Host plant-induced changes in metabolism and osmotic regulation gene expression in Diaphorina citri adults.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a worldwide citrus pest. It transmits the pathogen Candidatus Liberibacter spp. of Huanglongbing (HLB), causing severe economic losses to the citrus industry. Severalgenera of plants in the Rutaceae family are the hosts of D. citri. However, the impact of these hosts on the metabolism and osmotic regulation gene expression of the pest remains unexplored. In this study, the contents of total sugars, sucrose, fructose, and glucose in young shoots, old leaves, and young leaves of 'Shatangju' mandarin and Murraya exotica were analyzed. Metabolomic analysis found that sucrose and trehalose were more abundant in the gut samples of D. citri adults fed on M. exotica when compared to what's in 'Shatangju' mandarin. A total of six aquaporin genes were identified in D. citri through the genome and transcriptome data. Subsequently, the expression patterns of these genes were investigated with respect to their developmental stage and tissue specificity. Additionally, the expression levels of osmotic regulation and trehalose metabolism genes in adults fed on different plants were evaluated. Our results provide useful information on the transfer of sugar between plants and D. citri. Our results preliminary revealed the sugar metabolism regulation mechanism in D. citri adults.

Characterization and transcriptomic analyses of the toxicity induced by toosendanin in Spodoptera frugipreda.

The fall armyworm, Spodoptera frugiperda, is a destructive agricultural pest that seriously threatens global food security. Insecticide resistance of this pest has gradually formed in recent years due to improper usage, and alternative methods are badly needed. Toosendanin (TSN) is a botanical compound with broad-spectrum insecticidal activities against many pests. However, the effects of TSN on S. frugiperda are still unclear. In this study, the growth inhibition phenomenon, including weight loss and prolonged developmental duration, in the larvae with TSN exposure was clearly observed. Compared to the control group, a total of 450 and 3314 differentially expressed genes (DEGs) were identified by RNA-Seq in the larvae groups treated with 10 and 20 mg/kg TSN, respectively. Furthermore, the DEGs involved in the juvenile hormone and ecdysone signal pathways and downstream processes, including detoxifying enzyme genes, chitin synthesis and metabolism genes, and cuticular protein genes, were found. Our findings suggest that TSN regulates the expression of key genes in juvenile hormone and ecdysone signal pathways and a series of downstream processes to alter the hormone balance and cuticle formation and eventually inhibit larval growth, which laid the foundation for the molecular toxicological mechanism research of TSN on S. frugiperda larvae.

The effects of carvacrol on development and gene expression profiles in Spodoptera frugiperda.

The fall armyworm, Spodoptera frugiperda, is a highly polyphagous agricultural pest that is widely distributed around the world and causes severe crop yield loss. Carvacrol showed adverse effects on many pests, such as larval death and growth inhibition. While the effects of carvacrol on S. frugiperda larvae are not yet known. In this study, the effects of carvacrol on S. frugiperda, including larval growth inhibition and mortality induction, were observed. The detoxification and digestive enzyme activities of larvae with 1.0 and 2.0 g/kg carvacrol treatments were analyzed. Carvacrol boosted the enzyme activities of carboxylesterase (CarE) and glutathione S-transferase (GST) while decreasing the activities of α-amylase (AMS), lipase (LIP), and trypsin. A total of 3422 differentially expressed genes were identified in the larvae treated with 2.0 g/kg carvacrol, of which the DEGs involved in xenobiotic detoxification, food digestion, and insecticidal targets were further examined. These results suggest that carvacrol could regulate growth and development by affecting the process of food digestion, and exert its toxicity on the larvae through interaction with a variety of insecticidal targets. While the altered expressions of detoxification enzymes might be related to the detoxification and metabolism of carvacrol. Our findings offer a theoretical foundation for the use of carvacrol for S. frugiperda control in the field.

Characterization of the physiological, histopathological, and gene expression alterations in Spodoptera frugiperda larval midguts affected by toosendanin exposure.

