Heliothis Virescens Ascovirus 3h Research Articles

Flexible changes to the Heliothis virescens ascovirus 3h (HvAV-3h) virion components affect pathogenicity against different host larvae species

ABSTRACT The pathogenicity of a virus to a specific host species is an inerratic and describable ability of a virus to cause infection but is generally shaped by a variety of abiotic and biotic factors. In this investigation, the variations in pathogenicity of Heliothis virescens ascovirus 3h (HvAV-3h) to five noctuid pests were assessed based on mass spectrometry analysis on the virion compositions. Twenty-nine common HvAV-3h proteins were shared across all hosts, and different flexible proteins were identified in the virions of each specific host. Different host proteins were identified as HvAV-3h virion-associated proteins, including different detoxification enzyme proteins. Furthermore, a relatively fixed relationship between viral replication and changes in host detoxification enzyme activity caused by deficiencies in various viral structural proteins was found in the host larvae using a correlation matrix analysis: the host larval carboxylesterase and cytochrome P450 monooxygenases generally had highly similar responses to the viruses blocked by different structural proteins’ antisera and their effects on viral DNA replication. Different interaction patterns for the virion structural proteins were found in different host larvae-produced virions, and the interactions between Spodoptera litura glutathione S-transferases and viral structural proteins were confirmed. The different host responses after viral infection could be the reason for the changes in viral pathogenicity, while the virus responses gradually adapted to the different hosts and there were flexible changes in the virion structures. IMPORTANCE Different pathogenic processes of a virus in different hosts are related to the host individual differences, which makes the virus undergoes different survival pressures. Here, we found that the virions of an insect virus, Heliothis virescens ascovirus 3h (HvAV-3h), had different protein composition when they were purified from different host larval species. These “adaptive changes” of the virions were analyzed in detail in this study, which mainly included the differences of the protein composition of virions and the differences in affinity between virions and different host proteins. The results of this study revealed the flexible changes of viruses to help themselves adapt to different hosts. Also, these interesting findings can provide new insights to improve our understanding of virus adaptability and virulence differentiation caused by the adaptation process.

Spodoptera litura larvae are attracted by HvAV-3 h-infected S. litura larvae-damaged pepper leaves.

Herbivore-induced plant volatiles (HIPVs) are important self-defense outputs of pepper plants to resist insect pests. Ascoviruses are pathogenic to the larvae of most lepidopteran vegetable pests. However, whether Heliothis virescens ascovirus 3 h (HvAV-3 h)-infected Spodoptera litura larvae can change pepper leaf HIPVs is not well understood. S. litura larvae preferred S. litura-infested leaves, and this preference was stronger with longer duration of S. litura infestation. In addition, S. litura larvae significantly chose pepper leaves damaged by HvAV-3 h-infected S. litura over the healthy pepper leaves. Results also showed that S. litura larvae preferred leaves mechanically damaged and treated with oral secretions from HvAV-3 h infected-S. litura larvae in a simulation test. We captured the volatiles emitted by leaves under 6 treatments. Results showed that the volatile profile changed with the different treatments. Testing of volatile blends, prepared to the proportion released showed that the blend from simulated HvAV-3 h-infected S. litura larvae-damaged plants was the most attractive to S. litura larvae. Further, we also found that some of the compounds significantly attracted S. litura larvae at specific concentrations. HvAV-3 h-infected S. litura can alter the release of HIPVs in pepper plants and thus become more attractive to S. litura larvae. We speculate that this may be due to alterations in the concentration of some compounds (such as geranylacetone and prohydrojasmon) affecting the behavior of S. litura larvae. This article is protected by copyright. All rights reserved.

An Ascovirus Utilizes Different Types of Host Larval Regulated Cell Death Mechanisms To Produce and Release Vesicles.

