茧蜂病毒感染Hi5细胞与亲环素A的相关性研究

茧蜂病毒（Microplitis bicoloratus bracovirus,MbBV）属于多分DNA病毒（polydnavirus,PDV）的一种,主要存在于膜翅目茧蜂科寄生蜂中,对于寄生蜂成功寄生宿主起着至关重要的作用。而亲环素A（cyclophilin A,CypA）是一种肽基脯氨酰顺反异构酶,参与免疫反应等多种细胞活动。主要探讨了茧蜂病毒在感染昆虫细胞的过程中,是否与CypA存在相关性。研究结果显示,在粉纹夜蛾(Trichoplusia ni Hübner)卵细胞系（High Five,Hi5）培养基中加入茧蜂病毒24 h后,通过PCR可扩增出与病毒基因大小一致的目的片段,表明Hi5细胞已被茧蜂病毒感染。在病毒感染细胞后,CypA的基因转录水平显著升高,其蛋白表达水平也有所增加;当沉默cypa基因或抑制CypA活性后,实时荧光定量PCR（real-time fluorescence quantitative PCR,qRT-PCR）结果显示,茧蜂病毒基因中的vank86基因转录水平显著下降;而过表达cypa基因,可使vank86基因转录水平上升。研究结果提示,茧蜂病毒在感染昆虫细胞的过程中,可能与CypA存在一定的相关性。     

Disruption effect of Microplitis bicoloratus polydnavirus EGF-like protein, MbCRP, on actin cytoskeleton in lepidopteran insect hemocytes

Microplitis bicoloratus is a braconid endoparasitic wasp associated with the polydnavirus named Microplitis bicoloratus bracovirus （MbBV）. Parasitism of Spodoptera litura larvae leads to an impaired cellular immune response and to the disappearance of the 42 kDa actin in host hemocytes. In this work, we investigated if the absence of actin in blood cells was related to MbBV infection. An MbBV gene similar to egf-like genes identified in another bracovirus was partially cloned and named Mbcrp1. The full-length gene, named Mbcrp, is transcribed throughout the course of parasitism in host hemocytes and the 30 kDa MbCRP protein was detected in hemocytes 6-7 d post-parasitization. The Mbcrp1 gene contains the cysteine-rich trypsin inhibitor-like （TIL） domain coding sequence and the expression of recombinant MbCRP1 inhibited the expression of the 42 kDa actin in Hi5 cells. The 34.1 kDa MbCRPl-green fluorescent protein fusion protein locate specifically in the cytoplasm. These results suggest that expression of MbCRP in lepidopteran insect cells is related to the disruption of the actin cytoskeleton.     

A Role for Innexin2 and Innexin3 Proteins from Spodoptera litura in Apoptosis

Gap junctions formed by two hemichannels from two neighboring cells are cell-to-cell communication channels; hemichannels are communication channels between intracellular and extracellular environments. Hemichannels are hexameric proteins formed by connexins, pannexins, innexins and vinnexins. Innexin-hemichannels (innexons) exist in the lepidopteran cell surface, but their component innexins and functions have not been reported. Recent studies by others have demonstrated that hemichannels, connexons and pannexons from vertebrates serve as regulators of apoptosis via inactivating the PI3K/Akt signaling pathway. Here, the apoptogenic properties of innexons are demonstrated using two innexin cDNAs, Spli-inx2 and Spli-inx3, which were isolated from hemocytes of lepidopteran Spodoptera litura. Alignment analysis revealed that these two genes belong to a conserved innexin family, as they contain the insect signature YYQWV motif at the beginning of the second transmembrane domain. Immunofluorescence showed that two fusion proteins, Inx2-V5 and Inx3-V5, were localized predominantly in the cell membrane, cytoplasm and also nuclei. Ectopic expression in Sf9 cells and over-expression of Inx2 and Inx3 in Spli221 cells promoted apoptosis. In the Spli221 cells, apoptotic cells presented remarkable membrane blebbing. This study also showed that Sf9 and Spli221 cells undergo low level apoptosis under normal culture conditions, but not Hi5 cells. In Hi5 stable cell lines, biotinylation was used to isolate surface proteins and confirm Inx2 and Inx3 localization in the cell membrane and also further data showed that Hi5 cells may activate the PI3K signaling pathway via phosphorylating molecular Akt downstream. This result suggests that innexon-promoted apoptosis may be involving the PI3K/Akt signaling pathway. These findings will facilitate further examinations of the apoptotic regulation by the PI3K/Akt signaling pathway and comparative studies of innexons, connexons, pannexons, and vinnexons.     