The fall armyworm, Spodoptera frugiperda, is a polyphagous pest worldwide and feeds on many grain and cash crops, which threatens the safety of agriculture and forestry production. Toosendanin (TSN) is a commercial insecticidal active ingredient used to manage various pests in the field and showed adverse effects against S. frugiperda, while the effects of TSN on the larval midguts are not yet known. In this study, the effects of 10 and 20 mg/kg TSN exposures on the larval midguts were analyzed. The structural changes of the larval midgut induced by TSN treatments were also determined by hematoxylin-eosin staining. Besides, TSN treatments also changed the enzyme activities of three digestive enzymes (α-amylase, lipase, and trypsin) and two detoxification enzymes (CarE and GST). A total of 2868 differentially expressed genes (DEGs) were identified by RNA-Seq in the larval midguts with 20 mg/kg TSN treatment, and the DEGs responsible for food digestion and detoxification were further examined. Our findings revealed the preliminary modes of action of TSN on the larval midguts of S. frugiperda, which provide a preliminary rationale for controlling S. frugiperda with TSN in the field.

The comparison of gut gene expression and bacterial community in Diaphorina citri (Hemiptera: Liviidae) adults fed on Murraya exotica and 'Shatangju' mandarin (Citrus reticulate cv. Shatangju).

BACKGROUND: Diaphorina citri Kuwayama is an important citrus pest. It serves as the vector for the transmission of Candidatus Liberibacter asiaticus (CLas), which induced a destructive disease, Huanglongbing, and caused huge economic losses. During the interaction between insects and plants, insects have evolved a series of mechanisms to adapt to various host plants. Murraya exotica and 'Shatangju' mandarin (Citrus reticulate cv. Shatangju) are the Rutaceae species from different genera that have been discovered as suitable hosts for D. citri adults. While the adaptation mechanism of this pest to these two host plants is unclear. RESULTS: In this study, RNA-seq and 16 S rDNA amplification sequencing were performed on the gut of D. citri adults reared on M. exotica and 'Shatangju' mandarin. RNA-seq results showed that a total of 964 differentially expressed genes were found in different gut groups with two host plant treatments. The impacted genes include those that encode ribosomal proteins, cathepsins, and mitochondrial respiratory chain complexes. According to 16 S rDNA sequencing, the compositions of the gut bacterial communities were altered by different treatments. The α and ß diversity analyses confirmed that the host plant changes influenced the gut microbial diversity. The functional classification analysis by Tax4Fun revealed that 27 KEGG pathways, mostly those related to metabolism, including those for nucleotide metabolism, energy metabolism, metabolism of cofactors and vitamins, amino acid metabolism, carbohydrate metabolism, xenbiotics biodegradation and metabolism, lipid metabolism, and biosynthesis of other secondary metabolites, were significantly altered. CONCLUSION: Our preliminary findings shed light on the connection between D. citri and host plants by showing that host plants alter the gene expression profiles and bacterial community composition of D. citri adults.

Effects of azadirachtin on detoxification-related gene expression in the fat bodies of the fall armyworm, Spodoptera frugiperda.

The fall armyworm, Spodoptera frugiperda, has become a worldwide pest and threatens world food production. A previous study indicated that azadirachtin, the most effective botanical insecticide for S. frugiperda, inhibits larval growth of the insect. The effect of azadirachtin on the tissues of the larvae, however, remains to be determined. In this study, the effects of azadirachtin on the structure of fat bodies were analyzed. Comparative transcriptomic analysis was conducted between controls and samples treated with 0.1 µg/g azadirachtin for 7 days to explore potential relevant mechanisms. The expression of 5356 genes was significantly affected after azadirachtin treatment, with 3020 up-regulated and 2336 down-regulated. Among them, 137 encode detoxification enzymes, including 53 P450s, 20 GSTs, 27 CarEs, 16 UGTs, and 12 ABC transporters. Our results indicated that azadirachtin could destroy fat body structure and change the mRNA levels of detoxification-related genes. The up-regulated genes encoding detoxification enzymes might be related to detoxifying azadirachtin. Our results elucidate a preliminary mechanism of azadirachtin detoxification in the fat bodies of S. frugiperda larvae.

