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.

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.

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.

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.

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.

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.

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.

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.

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.

Curcumin-induced autophagy and nucleophagy in Spodoptera frugiperda Sf9 insect cells occur via PI3K/AKT/TOR pathways.

Compounds from plants or microbes are important resources for new natural pesticides against a wide variety of pests. The growing attention on the role of autophagy (type II cell death) in regulation of insect toxicology has propelled researchers to investigate autophagic cell death pathways. Our previous study proved that the cytotoxic effect of curcumin in Spodoptera frugiperda cells is regulated by autophagy. However, the signaling pathways and molecular mechanisms had not been determined. The current study elucidates curcumin inhibition of survival signaling by blocking the activation of PI3K/AKT/TOR pathways to induce autophagy in S. frugiperda cells. The result demonstrates that nucleophagy associated with cell death following the curcumin treatment. Following the curcumin treatment, Atg8/LC3 immunostaining in both nucleus and cytoplasm was markedly increased. Further, messenger RNA expression level of Atg8 and Atg1 genes regulation by curcumin was examined using quantitative reverse transcription polymerase chain reaction, and the result exhibited increased level of expression after curcumin treatment in a time-dependent manner. Our current study provides new insights to the autophagy occurring via PI3K/AKT/TOR pathways in S. frugiperda Sf9 insect cells induced by curcumin. Taken together, our results show for the first time that curcumin induced nucleophagy in lepidopteron insect cell line.

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.

Natural ß-carboline alkaloids regulate the PI3K/Akt/mTOR pathway and induce autophagy in insect Sf9 cells.

The ß-carboline alkaloids are a large group of naturally occurring and synthetic indole alkaloids with remarkable pharmacological properties. Furthermore, these alkaloids have also been reported to be effective agents for controlling many pests and plant pathogenic nematodes. However, studies on these potential insecticidal components are scarce. The previous finding that these bioactive compounds can induce programmed cell death in cancer cell lines provided a new insight for exploration of their toxicological mechanisms on insects. In the present study, the cytotoxicity of five natural harmala alkaloids was measured, and the autophagy-inducing effect was confirmed in the Spodoptera frugiperda Sf9 cultured cell line. The results demonstrated that these alkaloids inhibited the proliferation of Sf9 cells in a dose- and time-dependent manner, and the unsaturated ß-carboline alkaloids, harmine and harmol, exhibited stronger autophagy induction activity based on monodansylcadaverineand LysoTracker Red staining. Many autophagy-related genes were increased after ß-carbolines treatment at the RNA level, and the protein expression of Sf-Atg8 was also confirmed to increase after treatment. In addition, the primary autophagic signaling pathway, the PI3K/Akt/mTOR pathway, was altered during the procedure. Furthermore, experiments with special inhibitors and activators were performed to confirm the effect of ß-carbolines on this pathway. The results suggested that the PI3K/Akt/mTOR pathway primarily regulated harmine-induced autophagy in insect cells, and this finding may potentially benefit the application of these promising bioactivity components.

Proteomic Profiling Analysis of Male Infertility in Spodoptera Litura Larvae Challenged with Azadirachtin and its Potential-Regulated Pathways in the Following Stages.

Biopesticides are considered as an alternative to synthetic pesticide with a focus on increasing agricultural productivity as well as maintaining the ecosystem. Prior to application, its potential mechanism should be clearly addressed. Here, the effects of azadirachtin on the reproductive behavior in male Spodoptera litura (Fabricius) are determined. To further explore its molecular mechanism, an iTRAQ (isobaric tags for relative and absolute quantitation) based approach is applied to identify the differentially expressed proteins regulated by azadirachtin at two developmental stages. The results demonstrate that many proteins in the pathway of focal adhesion are regulated to exert influences in detachment of cell attachment, the loss of cell-cell interactions and inducing apoptosis at pupal stage, and many proteins in adenosine monophosphate-activated protein kinase pathway are also changed at the adult stage after azadirachtin-treatment as larvae. Moreover, based on their important roles, it is suggested that some proteins, such as ACTB-G1, ste20-related adaptor protein alpha, and regulatory-associated protein of mTOR (mTORC1) could serve as potential target proteins of azadirachtin to induce male infertility. The results of this study could provide evidence to illuminate the mechanism of male infertility induced by azadirachtin and potential targets for the development of environmentally friendly pesticides.

DnaJ homolog subfamily A member1 (DnaJ1) is a newly discovered anti-apoptotic protein regulated by azadirachtin in Sf9 cells.

