Aminopeptidase N1 is involved in Bacillus thuringiensis Cry1Ac toxicity in the beet armyworm, Spodoptera exigua.
ABSTRACT: Understanding how insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) interact with their hosts is crucial to fully explain the molecular bases of Bt specificity and insecticidal activity. Previous studies support ATP binding cassette transporters (ABCC2/3) and one cadherin-like protein are Cry1Ac functional receptors in the beet armyworm (Spodoptera exigua). In this study, a combined one-dimensional gel electrophoresis and immunoblotting approach identified aminopeptidase N (APNs) as putative Cry1Ac binding proteins in the midgut brush border membrane of S. exigua larvae. Functional analyses by gene silencing of six different S. exigua APN genes (SeAPN1, SeAPN2, SeAPN3, SeAPN4, SeAPN5 and SeAPN6) showed that only suppression of SeAPN1 resulted in decreased larval susceptibility to Cry1Ac toxin. These results support that SeAPN1 plays important functional role in Cry1Ac toxicity in S. exigua.
Project description:Host-pathogen interactions result in complex relationship, many aspects of which are not completely understood. Vip proteins, which are Bacillus thuringensis (Bt) insecticidal toxins produced during the vegetative stage, are selectively effective against specific insect pests. This new group of Bt proteins represents an interesting alternative to the classical Bt Cry toxins because current data suggests that they do not share the same mode of action. We have designed and developed a genome-wide microarray for the beet armyworm Spodoptera exigua, a serious lepidopteran pest of many agricultural crops, and used it to better understand how lepidopteran larvae respond to the treatment with the insecticidal protein Vip3Aa. With this approach, the goal of our study was to evaluate the changes in gene expression levels caused by treatment with sublethal doses of Vip3Aa (causing 99% growth inhibition) at 8 and 24 h after feeding. Results indicated that the toxin provoked a wide transcriptional response, with 19% of the microarray unigenes responding significantly to treatment. The number of up- and down-regulated unigenes was very similar. The number of genes whose expression was regulated at 8 h was similar to the number of genes whose expression was regulated after 24 h of treatment. The up-regulated sequences were enriched for genes involved in innate immune response and in pathogen response such as antimicrobial peptides (AMPs) and repat genes. The down-regulated sequences were mainly unigenes with homology to genes involved in metabolism. Genes related to the mode of action of Bt Cry proteins were found, in general, to be slightly overexpressed. The present study is the first genome-wide analysis of the response of lepidopteran insects to Vip3Aa intoxication. An insight into the molecular mechanisms and components related to Vip intoxication will allow designing of more effective management strategies for pest control.
Project description:BACKGROUND: Insecticidal toxins from Bacillus thuringiensis bind to receptors on midgut epithelial cells of susceptible insect larvae. Aminopeptidases N (APNs) from several insect species have been shown to be putative receptors for these toxins. Here we report the cloning and expression analysis of four APN cDNAs from Spodoptera exigua. RESULTS: Suppression Subtractive Hybridization (SSH) was used to construct cDNA libraries of genes that are up-and down-regulated in the midgut of last instar larvae of beet armyworm, S. exigua exposed to B. thuringiensis Cry1Ca toxin. Among the clones from the SSH libraries, cDNA fragments coding for two different APNs were obtained (APN2 and APN4). A similar procedure was employed to compare mRNA differences between susceptible and Cry1Ca resistant S. exigua. Among the clones from this last comparison, cDNA fragments belonging to a third APN (APN1) were detected. Using sequences obtained from the three APN cDNA fragments and degenerate primers for a fourth APN (APN3), the full length sequences of four S. exigua APN cDNAs were obtained. Northern blot analysis of expression of the four APNs showed complete absence of APN1 expression in the resistant insects, while the other three APNs showed similar expression levels in the resistant and susceptible insects. CONCLUSION: We have cloned and characterized four different midgut APN cDNAs from S. exigua. Expression analysis revealed the lack of expression of one of these APNs in the larvae of a Cry1Ca-resistant colony. Combined with previous evidence that shows the importance of APN in the mode of action of B. thuringiensis toxins, these results suggest that the lack of APN1 expression plays a role in the resistance to Cry1Ca in this S. exigua colony.
