): 693-702, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33403027

RESUMO

FOXD1 has been reported to function as an oncogene in several types of cancer. This study evaluated the expression of FOXD1 and its role in head and neck squamous cell carcinoma (HNSCC). We mined the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases for expression profiles, clinical significance, and potential mechanisms of FOXD1in HNSCC. Our validation cohort consisted of FOXD1 mRNA expression in 162 paired HNSCC and adjacent normal tissues, as determined using quantitative real-time polymerase chain reaction. FOXD1 expression was upregulated in HNSCC in the public databases and in the validation cohort. The expression level of FOXD1 was associated with DNA amplification and methylation level. The areas under the curves (AUC) of TCGA cohort and the validation cohort were 0.855 and 0.843, respectively. Furthermore, higher FOXD1 expression was significantly associated with worse overall survival (hazard ratio [HR]: 1.849, 95% confidence interval [CI]: 1.280-2.670, P = 0.001) and a lower rate of recurrence-free survival (HR: 1.650, 95% CI: 1.058-2.575, P = 0.027) in patients with HNSCC. Moreover, gene set enrichment analysis showed that cases of HNSCC with FOXD1 overexpression were enriched in bladder cancer, cell cycle, DNA replication, glycosaminoglycan biosynthesis chondroitin sulfate, homologous recombination, glycan biosynthesis, nucleotide excision repair, p53 signaling pathway, pyrimidine metabolism, and spliceosome pathways. In summary, FOXD1 was significantly upregulated in HNSCC and was a good diagnostic biomarker and an independent predictor of poor survival and low rate of recurrence-free survival in patients with HNSCC.

14.
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
15.
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
16.
Toxins (Basel) ; 13(1)2020 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-33374143

RESUMO

Insecticidal proteins from Bacillus thuringiensis (Bt) are used widely in sprays and transgenic crops to control insect pests. However, evolution of resistance by pests can reduce the efficacy of Bt toxins. Here we analyzed resistance to Bt toxins Cry1Ac and Cry1Fa in the diamondback moth (Plutella xylostella), one of the world's most destructive pests of vegetable crops. We used CRISPR/Cas9 gene editing to create strains with knockouts of the ATP-binding cassette (ABC) transporter genes PxABCC2, PxABCC3, or both. Bioassay results show that knocking out either gene alone caused at most 2.9-fold resistance but knocking out both caused >10,320-fold resistance to Cry1Ac and 380-fold resistance to Cry1Fa. Cry1Ac resistance in the double knockout strain was recessive and genetically linked with the PxABCC2/PxABCC3 loci. The results provide insight into the mechanism of cross-resistance to Cry1Fa in diamondback moth. They also confirm previous work with this pest showing that mutations disrupting both genes cause higher resistance to Cry1Ac than mutations affecting either PxABCC2 or PxABCC3 alone. Together with previous work, the results here highlight the value of using single and multiple gene knockouts to better understand the independent and synergistic effects of putative Bt toxin receptors on resistance to Bt toxins.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Toxinas de Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/química , Endotoxinas/farmacologia , Proteínas Hemolisinas/química , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Mariposas/efeitos dos fármacos , Mariposas/genética , Animais , Bacillus thuringiensis , Sistemas CRISPR-Cas , Edição de Genes , Larva , Proteínas de Neoplasias
17.
Insect Biochem Mol Biol ; 127: 103486, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33069773

RESUMO

The cotton bollworm P450s of the clustered CYP6AE subfamily share high sequence identities but differ dramatically in their capacity to metabolize xenobiotics, especially esfenvalerate. Among them, CYP6AE17 has the highest sequence identity with CYP6AE18 but shows ~7-fold higher metabolic efficiency. CYP6AE11 is most active towards esfenvalerate but CYP6AE20 is inactive even though the enzymes share 54.8% sequence identity. Sequence analysis revealed the SRS1 (Substrate Recognition Site) and SRS6 between CYP6AE17 and CYP6AE18, and SRS1 between CYP6AE11 and CYP6AE20 are the most variable among all six SRSs. In order to identify the key factors that underlie the observed catalytic difference, we exchanged these SRS sequences between two pairs of P450s and studied the activity of the resulting hybrid mutants or chimeras. In vitro metabolism showed that the CYP6AE17/18 chimeras had 2- and 14-fold decreased activities and the CYP6AE18/17 chimeras had 6- and 10-fold increased activities to esfenvalerate. Meanwhile, after exchanging SRS1 with each other, the CYP6AE11/20 chimera folded incorrectly but the CYP6AE20/11 chimera gained moderate activity to esfenvalerate. Molecular modelling showed that amino acids variants within SRS1 or SRS6 change the shape and chemical environment of the active sites, which may affect the ligand-binding interactions. These results indicate that the protein structure variation resulting from the sequence diversity of SRSs promotes the evolution of insect chemical defense and contributes to the development of insect resistance to pesticides.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Inseticidas/metabolismo , Mariposas/genética , Nitrilas/metabolismo , Piretrinas/metabolismo , Sequência de Aminoácidos , Animais , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Mariposas/metabolismo , Nitrilas/farmacologia , Piretrinas/farmacologia , Alinhamento de Sequência
18.
Pestic Biochem Physiol ; 170: 104699, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980057