Integrated miRNA and transcriptome profiling to explore the molecular mechanism of Spodoptera frugiperda larval midgut in response to azadirachtin exposure.

As a destructive agricultural pest, Spodoptera frugiperda has spread worldwide in the past few years. Azadirachtin, an environmentally friendly and most promising compound, showed adverse effects, including mortality and growth inhibition, against S. frugiperda. While the effects of azadirachtin on the midgut of this pest remain to be determined. In this study, structural damage was observed in the larval midguts of S. frugiperda with azadirachtin exposure. RNA-seq on the larval midguts with different azadirachtin treatments was performed. Compared to the control group, a total of 3344 and 4759 differentially expressed genes (DEGs) were identified in the midguts with 0.1 and 0.5 µg/g azadirachtin exposure, respectively. Among them, the DEGs encoding detoxification enzymes/proteins, immune-related proteins, digestion and absorption-related proteins, and transcript factors were further analyzed. High-throughput sequencing was also used for the identification of differentially expressed microRNAs in different treatments. A total of 153 conserved miRNAs and 147 novel miRNAs were identified, of which 11 and 29 miRNAs were affected by 0.1 and 0.5 µg/g azadirachtin treatments, respectively. The integrated analysis found that 13 and 178 miRNA versus mRNA pairs were acquired in the samples with 0.1 and 0.5 µg/g azadirachtin treatments, respectively. The results of high-throughput sequencing were confirmed by real-time quantitative polymerase chain reaction (RT-qPCR). These results provide useful information for revealing the molecular mechanism of S. frugiperda larval midgut in response to azadirachtin.

Selection of reference genes for quantitative real-time PCR normalization in the coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae).

The coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), is a major destructive pest of Coffea arabica L. (Gentianales: Rubiaceae), widely planted in many Asian countries, including China. Quantitative real-time polymerase chain reaction (qRT-PCR) is a common method for quantitative analysis of gene transcription levels. To obtain accurate and reliable qRT-PCR results, it is necessary to select suitable reference genes to different experimental conditions for normalizing the target gene expression. However, the stability of the expression of reference genes in X. quadripes has rarely been studied. In this study, the expression stability of nine candidate reference genes were investigated under biotic and abiotic conditions for use in qRT-PCR's normalization. By integrating the results of four algorithms of NormFinder, BestKeeper, geNorm, and RefFinder, the optimal reference gene combinations in different experimental conditions were performed as follows: RPL10a and EIF3D were the optimal reference genes for developmental stage samples, EIF4E, RPL10a, and RPS27a for tissue samples, V-ATP and EF1α for the sex samples, EIF3D and V-ATP for temperature treatment, RPS27a and RPL10a for insecticide stress, and RPL10a, RPS27a, and EF1α for all the samples. This study will help to obtain the stable internal reference genes under biotic and abiotic conditions and lay the foundation for in-depth functional research of target genes or genomics on olfactory molecular mechanisms, temperature adaptability, and insecticide resistance in X. quadripes.

Genome-wide identification, characterization and functional analysis of the chitianse and chitinase-like gene family in Diaphorina citri.