Project description:Crystal toxin Cry1Ca from Bacillus thuringiensis has an insecticidal spectrum encompassing lepidopteran insects that are tolerant to current commercially used B. thuringiensis crops (Bt crops) expressing Cry1A toxins and may be useful as a potential bioinsecticide. The mode of action of Cry1A is fairly well understood. However, whether Cry1Ca interacts with the same receptor proteins as Cry1A remains unproven. In the present paper, we first cloned a cadherin-like gene, SeCad1b, from Spodoptera exigua (relatively susceptible to Cry1Ca). SeCad1b was highly expressed in the larval gut but scarcely detected in fat body, Malpighian tubules, and remaining carcass. Second, we bacterially expressed truncated cadherin rSeCad1bp and its interspecific homologue rHaBtRp from Helicoverpa armigera (more sensitive to Cry1Ac) containing the putative toxin-binding regions. Competitive binding assays showed that both Cry1Ca and Cry1Ac could bind to rSeCad1bp and rHaBtRp, and they did not compete with each other. Third, Cry1Ca ingestion killed larvae and decreased the weight of surviving larvae. Dietary introduction of SeCad1b double-stranded RNA (dsRNA) reduced approximately 80% of the target mRNA and partially alleviated the negative effect of Cry1Ca on larval survival and growth. Lastly, rSeCad1bp and rHaBtRp differentially enhanced the negative effects of Cry1Ca and Cry1Ac on the larval mortalities and growth of S. exigua and H. armigera. Thus, we provide the first lines of evidence to suggest that SeCad1b from S. exigua is a functional receptor of Cry1Ca.
Project description:Study and research of Bt (Bacillus thuringiensis) transgenic plants have opened new ways to combat insect pests. Over the decades, however, insect pests, especially the Lepidopteran, have developed tolerance against Bt delta-endotoxins. Such issues can be addressed through the development of novel toxins with greater toxicity and affinity against a broad range of insect receptors. In this computational study, functional domains of Bacillus thuringiensis crystal delta-endotoxin (Cry1Ac) insecticidal protein and vegetative insecticidal protein (Vip3Aa) have been fused to develop a broad-range Vip3Aa-Cry1Ac fusion protein. Cry1Ac and Vip3Aa are non-homologous insecticidal proteins possessing receptors against different targets within the midgut of insects. The insecticidal proteins were fused to broaden the insecticidal activity. Molecular docking analysis of the fusion protein against aminopeptidase-N (APN) and cadherin receptors of five Lepidopteran insects (Agrotis ipsilon, Helicoverpa armigera, Pectinophora gossypiella, Spodoptera exigua, and Spodoptera litura) revealed that the Ser290, Ser293, Leu337, Thr340, and Arg437 residues of the fusion protein are involved in the interaction with insect receptors. The Helicoverpa armigera cadherin receptor, however, showed no interaction, which might be due to either loss or burial of interactive residues inside the fusion protein. These findings revealed that the Vip3Aa-Cry1Ac fusion protein has a strong affinity against Lepidopteran insect receptors and hence has a potential to be an efficient broad-range insecticidal protein.
Project description:The beet armyworm, Spodoptera exigua (Hübner), is a serious pest worldwide that causes significant losses in crops. Unfortunately, genetic resources for the beet armyworm is extremely scarce. To improve these resources we sequenced the transcriptome of S. exigua representing all stages including eggs, 1(st) to 5(th) instar larvae, pupae, male and female adults using the Illumina Solexa platform. We assembled the transcriptome with Trinity that yielded 31,414 contigs. Of these contigs, 18,592 were annotated as protein coding genes by Blast searches against the NCBI nr database. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step towards developing an efficient RNAi-mediated pest control technique is to find suitable target genes. To screen for effective target genes in the beet armyworm, we selected nine candidate genes. The sequences of these genes were amplified using the RACE strategy. Then, siRNAs were designed and chemically synthesized. We injected 2 µl siRNA (2 µg/µl) into the 4(th) instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (?20-94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and arf1 caused a significantly high level of mortality compared to the negative control (P<0.05). About 80% of the surviving insects in the siRNA-treated group of five genes (PGCP, chitinase1, tubulin1, tubulin2 and helicase) showed retarded development. In chitinase1-siRNA and chitinase7-siRNA administered groups, 12.5% survivors exhibited "half-ecdysis". In arf1-siRNA and arf2-siRNA groups, the body color of 15% became black 48 h after injections. In summary, the transcriptome could be a valuable genetic resource for identification of genes in S. exigua and this study provided putative targets for RNAi pest control.
Project description:The beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), is a serious polyphagous insect pest worldwide. This species is known as a long-distance migrant, and previous studies on its migration have been mostly carried out in regions where it can overwinter. However, what pattern of seasonal migration this species exhibits in regions where it cannot overwinter (i.e., the 'summer breeding region') remains unknown. Here, we present data from 14-years of monitoring on a small remote island located in the center of the Bohai Strait, in northern China, by means of searchlight trapping and ovarian dissection. We found that the population size of this overseas migration varied significantly among years, with very large migrations in 2005, 2007, 2009, and 2014 that resulted in annual total catches of more than ten thousand individuals. In addition, nightly catches exhibited a significant inter-month variability, with the vast majority of S. exigua moths being trapped in August and September, (81.1 ± 3.6%), making S. exigua one of the most frequently encountered species in that period. The mean time from the earliest trap capture to the latest capture within a given year was 113 ± 22 d (range 57 d  to 138 d ). The sex ratio (females: males) was significantly less than 1:1 in each month, but the proportion of females showed an upward trend from June to October. The majority of trapped females in summer were mated (94.4 ± 10.7% in June, 80.0 ± 6.4% in July) and sexually mature (88.9 ± 11.1% in June, 61.8 ± 12.3% in July), suggesting the onset of mating and/or sexual maturation does not terminate the migration behavior in this species. These findings provide a good starting point for study of the trans-regional migration of S. exigua across different climate zones.