Ascoviruses are insect-specific viruses that are thought to utilize the cellular apoptotic processes of host larvae to produce numerous virion-containing vesicles. In this study, we monitored the in vivo infection processes of Heliothis virescens ascovirus 3h (HvAV-3h) to illustrate the regulated cell death (RCD) of host cells. Transmission electron microscopic observations did not reveal any morphological markers of apoptosis in the fat bodies or hemocytes of HvAV-3h-infected Helicoverpa armigera or Spodoptera exigua larvae. However, several hemocytes showed the morphological criteria for necrosis and/or pyroptosis. Further in vitro biochemical tests were performed to confirm the RCD type of host cells after infection with HvAV-3h. Different morphological characteristics were found between the early (prior to 24 hours post-infection, [hpi]) and later (48 to 120 hpi) stages in both HvAV-3h infected larval fat bodies and hemocytes. In the early stages, the virions could only be found in several adipohemocytes, and the fat bodies were cleaving their contained lipid inclusions into small lipid dots. In the later stage, both fat bodies and hemocytes were filled with numerous virions. According to the morphological characteristics of HvAV-3h infected larval fat bodies or hemocytes, the pathogenic characteristics and infection patterns of HvAV-3h in the host larvae were described, and the systematic pathogenic mode of ascovirus infection was refined in this study. This study details the complete infection process of ascoviruses, which provides insights into the relationship between a pathogenesis of an insect virus and the RCD of different host tissues at different stages of infection. IMPORTANCE Viruses and other pathogens can interrupt host cellular apoptosis to gain benefits, such as sufficient resources and a stable environment that enables them to complete their replication and assembly. It is unusual for viruses to code proteins with homology to caspases, which are commonly recognized as apoptosis regulators. Ascoviruses are insect viruses with special cytopathology, and they have been hypothesized to induce apoptosis in their host larvae via coding a caspase-like protein. This enables them to utilize the process of cellular apoptosis to facilitate vesicle formation and replication. However, our previous studies revealed different trends. The fat bodies and hemocytes of Heliothis virescens ascovirus 3h (HvAV-3h)-infected larvae did not show any morphological markers of apoptosis but did display necrosis and/or pyroptosis morphological characteristics. The pathogenic characteristics and infection patterns of HvAV-3h in the host larvae were described, which can help us understand the relationship between the pathogenesis of an insect virus and host RCD.

Insight into the regulation of the Nrf2 pathway in response to ascovirus infection in Spodoptera exigua.

Ascoviruses are a type of entomopathogenic microorganism with high biological pest control potential and are expected to contribute to the natural control of lepidopteran pests. However, knowledge of the molecular mechanism underlying the biocidal activity of ascovirus on its host insects remains limited. In this study, the relative enzyme activity of superoxide dismutase and peroxidase, as well as the expression level of Spodoptera exigua peroxidase (SePOD), were found to be significantly increased at 6h post infection with Heliothis virescens ascovirus 3h (HvAV-3h). H2 O2 accumulation and enhanced expression of NADPH Oxidase (SeNOX) were also observed. In addition, Nuclear Factor erythroid 2-Related Factor 2 (SeNrf2) and muscle aponeurosis fibromatosis (SeMaf) were overexpressed following infection with HvAV-3h. Silencing of SeNrf2 decreased the expression of SePOD, whereas the mortality of SeNrf2-silenced larvae and viral genome copy number also increased. Further RNA interference of SeNOX significantly decreased expression of SeNrf2 and SePOD and therefore increased the mortality and viral genome copy number of the ascovirus-infected host. The HvAV-3h activated Nrf2/ARE pathway of S. exigua and reactive oxygen species were found to respond to ascovirus infection by regulating alterations in antioxidant enzyme genes mediated by the host Nrf2/ARE pathway. These findings enhance our knowledge of ascovirus-host interactions and lay the foundation for the application of ascoviruses in biological pest control. © 2022 Society of Chemical Industry.

Comprehensive identification of protein orthologs in the family Ascoviridae facilitates an understanding of phylogenomics, protein conservation, and phosphorylation.

Analysis of orthology is important for understanding protein conservation, function, and phylogenomics. In this study, we performed a comprehensive analysis of gene orthology in the family Ascoviridae based on identification of 366 protein homologue groups and phylogenetic analysis of 34 non-single-copy proteins. Our findings revealed 90 newly annotated proteins, five newly identified core proteins for the family Ascoviridae, and 14 core proteins for the genus Ascovirus. A phylogenomic tree of 11 Ascoviridae members was constructed based on a concatenation of 35 of the 45 ortholog groups. In combination with phosphoproteomic results and conservation estimations, 30 conserved phosphorylation sites on 17 phosphoproteins were identified from a total of 176 phosphosites on 57 phosphoproteins from Heliothis virescens ascovirus 3h (HvAV-3h), providing potential research targets for investigating the role of these protein in the regulation of viral infection. This study will facilitate genome annotation and comparison of further Ascoviridae members as well as functional genomic investigations.