BACKGROUND: Insect chitinases play vital roles in postembryonic development, especially during the molting process, and are potential targets for the RNA interference (RNAi)-based insecticidal strategy. Systematic functional analyses of chitinase genes have already been conducted on numerous insect pests, but similar analyses have not been carried out on Diaphorina citri. RESULTS: Eleven chitinase/chitinase-like genes and one endo-ß-N-acetylglucosaminidase (ENGase) gene were identified in the Diaphorina citri genome using various bioinformatic tools. Transcriptomes of the integument and midgut from fifth-instar nymphs and freshly-emerged adults of Diaphorina citri were generated and sequenced. Potential functions of 12 chitinase/chitinase-like genes were examined during nymph-adult transitions. Four chitinase genes, including DcCht5, DcCht7, DcCht10-1 and DcCht10-2, were mainly expressed in the integument of fifth-instar nymphs. These four genes were also up-regulated significantly under 20-hydroxyecdysone (20E) treatments. RNAi-mediated knockdown of these four genes suggests that they are essential for nymph-adult transition. CONCLUSION: Our results demonstrated essential roles of the chitinase/chitinase-like genes during the nymph-adult transition in Diaphorina citri, which are potentially useful targets for controlling the Diaphorina citri pest. © 2022 Society of Chemical Industry.

Effects of camptothecin on histological structures and gene expression profiles of fat bodies in Spodoptera frugiperda.

Spodoptera frugiperda is a serious threat to global food production. Our previous study demonstrated that Camptothecin (CPT), a bioactive secondary metabolite from Camptotheca acuminata (Decne: Nyssaceae), exhibits adverse impact on the larval midgut of S. frugiperda and inhibits insect growth. However, effects of CPT on fat bodies of S. frugiperda larvae have not been examined yet. In the present study, we found that histological structures of fat bodies of S. frugiperda larvae were damaged in insects treated with CPT. Comparative transcriptomic analyses among different fat body samples from controls and insects treated with 1.0 and 5.0 µg/g CPT were performed. A total of 4212 and 5044 differentially expressed genes (DEGs) were identified in the samples treated with 1.0 and 5.0 µg/g CPT, respectively. Our data indicated that the pathways of detoxification, immune response, fatty acids, chitin, and hormone biosynthesis in fat bodies were affected by CPT treatments based on DEGs. These results provided a comprehensive view of the damage and gene expression changes in fat bodies of S. frugiperda after CPT exposure, which shall be useful to reveal the mechanism of CPT toxicity against S. frugiperda in future.

Growth inhibition of Spodoptera frugiperda larvae by camptothecin correlates with alteration of the structures and gene expression profiles of the midgut.

BACKGROUND: Spodoptera frugiperda is a serious pest that causes devastating losses to many major crops, including corn, rice, sugarcane, and peanut. Camptothecin (CPT) is a bioactive secondary metabolite of the woody plant Camptotheca acuminata, which has shown high toxicity to various pests. However, the effect of CPT against S. frugiperda remains unknown. RESULTS: In this study, bioassays have been conducted on the growth inhibition of CPT on S. frugiperda larvae. Histological and cytological changes were examined in the midgut of larvae fed on an artificial diet supplemented with 1.0 and 5.0 µg/g CPT. The potential molecular mechanism was explored by comparative transcriptomic analyses among midgut samples obtained from larvae under different treatments. A total of 915 and 3560 differentially expressed genes (DEGs) were identified from samples treated with 1.0 and 5.0 µg/g CPT, respectively. Among the identified genes were those encoding detoxification-related proteins and components of peritrophic membrane such as mucins and cuticle proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that part of DEGs were involved in DNA replication, digestion, immunity, endocrine system, and metabolism. CONCLUSIONS: Our results provide useful information on the molecular basis for the impact of CPT on S. frugiperda and for future studies on potential practical application.

Identification of azadirachtin responsive genes in Spodoptera frugiperda larvae based on RNA-seq.

The fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) is a polyphagous pest with 353 plant species as its hosts, including maize, sorghum, cotton, and rice. Azadirachtin is one of the most effective botanical insecticides. The effect of azadirachtin against S. frugiperda remains to be determined. Here we report strong growth inhibition of azadirachtin on S. frugiperda larvae under either 1.0 or 5.0 µg/g azadirachtin. To explore the relevant mechanisms, the larvae fed with normal artificial diet and with 1.0 µg/g azadirachtin exposure for 3 days were collected as samples for RNA-Seq. RNA-Seq on S. frugiperda larvae under different treatments identified a total of 24,153 unigenes, including 3494 novel genes, were identified. Among them, 1282 genes were affected by 1.0 µg/g azadirachtin exposure, with 672 up-regulated and 610 down-regulated. The impacted genes include 61 coding for detoxification enzymes (31 P450s, 7 GSTs, 11 CarEs, 7 UGTs and 5 ABC transporters), 31 for cuticle proteins, and several proteins involved in insect chitin and hormone biosynthesis. Our results indicated that azadirachtin could regulate the growth of S. frugiperda by affecting insect chitin and hormone biosynthesis pathway. The enhanced expression of detoxification enzymes might be related to detoxifying azadirachtin. These findings provided a foundation for further delineating the molecular mechanism of growth regulation induced by azadirachtin in S. frugiperda larvae.

Effects of the entomopathogenic fungus Clonostachys rosea on mortality rates and gene expression profiles in Diaphorina citri adults.

Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a serious pest of citrus. The insect also transmits Candidatus Liberibacter asiaticus, the pathogen of a devastating citrus disease called Huanglongbing. Clonostachys rosea is a versatile fungus that possesses nematicidal and insecticidal activities. The effect of C. rosea against D. citri remains unclear. Here we examined the pathogenicity of C. rosea against D. citri adults. A mortality rate of 46.67% was observed in adults treated with 1 × 108 conidia/mL spore suspension. Comparative transcriptomic analyses identified 259 differentially-expressed genes (DEGs) between controls and samples treated with fungi. Among the DEGs, 183 were up-regulated and 76 down-regulated. Genes with altered expression included those involved in immunity, apoptosis and cuticle formation. Our preliminary observation indicated that C. rosea is virulent against ACP adults and has the potential as a biological control agent for ACP management in the field.

Transcriptome analysis of putative detoxification genes in the Asian citrus psyllid, Diaphorina citri.

BACKGROUND: The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a notorious pest that transmits the causal agent of huanglongbing (also called citrus greening disease). Resistance to insecticide in this destructive pest poses a serious threat to the citrus industry. To date, no systemic studies on genes coding for detoxification enzymes has been carried out on D. citri. RESULTS: Multiple transcriptomes were generated through deep sequencing of RNA libraries. Candidate genes potentially involved in detoxification including cytochrome P450 monooxygenases (CYPs), glutathione S-transferases (GSTs), and esterases (ESTs) were systematically identified by searching the transcriptomes and a draft genome assembly. A total of 49, 14 and 20 genes were found encoding CYPs, GSTs, and ESTs, respectively, in D. citri. The total numbers of candidate detoxification genes were much smaller than the counterparts reported in other insect species, which may reflect the strict oligophagy of this insect species. Developmental stage- and tissue-specific expression patterns of the identified genes as well as their responses to insecticide treatments identified a small set of genes that could participate in detoxifying plant secondary metabolites and insecticides. CONCLUSION: Our studies represent the most comprehensive investigation to date on identification, characterization and expression profiling of detoxification genes in D. citri. The information revealed in this study shall be useful in designing strategies to manage this important insect pest. © 2020 Society of Chemical Industry.

Pro-Apoptotic Function Analysis of the Reaper Homologue IBM1 in Spodoptera frugiperda.

As an important type of programmed cell death, apoptosis plays a critical role in lepidopteran insects in response to various internal and external stresses. It is controlled by a network of genes such as those encoding the inhibitor of apoptosis proteins. However, there are few studies on apoptosis-related genes in Spodoptera frugiperda. In this study, an orthologue to the Drosophila reaper gene, named Sf-IBM1, was identified from S. frugiperda, and a full-length sequence was obtained by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends PCR (RACE-PCR). The expression pattern of Sf-IBM1 was determined in different developmental stages and various tissues. Apoptotic stimuli including azadirachtin, camptothecin, and ultraviolet radiation (UV) induced the expression of Sf-IBM1 at both transcript and protein levels. Overexpression of Sf-IBM1 induced apoptosis in Sf9 cells, and the Sf-IBM1 protein was localized in mitochondria. The apoptosis induced by Sf-IBM1 could be blocked by the caspase universal inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) and Sf-IAP1. Our results provide valuable information that should contribute to a better understanding of the molecular events that lead to apoptosis in lepidopterans.