Project description:Three cell lines (QAU-Se-E-1, -2 and -3, or Se-1, -2 and -3 for short) were established from eggs of beet armyworm (Spodoptera exigua) that have been passaged stably for more than 60 times in TNM-FH medium supplemented with 10 % fetal bovine serum. The cell lines consisted of round and spindle-shaped cells. The round cells accounted for 96.82, 84.34 and 83.16 % of the cells in the three cell lines, respectively, with cell diameters of 16.21 ± 0.72, 15.63 ± 0.58 and 13.06 ± 0.44 ?m. Random amplified polymorphic DNA and analysis of the CO I gene showed that the three cell lines were all derived from S. exigua. Growth curves at passage 30 were determined and the results showed that the cell population doubling times were 59.03, 49.08 and 49.91 h, respectively. The three cell lines can be infected by S. exigua multiple nucleopolyhedrovirus (SeMNPV). Se-3 was extremely susceptible to the virus with an infection rate of 97.52 % 4 days after the inoculation and produced 2.02 × 10(6) OBs per mL of culture. Flow cytometry analysis showed that some of Se-1 and Se-2 cells had apoptosis after infection, whereas Se-3 cells did not. Bioassays showed that the virulence of the SeMNPV proliferated from Se-3 was similar to that from the insect with LC50 of 5.55 × 10(5) and 2.64 × 10(5) OBs/mL. Therefore, the cell lines can be used to study the SeMNPV-host interactions and mechanisms underlying the interactions.
Project description:The beet armyworm, Spodoptera exigua (Hubner), is one of the major crop pests and is a target for current pest control approaches using insecticides. In this study three cuticular protein genes CPG316, CPG860 and CPG4855 have been cloned from 0?h pupal integument of S. exigua through race PCR Strategy. The deduced amino acid sequences were found to contain the RR-2 consensus region of other insect cuticular proteins and construct phylogenetic trees for each protein. Using quantitative RT-PCR, the developmental expression of the three genes through several larval and the early pupal stages was studied. All three genes contribute to the endocuticle although CPG316 may have a different role from the other two genes. All three newly isolated genes were analyzed and their functions were determined by using direct injection of the dsRNA into early 5th instar larvae. All genes are expressed in the larvae and early pupae but in different patterns. Furthermore, phenotypic results show that these genes have differing effects on the development of cuticle, its flexibility and a big role in metamorphosis in both larval and pupal stages.
Project description:Cellular immunity in insects is accompanied by change in hemocyte shape. This study hypothesizes that cytoskeletal rearrangement is accompanied by transmembrane water transport to change cell volume, thus changing cell shape. A water-transporting pore (=aquaporin:AQP) has been identified in the beet armyworm, Spodoptera exigua. Its expression was detected in all developmental stages and tissues, although its transcription levels were different between biotic and abiotic conditions. Heterologous expression of Se-AQP in Sf9 cells showed that Se-AQP was localized on cell membrane. RNA interference (RNAi) using double-stranded RNA effectively suppressed its transcript levels. Under different ionic concentrations, hemocytes of RNAi-treated larvae did not change cell volume presumably due to malfunction in water transportation. Se-AQP might participate in glycerol transport because up-regulation of hemolymph glycerol titer after rapid cold-hardening was prevented by RNAi treatment against Se-AQP expression. The inhibitory effect of RNAi treatment on change of cell shape significantly impaired cellular immune responses such as phagocytosis and nodule formation upon bacterial challenge. RNAi treatment also significantly interfered with immature development of S. exigua. These results indicate that Se-AQP plays a crucial role in cell shape change that is required for cellular immunity and other physiological processes.
Project description:Insect resistance to <i>Bacillus thuringiensis</i> (Bt) insecticidal proteins has rapidly evolved with the expansion of the planting area of transgenic Bt crops. Pyramiding RNA interference (RNAi) and Bt in crops is urgently needed to counter the rapid increase in pest resistance. The ideal "pyramid" strategy simultaneously targets different action pathways that exert synergetic effects on each other. Here, we identified a dephosphatase, namely, <i>Helicoverpa armigera</i> calcineurin (HaCAN), which might enhance the insecticidal activity of Cry1Ac against <i>Helicoverpa armigera</i> by regulating immune gene expression via dephosphatase activity, but not by acting as a receptor. Notably, blocking enzyme activity or knocking down endogenous HaCAN significantly promoted the enhancement in Cry1Ac toxicity to insect larvae and cells. Correspondingly, the increase in HaCAN activity reduced the cytotoxicity of Cry1Ac as shown by the heterologous expression of HaCAN. Our results provide a probable that HaCAN is an important candidate gene for pyramiding RNAi and Cry1Ac crops to control cotton bollworm.