Mitochondria are involved in apoptosis induced by ultraviolet radiation in lepidopteran Spodoptera litura cell line

Abstract Mitochondria are involved in apoptosis of mammalian cells and even single‐cell organisms, but mitochondria are not required in apoptosis in cultured Drosophila cells such as S2 and BG2 cell lines. It is not very clear whether mitochondria are involved in apoptosis in other insect cells such as lepidopteran cell lines. Thus, we determined to elucidate the role of mitochondria in apoptosis induced by ultraviolet radiation in Spodoptera litura (Lepidoptera: Noctuidae) cell line (SL‐ZSU‐1). The Western blot results suggested that cytochrome c in the ultraviolet‐treated SL‐1 cells was released from the mitochondria to cytosol as early as 4 h after the induction of ultraviolet radiation and increased in the cytosolic fractions in a time‐dependent manner. Flow cytometric analysis of mitochondrial membrane potential (ΔΨm) of SL‐ZSU‐1 cell treated with ultraviolet‐C (UV‐C) light indicated the decrease in mitochondrial membrane potential was dependent on the times of ultraviolet treatment. Both of them are different from apoptosis in cultured Drosophila melanogaster cell lines (S2 and BG2) and it appears evident mitochondria are involved in apoptosis of the studied lepidopteran cells.     

MOLECULAR CHARACTERIZATION OF AUTOPHAGY‐RELATED GENE 5 FROM Spodoptera exigua AND EXPRESSION ANALYSIS UNDER VARIOUS STRESS CONDITIONS

Autophagy is not only involved in development, but also has been proved to attend immune response against invading pathogens. Autophagy protein 5 (ATG5) is an important autophagic protein, which plays a crucial role in autophagosome elongation. Although ATG5 has been well studied in mammal, yeast, and Drosophila, little is known about ATG5 in lepidopteran insects. We cloned putative SeAtg5 gene from Spodoptera exigua larvae by the rapid amplification of cDNA ends method, and its characteristics and the influences of multiple exogenous factors on its expression levels were then investigated. The results showed that the putative S. exigua SeATG5 protein is highly homologous to other insect ATG5 proteins, which has a conserved Pfm domain and multiple phosphorylation sites. Next, fluorescence microscope observation showed that mCherry‐SeATG5 was distributed in both nucleus and cytoplasm of Spodoptera litura Sl‐HP cells and partially co‐localized with BmATG6‐GFP, but it almost has no significant co‐localization with GFP‐HaATG8. Then, the Western blot analysis demonstrated that GFP‐SeATG5 conjugated with ATG12. Moreover, real‐time PCR revealed that its expression levels significantly increased at the initiation of pupation and the stage of adult. In addition, the expression levels of SeAtg5 can be enhanced by the starvation, UV radiation, and infection of baculovirus and bacterium. However, the expression levels of SeAtg5 decreased at 24 h post treatments in all these treatments except in starvation. These results suggested that SeATG5 might be involved in response of S. exigua under various stress conditions.     

RECOVERY RESPONSE OF HETERORHABDITIS BACTERIOPHORA AND STEINERNEMA CARPOCAPSAE TO DIFFERENT NON‐SYMBIOTIC MICROORGANISMS*

Abstract Axenic Steinernema carpocapsae Agriotos (A24) and Heterorhabditis bacteriophora H06 dauer juveniles were exposed to Spodoptera litura insect cell cultures, and the cell‐free filtrates or cells of different non‐symbiotic microorganism cultures, including Bacillus subtilis, B. thuringiensis, Pseudomonas fluorescens, Micromonospora purpurea, Rhizopus delemar, Pseudomonas aeruginosa, Streptomyces venezuelae, Streptomyces antibioticus, Penicillium citrnum, Ganoderma lucidum, Agaricus bisporus, Pleurotus ostreatus, Rhizobium legumiunosarum, and Photobacterium phosphoreum. None of these cell‐free filtrates or cultures, or insect cell culture triggered recovery of H. bacteriophora H06. However, cell‐free filtrate of P. phosphoreum induced recovery of S. carpocapsae A24, although the cell culture of this bacterium kill the A24 dauer juveniles before recovery. S. litura insect cells provided the nutrients for axenic S. carpocapsae A24 nematode growth and next generation of dauer juveniles were observed. These results further demonstrated that food signals were much more specific to H. bacteriophora than to S. carpocapsae.     

