Near-freezing temperature suppresses avocado (Persea americana Mill.) fruit softening and chilling injury by maintaining cell wall and reactive oxygen species metabolism during storage.

To enhance the postharvest quality of avocado (Persea americana Mill.) fruit, this study investigates alterations in cell wall metabolism and reactive oxygen species (ROS) metabolism during near-freezing temperature (NFT) storage, and explores their impact on fruit softening. The fruit was stored at 25 °C, 5 °C, 2 °C, and NFT, respectively. NFT storage retarded firmness loss and chilling injury in comparison with 25 °C, 5 °C, and 2 °C. NFT storage delayed the decrease of ionic-soluble pectin (ISP) and cellulose (CLL) contents by suppressing cell wall degradation enzyme activities. Correlation analysis showed that cell wall degradation enzyme activities were positively correlated to rates of ethylene release and respiration. Moreover, NFT storage maintained higher levels of DPPH and ABTS scavenging abilities, activities of superoxide dismutase, peroxidase, and catalase, as well as ascorbate-glutathione cycle (ascorbic acid, glutathione, glutathione disulfide, ascorbate peroxidase, cycle-related enzymes), thereby inhibited the increase of ROS content, malondialdehyde content, and cell membrane permeability. Fruit firmness and chilling injury were correlated with the contents of hydrogen (H2O2), superoxide anion (O2.-), ISP, and CLL. These results suggested that NFT could suppress fruit softening and chilling injury by inhibiting cell wall degradation through delaying respiration and ethylene production and suppressing ROS production via activation of antioxidant systems, thereby maintaining quality and prolonged storage life during avocado fruit storage.

Penicilloneines A and B, Quinolone-Citrinin Hybrids from a Starfish-Derived Penicillium sp.

Penicilloneines A (1) and B (2) are the first reported quinolone-citrinin hybrids. They were isolated from the starfish-derived fungus Penicillium sp. GGF16-1-2, and their structures were elucidated using spectroscopic, chemical, computational, and single-crystal X-ray diffraction methods. Penicilloneines A (1) and B (2) share a common 4-hydroxy-1-methyl-2(1H)-quinolone unit; however, they differ in terms of citrinin moieties, and these two units are linked via a methylene bridge. Penicilloneines A (1) and B (2) exhibited antifungal activities against Colletotrichum gloeosporioides, with lethal concentration 50 values of 0.02 and 1.51 µg/mL, respectively. A mechanistic study revealed that 1 could inhibit cell growth and promote cell vacuolization and consequent disruption of the fungal cell walls via upregulating nutrient-related hydrolase genes, including putative hydrolase, acetylcholinesterase, glycosyl hydrolase, leucine aminopeptidase, lipase, and beta-galactosidase, and downregulating their synthase genes 3-carboxymuconate cyclase, pyruvate decarboxylase, phosphoketolase, and oxalate decarboxylase.

New polyketides from the starfish-derived symbiotic fungus Penicillium sp. GGF16-1-2.

One new rare carbon-bridged citrinin dimer quinocitrindimer C (1) as a pair of epimers, two new polyketide penicilliodes D (3) and E (4) together with nine known citrinin derivatives, were isolated from the fermentation broth of starfish-derived symbiotic fungus Penicillium sp. GGF16-1-2. Their structures and configurations were elucidated by comprehensively spectroscopic data analysis and electronic circular dichroism calculations. Eleven citrinin derivatives were tested by Colletotrichum gloeosporioides, and compound 2 played a significant antifungal activity against Colletotrichum gloeosporioides with LC50 value of 0.27 µg/ml.

Development of a multi-spectroscopy method coupling µ-FTIR and µ-Raman analysis for one-stop detection of microplastics in environmental and biological samples.