Distinct microbial communities among different tissues of citrus tree Citrus reticulata cv. Chachiensis.

Plant microbiota colonize all organs of a plant and play crucial roles including supplying nutrients to plants, stimulating seed germination, promoting plant growth, and defending plants against biotic and abiotic stress. Because of the economic importance, interactions between citrus and microbes have been studied relatively extensively, especially citrus-pathogen interactions. However, the spatial distribution of microbial taxa in citrus trees remains under-studied. In this study, Citrus reticulata cv. Chachiensis was examined for the spatial distribution of microbes by sequencing 16S rRNA genes. More than 2.5 million sequences were obtained from 60 samples collected from soil, roots, leaves, and phloem. The dominant microbial phyla from all samples were Proteobacteria, Actinobacteria and Acidobacteria. The composition and structure of microbial communities in different samples were analyzed by PCoA, CAP, Anosim and MRPP methods. Variation in microbial species between samples were analyzed and the indicator microbes of each sample group were identified. Our results suggested that the microbial communities from different tissues varied significantly and the microenvironments of tree tissues could affect the composition of its microbial community.

Comparative transcriptomic analyses revealed genes and pathways responsive to heat stress in Diaphorina citri.

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a serious pest that feeds on plant phloem sap of citrus trees, and transmits Candidatus Liberibacter asiaticus, a bacterium that induces the destructive disease called Huanglongbing. Increasing evidence suggests that high temperatures could affect various biological traits, including size, longevity, mortality, behavior and metabolism of D. citri. However, the relevant mechanisms of heat stress remain unclear. In this study, a large set of transcriptomic data derived from D. citri adults were generated and differentially expressed genes (DEGs) after heat stress were identified by RNA sequencing. A total of 118, 399 unigenes were obtained, from which 37, 665 were mapped to sequences from at least one database. Seven hundreds and twenty-two unigenes were affected by high temperature of 40 °C for 4 h, in which 486 up-regulated and 236 down-regulated, and part of heat shock proteins, antioxidant and detoxification genes and cathepsins were identified as the DEGs. KEGG pathway enrichment analysis demonstrated that part of genes involved in protein synthesis and processing, metabolism, immunity, and signal transduction were differentially expressed under heat stress. Furthermore, the mRNA expression levels of 20 DEGs were confirmed by qRT-PCR, which verified the accuracy of high-throughput sequencing. Our results revealed that the response of D. citri adults to high temperatures is associated with a range of changes involved in various physiological and biochemical processes. Our data provide a basis for future research to improve our understanding on the molecular mechanism for heat responses in D. citri adults.

Combined transcriptomic and proteomic analysis of harmine on Spodoptera frugiperda Sf9 cells to reveal the potential resistance mechanism.