A new cell line from Spodoptera exigua (Lepidoptera: Noctuidae) and its differentially expressed genes

A new cell line, designated IOZCAS‐Spex XI, was established from the pupal ovaries of Spodoptera exigua (Lepidoptera: Noctuidae) in TNM‐FH medium containing 10% foetal bovine serum. The spherical cells were predominant among the various cell types. The population‐doubling time during the logarithmic phase of growth was 81.7 h. It was confirmed that the cell line originated from S. exigua by DAF‐PCR technique. Analysis of susceptibility to baculovirus showed that the new cell line was susceptible to S. exigua nucleopolyhedrovirus (SeNPV), Autographa californica multiple NPV (AcMNPV) and slightly susceptible to S. litura NPV (SpltNPV), while not permissive to Helicoverpa armigera NPV and Hyphantria cunea NPV (HcNPV). Real‐Time PCR analysis was carried out to compare some differentially expressed genes between the cell line and the primary culture. The result showed that marked significant differences were observed in the expression of the genes of SUMO‐1 activating enzyme, BCCIP‐like protein, 10 kDa HSP, CypA, receptor for activated PKC, PDI‐like protein ERp57, ALDH, DEAD box ATP‐dependent RNA helicase‐like protein (P < 0.01), while a significant difference was obtained in the expression of GST gene between the cell line and the primary culture (P < 0.05).     

Highly passage of <Emphasis Type="Italic">Spodoptera litura</Emphasis> cell line causes its permissiveness to baculovirus infection

It is well known that the characteristics of cell lines possibly alter when cell lines are at high-passage number because of the environmental selection. We do not know whether non-permissive or low-permissive cell lines could become permissive or more permissive to virus infection after over-high passage. In the present studies, the alteration of the permissiveness of Spodoptera litura cell line Sl-zsu-1 to three baculovirus infection was investigated after over-high passage, and the possible mechanisms are also investigated. Vigorous apoptosis in Sl-zsu-1 cells was induced by both the recombinant Autographa californica multiple nucleopolyhedrovirus AcMNPV-GFP-actin and the celery looper Anagrapha falcifera multiple nucleopolyhedrovirus AfMNPV, suggesting the replication of the two viruses was blocked by apoptosis. However, the cells infected by S. litura multicapsid nucleopolyhedrovirus SpltMNPV did not undergo apoptosis, but the SpltMNPV titre of the supernatant was not detectable, suggesting this cell line was low-permissive for this virus infection and other factor(s) involved in blockage of the virus replication except apoptosis. However, when Sl-zsu-1 cells had been subcultured continuously for more than 4 years (high-passage cell), which was named as Sl-HP cell line afterwards, no significant apoptosis was induced by the three baculovirus in Sl-HP cells, and many replicated virions or nucleocapsids were observed in the cells. But the permissiveness of Sl-HP cells to the three viruses was very different according to the titre of viruses in the cell cultures. Interestingly, the DNA extracted from SpltMNPV could induce vigorous apoptosis of Sl-HP cells. Altogether, Sl-zsu-1 cell line becomes more permissive to baculovirus infection after over-high passage and multiple paths can block the baculovirus infectivity.     