Current techniques for microplastics (MPs) analysis are diverse. However, most techniques have individual limitations like the detection limit of spatial resolution, susceptibility, high cost, and time-consuming detection. In this study, we proposed a multi-spectroscopy method coupling µ-FTIR and µ-Raman analysis for one-stop MPs detection, in which barium fluoride was used as the substrate alternative to the filter membrane. Compared with commonly used filter membranes (alumina, silver, PTFE and nylon membranes), the barium fluoride substrate showed better spectroscopic detection performance on microscopic observation, broader transmittable wavenumber range for µ-FTIR (750-4000 cm-1) and µ-Raman (250-4000 cm-1) detection, thus suitable for the multi-spectroscopy analysis of spiked samples. Further, the real environmental and biological samples (indoor air, bottled water and human exhaled breath) were collected and detected to verify the applicability of the developed multi-spectroscopy method. The results indicated that the average content of detected MPs could be increased by 30.4 ± 29.9 % for indoor air, 17.1 ± 13.2 % for bottled water and 38.4 ± 16.0 % for human exhaled breath, respectively in comparison with widely used µ-Raman detection, which suggested that MPs exposure might be underestimated using single spectroscopy detection. Moreover, the majority of underestimated MPs were colored and smaller sized (<50 µm) MPs, which could pose higher risks to human body. In addition, the proposed method consumed lower sample pre-treatment costs and was environmental-friendly since the barium fluoride substrate could be used repeatedly after being cleaned by organic solvent with reliable results (n = 10, CV = 10 %, ICC = 0.961), which reduced the cost of MPs detection by at least 2.49 times compared with traditional methods using silver membrane.

First report of Botryosphaeria dothidea causing fruit rot on Chinese olive (Canarium album Raeusch.) in Guangdong province of China.

Chinese olive (Canarium album Raeusch.) is a traditional Chinese medicinal plant, mainly cultivated in Guangdong and Fujian provinces in China (Lai et al. 2022). In October 2023, Chinese olive fruit spots were observed in all the Chinese olive orchards surveyed in Chaozhou city (23.75°N, 116.67°E) of Guangdong, with an incidence up to 15%. Early disease symptoms on fruits appeared as circular or irregular, dark brown to black spots with yellowish lesions, and later the spots slowly coalesced to form large necrotic areas, which seriously affected the fruit marketability. To isolate the causal agent, small pieces (~0.3 mm2) of fruit tissue were excised from the lesion margins, and surface-disinfested with 75% (v/v) ethanol for 1 min, followed by 1% NaClO for 3 min, and rinsed three times with sterile water. The pieces were then placed on potato-dextrose-agar (PDA) and incubated at 27°C. Ultimately, four fungal isolates were obtained with similar morphology phenotypes, colonies initially appeared white with irregular margins and after 4-6 days turned dark gray gradually with dense aerial myceliu. Microscopy revealed conidia were single-celled, hyaline, aseptate, fusiform to subclavate, and measured 18.1-22.5 µm × 6.4-9.3 µm (19.8 × 7.4 m on average, n = 30), which were consistent with those descriptions of Botryosphaeria dothidea (Vasic et al. 2013; Zhang et al. 2023). To further identity the isolates, partial sequences of ribosomal transcribed spacer (ITS), translation elongation factor 1-α (TEF1-α), and ß-tubulin (TUB2) genes were amplified using primers ITS1/ITS5, TEF-F/R, TUB2-F/R, respectively (Xu et al., 2023; Hong et al. 2006). The sequences of four isolates were identical, and those of representative strain GDCZ-1 were deposited in GenBank (ITS, OR584295; TEF1-α, OR685157; TUB2, OR685158). Using Neighbor-Joining algorithm, phylogenetic tree based on concatenated sequences of ITS, TEF1-α, and TUB2 showed that GDCZ-1 clustered with B. dothidea. To fulfill Koch's postulates, pathogenicity tests were performed on healthy Chinese olive fruits using the needle-prick inoculation method. The fruits were wounded with a sterile needle at the equatorial area (depth of 3-4 mm), and inoculated with 10 µL of spore suspension (106 /mL). The control fruits were inoculated with sterile water. Inoculated fruits were placed in sterile plastic containers to maintain high relative humidity (almost 100%) and incubated at 27°C. After 4 days, the inoculated fruits showed similar symptoms with those observed in the field infected fruits, while the control remained asymptomatic. Pathogen re-isolated from the inoculated fruits showed identical morphological characteristics to the original isolate GDCZ-1. As far as we know, fruit rot caused by Alternaria alternata has been recently reported on C. album in China (Shao et al. 2024). To our knowledge, this is the first report of B. dothidea causing fruit rot disease on C. album in Guangdong. Our report will provide crucial information for studying the epidemiology and management of this disease.