RESUMO

The resistance to dieldrin gene (Rdl) encodes a subunit of the insect γ-amino butyric acid (GABA) receptor, and the encoded Rdl subunit is a major target site for cyclodiene and phenylpyrazole insecticides. Since the substitution of a single amino acid (Ala to Ser/Gly at position 302) of the Drosophila melanogaster Rdl gene was first identified to confer high level resistance to dieldrin, mutations at the equivalent positions have been reported to confer resistance to dieldrin and/or fipronil in a wide range of different insects. In the cotton bollworm Helicoverpa armigera, there are two Rdl homologs (HaRdl-1 and HaRdl-2) in close proximity on the Z chromosome, which as wild-type sequences, encode alanine and serine respectively at amino acid position 302. In the present study, we used the CRISPR/Cas9 gene editing approach to knock out HaRdl-1 and HaRdl-2 and establish two homozygous knockout strains (ΔRdl-1 and ΔRdl-2). The ΔRdl-1 strain showed low levels of resistance (8.0- to 9.3-fold) to three cyclodiene insecticides (endosulfan, aldrin and dieldrin) compared with the background SCD strain. In contrast, toxicity of the three cyclodiene insecticides to the ΔRdl-2 strain increased significantly (3.6- to 6.3-fold) when compared with the SCD strain. Genetic analysis indicated the obtained resistance to endosulfan and dieldrin in the ΔRdl-1 strain was sex-linked, which is consistent with the fact that HaRdl-1 locus is located on the Z chromosome. The above results demonstrate that both HaRdl-1 and HaRdl-2 are important determinants for the susceptibility of H. armigera SCD strain to the three cyclodiene insecticides, but have opposite effects. It was also found that HaRdl-1 and HaRdl-2 are involved, to some extent, in mediating sensitivity of H. armigera to avermectin and fipronil respectively. We speculate that the HaRdl-1 and HaRdl-2 subunits have different pharmacological properties, which contribute to the differential sensitivities of H. armigera to the tested cyclodienes and other insecticides.


Assuntos
Proteínas de Drosophila/genética , Inseticidas/farmacologia , Inseticidas/toxicidade , Mariposas/genética , Animais , Dieldrin/toxicidade , Drosophila melanogaster/genética , Resistência a Inseticidas/efeitos dos fármacos , Resistência a Inseticidas/genética , Receptores de GABA/genética , Receptores de GABA-A/genética , Genética Reversa
19.
Sci Rep ; 10(1): 14997, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929113

RESUMO

Injectable dynamic hydrogels play a key role in cell transplantation to protect the cells from shear stress during injection. However, it still remains challenging to design dynamic hydrogels with fast gelation and high stability for protecting cells under flow due to the slow formation and exchange of most dynamic bonds. Here, a novel dual-crosslinked hydrogel system with fast dynamic crosslinks is developed by using methacrylate chitosan (CHMA) and aldehyde functionalized hyaluronate (oxidized HA, OHA). Based on the cooperation of electrostatic interaction between cationic amino of chitosan and anionic carboxyl of HA and Schiff-based crosslinking through amino and aldehyde groups, the dynamic CHMA-OHA hydrogel shows rapid gelation and high injectability. Further, the CHMA-OHA hydrogel is photopolymerized for achieving a high modulus and stability. Importantly, such hydrogels loaded with stem cells remains a cell viability (~ 92%) after extrusion. These results indicate that the CHMA-OHA hydrogel is a promising tissue engineering biomaterial for therapeutic cell delivery and 3D printing of encapsulated cell scaffolds.


Assuntos
Ácido Hialurônico/química , Hidrogéis/química , Técnicas de Cultura de Tecidos/métodos , Animais , Sobrevivência Celular , Quitosana/química , Células-Tronco Mesenquimais/citologia , Metacrilatos/química , Ratos , Reologia , Bases de Schiff/química
20.
J Ophthalmol ; 2020: 7392165, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32774909

RESUMO

Purpose: To evaluate whether clinical measures of postoperative binocular functions could predict the long-term stability of postoperative ocular alignment in children with intermittent exotropia. Methods: A retrospective study was performed in thirty-nine children (median: 7 years) who have been surgically treated from intermittent exotropia without overcorrection (less than 10 prism diopters [pd] of exodeviation at 1 month postoperatively). Angles of deviation and binocular functions were measured preoperatively and at 1 month, 6 months, and the final follow-up visit (≥24 months) postoperatively. We examined the relationships between postoperative drift (change of ocular alignment) and binocular functions (sensory fusion, fusional convergence amplitude, and stereoacuity). Results: The surgical success rate (esophoria/tropia ≤5 pd to exophoria/tropia ≤10 pd) dropped to 76.9% at 6 months after surgery and to 53.8% at individuals' last visit (mean: 37 months). The mean exodrift was 7.7 ± 9.2 pd from the postoperative month 1 to the final visit (p < 0.001) on distance fixation. Distance stereoacuity, central fusion, and fusional convergence amplitude significantly improved following surgery (p < 0.05). However, no significant correlation was found between their binocular functions measured at the beginning of each follow-up period and the postoperative drift (all p > 0.13). Conclusion: Our findings suggest that the clinical measures of sensory fusion, fusional convergence amplitude, and stereoacuity cannot serve as a robust predictor for the long-term stability of postoperative ocular alignment in patients who underwent successful surgery without overcorrection at 1 month postoperatively.

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