RNA interference of β1 integrin subunit impairs development and immune responses of the beet armyworm, Spodoptera exigua

Integrin is a cell surface protein that is composed of α and β heterodimer and mediates cell interaction with extracellular matrix or other cells including microbial pathogens. A full length cDNA sequence (2862 bp) of a β1 subunit integrin (βSe1) was cloned from the beet armyworm, Spodoptera exigua. Phylogenetic analysis showed that βSe1 was clustered with other insect β integrin subunits with the highest amino acid sequence identity (98.3%) to β1 of Spodoptera litura. Structural analysis of the deduced amino acid sequence indicated that βSe1 possessed all functional domains known in other insect β1 integrins. RT-PCR analysis showed that βSe1 was expressed in all developmental stages and all tested tissues of S. exigua. Its expression was further upregulated in hemocytes by injections of various microbes from quantitative RT-PCR analysis. Injection of double-stranded βSe1 RNA (dsRNA^βSe1) into late instar S. exigua suppressed βSe1 expression and resulted in significant reduction in pupal weight. The dsRNA^βSe1 injection significantly impaired hemocyte-spreading and nodule formation of S. exigua in response to bacterial challenge. Furthermore, oral ingestion of dsRNA^βSe1 induced reduction of βSe1 expression in midgut and resulted in significant mortality of S. exigua during immature development. These results suggest that βSe1 plays crucial roles in performing cellular immune responses as well as larval development in S. exigua.     

Using cell size kinetics to determine optimal harvest time for <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> and <Emphasis Type="Italic">Trichoplusia ni</Emphasis> BTI-TN-5B1-4 cells infected with a baculovirus expression vector system expressing enhanced green fluorescent protein

Infecting insect cells with a baculovirus expression vector system (BEVS) is an increasingly popular method for the production of recombinant proteins. Due to the lytic nature of the system, however, determining the optimal harvest time is critical for maximizing protein yield. We found that measuring the change in average diameter during the progress of infection with an automated cell analysis system (Cedex HiRes, Innovatis AG) could be used to determine the time of maximum protein production and, thus, optimal harvest time. As a model system, we use insect cells infected with a baculovirus expressing enhanced green fluorescent protein (EGFP). We infected two commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-TN-5B1-4 (Hi5) with an Autographa californica nuclear polyhedrosis virus (AcNPV) encoding EGFP at various multiplicities of infection (MOI). We monitored the progress of infection with regard to viability, viable cell density and change in average cell diameter with a Cedex HiRes analyzer and compared the results to the EGFP produced. Peak protein production was reached one to two days after the point of maximum average diameter in all conditions. Thus, optimal harvest time could be determined by monitoring the change in average cell diameter during the course of an infection of a cell culture.     

Lethal and sub‐lethal effects of spinosad on the life‐history traits of army worm,Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), and its fitness cost of resistance

Spodoptera litura is one of the most destructive polyphagous insect pests, with more than 120 host‐plant species. In our present study, a field‐collected population of S. litura when selected with spinosad for 11 consecutive generations resulted in the development of 3921‐fold resistance to spinosad as compared to the susceptible strain. The spinosad‐resistant strain of S. litura had a relatively high fitness cost (0.17) as compared to the susceptible strain. Furthermore, the lethal and sub‐lethal effects of different concentrations of spinosad were checked on the susceptible strain at different levels; i.e., LC₄₀, LC₃₀, LC₂₀ and LC₁₀, which revealed that the impact of spinosad on the life‐history traits of S. litura increased with the increase in concentration of spinosad. A significant impact of spinosad was recorded on the larval duration, pre‐pupal weight, pupal duration, pupal weight, reproductive potential and adult emergence. The outcomes of the current research clearly indicate that fitness cost of spinosad and its sub‐lethal effects have a significant impact on population dynamics of S. litura, for which it can be incorporated in integrated pest management.     

Cytochrome c and insect cell apoptosis

Abstract The role of cytochrome c in insect cell apoptosis has drawn considerable attention and has been subject to considerable controversy. In Drosophila, the majority of studies have demonstrated that cytochrome c may not be involved in apoptosis, although there are conflicting reports. Cytochrome c is not released from mitochondria into the cytosol and activation of the initiator caspase Dronc or effector caspase Drice is not associated with cytochrome c during apoptosis in Drosophila SL2 cells or BG2 cells. Cytochrome c failed to induce caspase activation and promote caspase activation in Drosophila cell lysates, but remarkably caused caspase activation in extracts from human cells. Knockdown of cytochrome c does not protect cells from apoptosis and over‐expression of cytochrome c also does not promote apoptosis. Structural analysis has revealed that cytochrome c is not required for Dapaf‐1 complex assembly. In Lepidoptera, the involvement of cytochrome c in apoptosis has been demonstrated by the accumulating evidence. Cytochrome c release from mitochondria into cytosol has been observed in different cell lines such as Spodoptera frugiperda Sf9, Spodoptera litura Sl‐1 and Lymantria dispar LdFB. Silencing of cytochrome c expression significantly affected apoptosis and activation of caspase and the addition of cytochrome c to cell‐free extracts results in caspase activation, suggesting the activation of caspase is dependent on cytochrome c. Although Apaf‐1 has not been identified in Lepidoptera, the inhibitor of apoptosome formation can inhibit apoptosis and caspase activation. Cytochrome c may be exclusively required for Lepidoptera apoptosis.     