Comparative physiological and transcriptome analysis provide insights into the inhibitory effect of osthole on Penicillium choerospondiatis.

Blue mold induced by Penicillium choerospondiatis is a primary cause of growth and postharvest losses in the fruit of Phyllanthus emblica. There is an urgent need to explore novel and safe fungicides to control this disease. Here, we demonstrated osthole, a natural coumarin compound isolated from Cnidium monnieri, exhibited a strong inhibitory effect on mycelia growth, conidial germination rate and germ tube length of P. choerospondiatis, and effectively suppressed the blue mold development in postharvest fruit of P. emblica. The median effective concentration of osthole was 9.86 mg/L. Osthole treatment resulted in cellular structural disruption, reactive oxygen species (ROS) accumulation, and induced autophagic vacuoles containing cytoplasmic components in fungal cells. Transcriptome analysis revealed that osthole treatment led to the differentially expressed genes mainly enriched in the cell wall synthesis, TCA cycle, glycolysis/ gluconeogenesis, oxidative phosphorylation. Moreover, osthole treatment led to increase genes expression involved in peroxisome, autophagy and endocytosis. Particularly, the autophagy pathway related genes (PcATG1, PcATG3, PcATG15, PcATG27, PcYPT7 and PcSEC18) were prominently up-regulated by osthole. Summarily, these results revealed the potential antifungal mechanism of osthole against P. choerospondiatis. Osthole has potentials to develop as a natural antifungal agent for controlling blue mold disease in postharvest fruits.

MicroRNA-Mediated Host Immune Genes Manipulation Benefits AcMNPV Proliferation in Spodoptera frugiperda.

Spodoptera frugiperda is a highly destructive migratory pest that threatens various crops globally. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an effective biocontrol agent against lepidopteran pests. Here, we explored the molecular mechanisms underlying the immune response to AcMNPV infection in S. frugiperda. RNA-seq and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses identified the Toll, IMD, and apoptosis pathways as primary immune responses. Investigation into AcMNPV-induced apoptosis in the S. frugiperda cell line (Sf9) revealed that the Toll pathway activated the JNK via the TRAF6 (TNF receptor-associated factor 6) adapter. In addition, AcMNPV-induced the differential expression of several host-encoded microRNAs (miRNAs), with significant negative regulatory effects, on S. frugiperda antiviral immune genes. RNAi and miRNA-mimic mediated silencing of these genes resulted in increased AcMNPV proliferation. Our findings reinforce the potential of AcMNPV as a potent biocontrol agent and further our understanding of developing biotechnology-based targeted pest control agents.

Eurycomanone (EN) Activates Transcription Factor FoxO by Inhibiting the Insulin Signaling Pathway to Suppress the Development of Spodoptera frugiperda.

The insulin-like signaling (IIS) pathway is essential for insect growth and development. In this study, we showed that eurycomanone (EN) is an active compound with growth inhibitory activity against Spodoptera frugiperda larvae. Experiments in cells and RNA-seq analysis in the midgut showed that EN targeted the IIS pathway in S. frugiperda to activate the transcription factor SfFoxO (S. frugiperda forkhead boxO) to regulate mRNA levels associated with nutrient catabolism. Additionally, mass spectrometry imaging revealed that EN was distributed in the larval gut and enriched in the inner membrane of the gut. Immunofluorescence, western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results showed that EN induced program cell death (PCD) in the larvae midgut. Thus, EN targeted the insulin receptor to inhibit the IIS signaling pathway, exerting inhibitory activity on the growth and development of S. frugiperda larvae. Our results suggest that EN has great potential as a botanical pesticide, and the IIS signaling pathway may be an effective target for botanical pesticides.

Potential Biochemical Pesticide-Synthesis of Neofuranocoumarin and Inhibition the Proliferation of Spodoptera frugiperda Cells through Activating the Mitochondrial Pathway.