BACKGROUND: Azadirachtin, one of the most promising botanical insecticides, has been widely used for pest control. Azadirachtin induces apoptosis in insect cell lines, including Sf9, SL-1 and BTI-Tn-5B1-4. Mitochondrial and lysosomal pathways are likely involved in the azadirachtin-induced apoptosis, however, detailed molecular mechanisms remain largely undefined. RESULTS: Azadirachtin-induced apoptosis in Sf9 cells was verified by morphological observation, Hoechst 33258 staining, and a Caspase-3-based analysis. Comparative two-dimensional gel electrophoresis (2-DE) coupled with a linear ion trap quadrupole (LTQ)-MS/MS analysis identified 12 prominent, differentially expressed proteins following azadirachtin treatment. These differentially expressed genes are involved in regulating cytoskeleton development, signal transduction, gene transcription, and cellular metabolism. Knockdown gene expression of a gene encoding a DnaJ homolog enhanced apoptosis induced by azadirachtin in Sf9 cells. CONCLUSION: Azadirachtin treatment induces apoptosis in Sf9 cells and affects expression of multiple genes with functions in cytoskeleton development, signal transduction, gene regulation, and cellular metabolisms. Azadirachtin induces apoptosis at least partially by down-regulation of Sf-DnaJ in Sf9 cells.

Cytotoxic and Apoptotic Activity of the Novel Harmine Derivative ZC-14 in Sf9 Cells.

Harmine, one of the natural ß-carboline alkaloids extracted from Peganum harmala L., exhibits broad spectrum but limited insecticidal ability against many pests. So there is an urgent need to synthesize novel derivatives with high efficiency. In the present study, a new synthetic compound, [1-(2-naphthyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl) ß-carboline] (ZC-14), showed a strong proliferation inhibition effect against the Spodoptera frugiperda Sf9 cell line in a dose-dependent manner. Simultaneously, apoptosis induced by 7.5 µg/mL ZC-14 was confirmed with physiological and biochemical evidence, including typical apoptosis characteristics with shrinkage, apoptotic bodies, nuclear condensation/fragmentation, a clear DNA ladder, and a series of apoptotic rates. In addition, mitochondria were confirmed to be involved in apoptosis induced by ZC-14 accompanied with the loss of mitochondrial membrane potential (Δψm), the release of cytochrome c from mitochondria into the cytosol and increased expression of cleaved-caspase-3. However, harmine could not induce apoptosis at the same concentration. In summary, these data indicated that compound ZC-14 has a higher cytotoxicity than harmine against Sf9 cells. Besides, it exhibited an anti-proliferative effect in Sf9 cells via inducing apoptosis in which the mitochondrial apoptotic pathway plays a crucial role.

Curcumin induces autophagic cell death in Spodoptera frugiperda cells.

The increasing interest in the role of autophagy (type II cell death) in the regulation of insect toxicology has propelled study of investigating autophagic cell death pathways. Turmeric, the rhizome of the herb Curcuma longa (Mañjal in Tamil, India and Jianghuáng in Chinese) have been traditionally used for the pest control either alone or combination with other botanical pesticides. However, the mechanisms by which Curcuma longa or curcumin exerts cytotoxicity in pests are not well understood. In this study, we investigated the potency of Curcuma longa (curcumin) as a natural pesticide employing Sf9 insect line. Autophagy induction effect of curcumin on Spodoptera frugiperda (Sf9) cells was investigated using various techniques including cell proliferation assay, morphology analysis with inverted phase contrast microscope and Transmission Electron Microscope (TEM) analysis. Autophagy was evaluated using the fluorescent dye monodansylcadaverine (MDC). Cell death measurement was examined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) within the concentrations of 5-15µg/mL. Curcumin inhibited the growth of the Sf9 cells and induced autophagic cell death in a time and dose dependent manner. Staining the cells with MDC showed the presence of autophagic vacuoles while increased in a dose and time dependent manner. At the ultrastructural level transmission electron microscopy, cells revealed massive autophagy vacuole accumulation and absence of chromatin condensation. Protein expression levels of ATG8-I and ATG8-II, well-established markers of autophagy related protein were elevated in a time dependent manner after curcumin treatment. The present study proves that curcumin induces autophagic cell death in Sf9 insect cell line and this is the first report of cytotoxic effect of curcumin in insect cells and that will be utilized as natural pesticides in future.

A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9.

The induction of apoptosis by azadirachtin, a well-known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 µg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 µg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC50 at 48 and 72 h was 2.727 × 10(-6) and 6.348 × 10(-9) µg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase-1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase-dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds.

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.