The fall armyworm, Spodoptera frugiperda, is an important invasive pest and exhibits resistance to many insecticides. Harmine, a remarkable, natural ß-carboline alkaloid, exhibits a variety of bioactivities and induces programmed cell death in Sf9 cells. In the present study, transcriptomic and proteomic analyses were combined to investigate the effects of harmine on Sf9 cells. A sublethal dose, 0.05 mM, was selected and the transcriptomic analysis revealed 2463 upregulated and 689 downregulated genes after harmine treatment. The most frequently enriched pathways of differentially expressed genes (DEGs) were mainly involved in drug and xenobiotic metabolism. The proteomics analysis revealed 36 upregulated and 77 downregulated proteins, and the results showed a nonlinear relationship with mRNA expression. All the genes related to detoxification and resistance in the transcriptome and DEGs were identified and annotated. Complete open reading frames of 27 cytochrome P450s (CYPs), 27 glutathione S-transferases (GSTs), 11 carboxylesterases (CarEs), 10 UDP-glucuronosyltransferases (UGTs) and 29 heat shock proteins (HSPs) were assembled and verified using qRT-PCR. Harmine exhibited a completely different detoxification mechanism from normal pesticides. The Sigma and Delta class GSTs and UGTs might play important roles, rather than CYP6 and CYP9 clans, CarEs or HSPs. BIOLOGICAL SIGNIFICANCE: Harmine, a natural ß-carboline alkaloid, inhibits the cell proliferation and induces programmed cell death of Sf9 cells, which derived from Spodoptera frugiperda, an important world invasive pest. In the article, the combined transcriptomic and proteomic analysis is used to explore the potential solution for its resistance management. These results supporting that harmine can be applied as a novel adjuvant or pesticide. In addition, the systematically identified resistance-related genes in fall armyworm provide the foundation for potential resistance monitoring and management.

Stability of selected reference genes in Sf9 cells treated with extrinsic apoptotic agents.

As a tightly controlled cell death process, apoptosis eliminates unwanted cells and plays a vital role in multicellular organisms. Previous study have demonstrated that apoptosis occurred in Spodoptera frugiperda cultured Sf9 cells, which triggered by diverse apoptotic stimuli, including azadirachtin, camptothecin and ultraviolet. Due to its simplicity, high sensitivity and reliable specificity, RT-qPCR has been used widespread for analyzing expression levels of target genes. However, the selection of reference genes influences the accuracy of results profoundly. In this study, eight genes were selected for analyses of their suitability as references for normalizing RT-PCR data in Sf9 cells treated with apoptotic agents. Five algorithms, including NormFinder, BestKeeper, Delta Ct method, geNorm, and RefFinder, were used for stability ranking. Based on comprehensively analysis, the expression stability of selected genes varied in cells with different apoptotic stimuli. The best choices for cells under different apoptosis conditions were listed: EF2 and EF1α for cells treated with azadirachtin; RPL13 and RPL3 for cells treated with camptothecin; EF1α and ß-1-TUB for cells irradiated under ultraviolet; and EF1α and EF2 for combinational analyses of samples. Our results not only facilitate a more accurate normalization for RT-qPCR data, but also provide the reliable assurance for further studies of apoptotic mechanisms under different stimulus in Sf9 cells.

Harmine induced apoptosis in Spodoptera frugiperda Sf9 cells by activating the endogenous apoptotic pathways and inhibiting DNA topoisomerase I activity.

Harmine, a useful botanical compound, has demonstrated insecticidal activity against some pests. However, harmine's mechanism of action has not been thoroughly elucidated to date. To preliminarily explore harmine's insecticidal mechanisms, the cytotoxicity of harmine against Spodoptera frugiperda Sf9 cells was comprehensively investigated. Our results indicated that harmine induced apoptosis in Sf9 cells, as evidenced by cellular and nuclear morphological changes, DNA laddering and increases in caspase-3-like activities. In addition, activation of the mitochondrial apoptotic pathway by harmine was confirmed by the generation of ROS, opening of mitochondrial permeability transition pores (MPTPs), increase in cytosolic Ca2+, changes in mRNA expression levels of genes involved in the mitochondrial apoptotic pathway and increase and release of Cytochrome c. Furthermore, lysosomal membrane permeabilization, release of cathepsin L from the lysosome into the cytosol and cleavage of caspase-3 were also triggered, which indicated that lysosomes were involved in this physiological process. Moreover, the effect of harmine on DNA topoisomerase I activity was investigated by in vivo and molecular docking experiments. These data not only verified that harmine induced apoptosis via comprehensive activation of the mitochondrial and lysosomal pathways and inhibition of DNA topoisomerase I activity in Sf9 cells but also revealed a mechanism of harmine insecticidal functions for pest control.