Furanocoumarins, the secondary metabolites of plants, are considered to be natural insecticides and fungicides because they prevent the invasion of plant pathogenic microorganisms and the predation of herbivorous insects. In this study, novel 2-arylfuranocoumarin derivatives were designed to synthesize by condensation, esterification, bromination, and Wittig reaction. The results showed an excellent photosensitive activity of 2-thiophenylfuranocoumarin (I34). Cell Counting Kit-8 detected that I34 could inhibit the proliferation of Spodoptera frugiperda (Sf9) cells in a time- and concentration-dependent manner under ultraviolet A (UV-A) light for 3 min. The inverted microscope revealed that cells treated with I34 swelled, the membrane was ruptured, and apoptotic bodies appeared. The flow cytometry detected that I34 could induce apoptosis of Sf9 cells, increase the level of intracellular reactive oxygen species (ROS), decrease the mitochondrial membrane potential, and block cell cycle arrest in the G2/M phase. Transmission electron microscopy detected cell mitochondrial cristae damage, matrix degradation, and mitochondrial vacuolation. Further enzyme activity detection revealed that the enzyme activities of apoptosis-related proteins caspase-3 and caspase-9 increased significantly (p < 0.05). Finally, Western blotting analysis detected that the phosphorylation level of Akt and Bad and the expression of the apoptosis inhibitor protein Bcl-XL were inhibited, cleaved-PARP and P53 were increased, and cytochrome C was released from the mitochondria into the cytoplasm. Moreover, under UV-A irradiation, I34 promoted the increase in ROS in Sf9 cells, activated the mitochondrial apoptotic signal transduction pathway, and finally, inhibited cell proliferation. Thus, novel furanocoumarins exhibit a potential application prospect as a biochemical pesticide.

Rare Carbon-Bridged Citrinin Dimers from the Starfish-Derived Symbiotic Fungus Penicillium sp. GGF16-1-2.

Four novel, rare carbon-bridged citrinin dimers, namely dicitrinones G-J (1-4), and five known analogs (5-9) were isolated from the starfish-derived fungus Penicillium sp. GGF 16-1-2. Their structures were elucidated by extensive spectroscopic analysis and quantum chemical calculations. Compounds 1-9 exhibited strong antifungal activities against Colletotrichum gloeosporioides with LD50 values from 0.61 µg/mL to 16.14 µg/mL. Meanwhile, all compounds were evaluated for their cytotoxic activities against human pancreatic cancer BXPC-3 and PANC-1 cell lines; as a result, compound 1 showed more significant cytotoxicities than the positive control against both cell lines. In addition, based on the analyses of the protein-protein interaction (PPI) network and Western blot, 1 could induce apoptosis by activating caspase 3 proteins (CASP3).

The botanical eurycomanone is a potent growth regulator of the diamondback moth.

Eurycomanone is a quassinoid compound that is derived from Eurycoma longifolia, and it is often used as an indicator to evaluate the active ingredients of Eurycoma longifolia. However, Eurycomanone has rarely been reported to have biological activity toward pests. In this study, we evaluated the antifeedant activity of eurycomanone against the diamondback moth(Plutella xylostella), with a non-selective AFC50(the concentration that corresponds to 50% antifeedant action) value and selective AFC50 of 17.5 mg/L and 14.2 mg/L, respectively, which were 2.1-fold (36.9 mg/L) and 2-fold (28.5 mg/L) lower than that of azadirachtin, respectively. In addition, eurycomanone was used to treat the roots of Brassica chinensis L. at a concentration of 100 µg/g for 72 h. The antifeedant index was found to reach 93% by tracking the leaves. After feeding with 20 µg/g eurycomanone, no pupae or eclosion were observed. To explore this mechanism, we used scanning electron microscopy to discover that eurycomanone could prevent the development of taste receptors on the maxillary palp of diamondback moth larvae. Additional electrophysiological measurements showed that eurycomanone exhibited excitatory action to the central taste neurons of diamondback moth and significantly inhibited the GABAA receptor current. Eurycomanone exhibited significant activity as an antifeedant, inhibited growth and excelled at systemic absorption.