Unexpected link between insect innexins and apoptosis of HeLa cells

Little is known about how mammalian cells respond to the expression of innexins (Inxs), which are known to mediate cell‐to‐cell communication that causes apoptosis in the cells of the insect Spodoptera litura. The mammalian expression system, p3xFLAG tag protein, containing the CMV promoter, allowed us to construct two C‐terminally elongated innexins (Cte‐Inxs), SpliInx2 (Inx2‐FLAG), and SpliInx3 (Inx3‐FLAG), which were predicted to have the same secondary topological structures as the native SpliInx2 and SpliInx3. Here, we found that only the mRNAs of the two Cte‐Inxs were expressed under the control of the CMV promoter in HeLa cells. Unexpectedly, mRNA expression of the two Cte‐Inxs enhanced apoptosis of HeLa cells. The two Cte‐Inx mRNAs were associated with a significant decrease in Akt phosphorylation in HeLa cells undergoing apoptosis. Furthermore, Inx3‐FLAG mRNA expression in nonapoptotic HCT116 cells was also associated with a significant decrease in the levels of phosphorylated Akt. Intriguingly, expression of the mRNAs of the two Cte‐Inxs did not activate caspase 3, but it markedly reduced Bid levels in HeLa cells undergoing apoptosis. These results suggest that mRNA expression of the two Cte‐Inxs may activate a Bid‐dependent apoptotic pathway in HeLa cells. Our study demonstrates that invertebrate gap junction mRNAs can function in vertebrate cancer cells as tumor suppressors.     

Identification and functional characterization of doublesex gene in the testis of Spodoptera litura

Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at Go generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.     

CRISPR/Cas9‐mediated ebony knockout results in puparium melanism in Spodoptera litura

Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.     

Genome profiling implies high genetic diversity in microsporidia isolated from the common cutworm, <Emphasis Type="Italic">Spodoptera litura</Emphasis> (Lepidoptera: Noctuidae), in Vietnam

In order to evaluate the potential application of microsporidia as a microbial control agent against lepidopteran insect pests, microsporidian infection in a field population of the common cutworm, Spodoptera litura (Fabricius), was surveyed in vegetable crop fields in Can Tho City, Vietnam, in March 2007. The infection rate of microsporidia was 46.7% (99/212 individuals) in adult S. litura, and 16 samples of infected adults were used to characterize the microsporidia at the molecular level. Analysis of the small subunit ribosomal RNA (SSU rRNA) sequences indicated that microsporidian strains isolated from S. litura were closely related to Nosema bombycis from the silkworm, Bombyx mori (Linnaeus); however, phylogenetic analysis based on genome profiling produced a different result from the SSU rRNA sequences. Temperature gradient gel electrophoresis profiles of 12 microsporidian strains from S. litura were closely related to N. bombycis strains, while the profiles of three microsporidian strains formed a different cluster. The Vietnamese strains did not form a single group, but were classified into at least three groups. These results suggested that the microsporidia isolated from S. litura in the Mekong Delta, Vietnam, are genetically diverse.     

Cover Caption

The common cutworm, Spodoptera litura (Lepidoptera: Noctuidae) is one of the most destructive phytophagous pests of crops. Body coloration affects how animals interact with the environment, and pigmentation also influences behavior and immunity. To explore the mechanisms that drive pigmentation, the CRISPR/Cas9 genome editing system was applied to target the ebony gene in the non‐model insect S. litura. The mutants showed the typically deep coloration during the pupal and adult stage. This study clearly demonstrates the function of ebony in the body coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests (see pages 1011–1019). Photo provided by Hong‐Lun Bi.