Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR) could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this present study, the fragments of eight candidate reference genes were cloned and identified from the pest Spodoptera litura. In addition, the expression stability of these genes in different samples from larvae of control and azadirachtin treatments were evaluated by the computational methods of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. According to our results, two of the reference genes should be the optimal number for RT-qPCR analysis. Furthermore, the best reference genes for different samples were showed as followed: EF-1α and EF2 for cuticle, β-Tubulin and RPL7A for fat body, EF2 and Actin for midgut, EF2 and RPL13A for larva and RPL13A and RPL7A for all the samples. Our results established a reliable normalization for RT-qPCR experiments in S. litura and ensure the data more accurate for the mechanism analysis of azadirachtin.
As an important botanical pesticide, azadirachtin demonstrates broad insecticidal activity against many agricultural pests. The results of a previous study indicated the toxicity and apoptosis induction of azadirachtin in Spodoptera frugiperda Sf9 cells. However, the lack of genomic data has hindered a deeper investigation of apoptosis in Sf9 cells at a molecular level. In the present study, the complete transcriptome data for Sf9 cell line was accomplished using Illumina sequencing technology, and 97 putative apoptosis-related genes were identified through BLAST and KEGG orthologue annotations. Fragments of potential candidate apoptosis-related genes were cloned, and the mRNA expression patterns of ten identified genes regulated by azadirachtin were examined using qRT-PCR. Furthermore, Western blot analysis showed that six putative apoptosis-related proteins were upregulated after being treated with azadirachtin while the protein Bcl-2 were downregulated. These data suggested that both intrinsic and extrinsic apoptotic signal pathways comprising the identified potential apoptosis-related genes were potentially active in S. frugiperda. In addition, the preliminary results revealed that caspase-dependent or caspase-independent apoptotic pathways could function in azadirachtin-induced apoptosis in Sf9 cells.
Background: Bactrocera dorsalis and B. correcta are economically important fruit fly pests of crops, vegetables, fruits, and nuts worldwide, especially in China. Nowadays in China, B. correcta is a second notorious pest of many fruits after B. dorsalis. Different botanicals have been tested against the B. dorsalis but in the case of B. correcta, no records were published. Methodology: This study evaluated the repellency of four botanicals (Seriphidium brevifolium, Piper nigrum, Azadirachta indica and quercetin) in acetone dilutions (5%, 2.5% and 1%) against the B. dorsalis and B. correcta at the laboratory conditions (25 ± 2 C, 60 ± 5% relative humidity, and a photoperiod of L:D 14:10 h). Results: The number of visits after 24-48 h, oviposition punctures, and pupae made by both species were lower on the treated mangoes in comparison to untreated mangoes. S. brevifolium, P. nigrum, A. indica and quercetin have significantly reduced the visits, ovipositional punctures, and pupae of both species. Among botanicals, the P. nigrum was the most effective repellent against B. correcta and as well as B. dorsalis. However, the harmful effects of these botanicals against natural enemies are still unknown. Fraceto LF. 2014. Application of nanotechnology for the encapsulation of botanical insecticides for sustainable agriculture: prospects and promises. Biotechnology Advances 32 (8):1550-1561
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