Discovery and Biomimetic Synthesis of a Phloroglucinol-Terpene Adduct Collection from Baeckea frutescens and Its Biogenetic Origin Insight.

A phloroglucinol-terpene adduct (PTA) collection consisting of twenty-four molecules featuring three skeletons was discovered from Baeckea frutescens. Inspired by its biosynthetic hypothesis, we synthesized this PTA collection by reductive activation of stable phloroglucinol precursors into highly reactive ortho-quinone methide (o-QM) intermediates and subsequently Diels-Alder cycloaddition. We also demonstrated, for the first time, the generation process of the active o-QM by performing dynamic NMR and HPLC-MS monitoring experiments. Moreover, the PTA collection showed significant antifeedant effect toward the Plutella xylostella larvae.

Unprecedented Quassinoids from Eurycoma longifolia: Biogenetic Evidence and Antifeedant Effects.

Six new quassinoids (1-6) were isolated from the roots of Eurycoma longifolia, and their structures with absolute configurations were determined unambiguously by spectroscopic analyses and single-crystal X-ray crystallographic experiments. Compounds 1 and 2 are the first members of a new class of quassinoids with an unusual C26 carbon skeleton. Compound 6 features a C20 cage-like scaffold with an unprecedented densely functionalized 2,5-dioxatricyclo[5.2.2.04,8]undecane core. The discovery of the two C26 quassinoids 1 and 2 has provided firm evidence for the better understanding the biogenetic process from C30 triterpenoid precursors to quassinoids. Compound 5 exhibited significant antifeedant activity on the diamondback moth (DBM) larvae and excellent systemic absorption and accumulated properties in Brassica chinensis.

Azadirachtin A inhibits the growth and development of Bactrocera dorsalis larvae by releasing cathepsin in the midgut.

Azadirachtin, a botanical insecticide with high potential, has been widely used in pest control. Azadirachtin has shown strong biological activity against Bactrocera dorsalis in toxicological reports, but its mechanism remains unclear. This study finds that azadirachtin A inhibits the growth and development of Bactrocera dorsalis larvae. The larval weights and body sizes of the azadirachtin-treated group were significantly less than those of the control group in a concentration-dependent manner. Further, pathological sections revealed that azadirachtin destroyed the midgut cell structure and intestinal walls, while TUNEL staining showed that azadirachtin could induce apoptosis of midgut cells, and Western blot analysis indicated that Bcl-XL expression was inhibited and cytochrome c (CytC) released into the cytoplasm. The results also imply azadirachtin-induced structural alterations in the Bactrocera dorsalis larvae midgut by activation of apoptosis. RNA-seq analysis of midgut cells found that 482 and 708 unique genes were upregulated and downregulated, respectively. These differentially expressed genes (DEGs) were enriched in apoptotic and lysosomal signaling pathways and included 26 genes of the cathepsin family. qRT-PCR verified the expression patterns of some DEGs, indicating that Cathepsin F was upregulated by 278.47-fold and that Cathepsin L and Cathepsin D were upregulated by 28.06- and 8.97-fold, respectively. Finally, association analysis between DEGs and DEMs (differentially expressed metabolites) revealed that azadirachtin significantly reduced the digestion and absorption of carbohydrates, proteins, fats, vitamins and minerals in the midgut. In conclusion, azadirachtin induces the release of cathepsin from lysosomes, causing apoptosis in the midgut. Ultimately, this leads to reduced digestion and absorption of nutrient metabolites in the midgut and inhibition of the growth and development of Bactrocera dorsalis larvae.

Comparative Analyses of the Transcriptome and Proteome of Comte de Paris and Smooth Cayenne to Improve the Understanding of Ethephon-Induced Floral Transition in Pineapple.

BACKGROUND/AIMS: Ethylene is usually used to induce floral transition in pineapple. However, its successful induction in plants categorized as Cayenne is difficult or completely ineffective, and information concerned is limited. The present study was undertaken to investigate the molecular mechanisms underlying this obstacle. METHODS: Transcriptome and proteome comparative analyses were performed to explore the important regulation and pathway variations after ethephon induction in the induction-easy 'Comte de Paris' (CP) and induction-hard 'Smooth Cayenne' (SC) cultivars via RNA-seq (RNA-sequencing) and iTRAQ (isobaric tags for relative and absolute quantification). RESULTS: CP and SC exhibited basic differences at the transcriptomic and proteomic levels before ethephon treatment, including the expression of genes and proteins related to ethylene signal transduction. After ethephon induction, the expression of genes and proteins involved in plant ethylene signal transduction and carbohydrate metabolism responded more strongly in CP than in SC. The expression of the floral meristem identity (FMI) genes AG, TFL and FT exhibited greater changes in CP, and more transcription factors responded in SC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that many differentially expressed genes (DEGs) in CP were annotated to terms and pathways involved in photoperiodism and shared components involved in carbohydrate metabolism and plant hormone signal transduction. CONCLUSION: These findings contribute to the understanding of the molecular mechanism underlying the variation between CP and SC in response to ethephon-mediated floral induction.

Identification and functional analysis of the GTPV bidirectional promoter region.

The goat pox chick embryo-attenuated virus (GTPV) has been developed as an effective vaccine that can elicit protective immune responses. It possesses a large genome and a robust ability to express exogenous genes. Thus, this virus is an ideal vector for recombinant live vaccines for infectious diseases in ruminant animals. In this study, we identified a novel bidirectional promoter region of GTPV through screening named PbVV(±). PbVV(±) is located between ETF-l and VITF-3, which are transcribed in opposite directions. A new recombinant goat pox virus (rGTPV) was constructed, in which duplicate PbVV(+) was used as a promoter element to enhance Brucella OMP31 expression, and duplicate PbVV(-) was used as a promoter element to regulate enhanced green fluorescent protein (EGFP) at the same time as the selection marker. PbVV(-) promoter activity was compared to that of the P7.5 promoter of vaccinia virus, as measured by EGFP expression; the fluorescence intensity of EGFP expressed in cells was confirmed by fluorescence microscopy and flow cytometry. PbVV(+) promoter activity was measured by Brucella OMP31 expression. Interaction with the anti-Brucella-OMP31 monoclonal antibody was confirmed by western blotting, and OMP31 mRNA expression was assessed by qRT-PCR. The results of this study will be useful for the further study of effective multivalent vaccines based on rGTPV. This study also provides a theoretical basis for overcoming the problem of low expression of exogenous genes.

Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells.

Outer membrane protein 25 (OMP25), a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK) signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308) and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10) were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK), and Jun-N-terminal kinase (JNK) from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

Induction of Autophagy and Apoptosis via PI3K/AKT/TOR Pathways by Azadirachtin A in Spodoptera litura Cells.

Azadirachtin is one of the most effective botanical insecticides and has been widely used in pest control. Toxicological reports show that azadirachtin can induce apoptosis in various insect cell lines. However, studies of azadirachtin-induced autophagy in cultured insect cells are lacking. This study reports that azadirachtin A significantly inhibits cell proliferation by inducing autophagic and apoptotic cell death in Spodoptera litura cultured cell line (SL-1 cell). Characteristic autophagolysosome and Atg8-PE (phosphatidylethanolamine) accumulation were observed by electron microscopy and western blotting, indicating that azadirachtin triggered autophagy in SL-1 cell. Furthermore, azadirachtin inhibited survival signaling by blocking the activation of PI3K, AKT and the down-stream target of rapamycin. Similar to the positive control of starvation, azadirachtin induced the activation of insulin receptor (InR) via a cellular feedback mechanism. In addition, the autophagy-related 5 (Atg5), a molecular switch of autophagy and apoptosis, was truncated (tAtg5) to trigger cytochrome c release into the cytoplasm under azadirachtin stress, which indicated that azadirachtin induced apoptosis through autophagy. Our findings suggest that azadirachtin primarily induced autophagy in SL-1 cell by dysregulating InR- and PI3K/AKT/TOR pathways, then stimulated apoptosis by activating tAtg5.