Heliothis virescens ascovirus 3h blocks the cell cycle of Spodoptera exigua fat body cells at G2 /M phase by downregulating cyclin B1 and cyclin-dependent kinase 1.

Ascoviruses are double-stranded DNA viruses that are pathogenic to noctuid larvae. In vitro infection causes the cells to fail to replicate and proliferate normally. However, the molecular mechanisms are unclear. In this study, the transmission electron microscopydata of infected-Spodoptera exigua (Hübner) fat body cells (SeFB, IOZCAS-SpexII-A cells) showed that virions were internalized in phagocytic vesicles, but not in the nucleus. FACS of cell-cycle progression was performed in SeFB cells infected with Heliothis virescens ascovirus 3h (HvAV-3h). The cell cycle phase distributions of the SeFB cells were G1  = 29.52 ± 1.10%, S = 30.33 ± 1.19%, and G2 /M = 40.06 ± 0.75%. The cell culture doubling time was approximately 24 h. The G1 , S, and G2 /M phases were each approximately 8 h. The unsynchronized or synchronized cells were arrested at G2 /M phase after infection with HvAV-3h. Our data also showed that cells with more than 4N DNA content appeared in the HvAV-3h-treated group. While the mRNA levels of cyclin B1 , cyclin H, and cyclin-dependent kinase 1 (CDK1) were downregulated after HvAV-3h infection, the mRNA expression levels of cyclin A, cyclin D, and cyclin B2 were not significantly changed. Western blotting results showed that the expression of cyclin B1 and CDK1 in infected SeFB cells within 24 h postinfection(hpi), and HvAV-3h infection inhibited the expression of cyclin B1 and CDK1 at 12-24 hpi. Overall, these data implied that HvAV-3h infection leads to an accumulation of cells in the G2 /M phases by downregulating the expression of cyclin B1 and CDK1.

3H-31, A Non-structural Protein of Heliothis virescens ascovirus 3h, Inhibits the Host Larval Cathepsin and Chitinase Activities

3h-31 of Heliothis virescens ascovirus 3h (HvAV-3h) is a highly conserved gene of ascoviruses. As an early gene of HvAV-3h, 3h-31 codes for a non-structural protein (3H-31) of HvAV-3h. In the study, 3h-31 was initially transcribed and expressed at 3h post-infection (hpi) in the infected Spodoptera exigua fat body cells (SeFB). 3h-31 was further inserted into the bacmid of Autographa californica nucleopolyhedrovirus (AcMNPV) to generate an infectious baculovirus (AcMNPV-31). In vivo experiments showed that budded virus production and viral DNA replication decreased with the expression of 3H-31, and lucent tubular structures were found around the virogenic stroma in the AcMNPV-31-infected SeFB cells. In vivo, both LD50 and LD90 values of AcMNPV-31 were significantly higher than those of the wild-type AcMNPV (AcMNPV-wt) in third instar S. exigua larvae. An interesting finding was that the liquefaction of the larvae killed by the infection of AcMNPV-31 was delayed. Chitinase and cathepsin activities of AcMNPV-31-infected larvae were significantly lower than those of AcMNPV-wt-infected larvae. The possible regulatory function of the chitinase and cathepsin for 3H-31 was further confirmed by RNAi, which showed that larval cathepsin activity was significantly upregulated, but chitinase activity was not significantly changed due to the RNAi of 3h-31. Based on the obtained results, we assumed that the function of 3H-31 was associated with the inhibition of host larval chitinase and cathepsin activities, so as to restrain the hosts in their larval stages.

A Spodoptera exigua serpin is involved in the immunity of hemolymph induced by Heliothis virescens ascovirus 3h

• A serpin-2.1 like gene from Spodoptera exigua was cloned and identified. • The expression patterns of Seserpin-2.1 like were explored. • After infection of HvAV-3h, the transcription of Seserpin-2.1 like was upregulated. • Expression of Seserpin-2.1 like reduced in hemolymph after infection of HvAV-3h. Serine protease inhibitors (serpins) are a broadly distributed protease superfamily in various organisms, which participate in the negative regulation of melanization in insects. Our pervious study demonstrated that induced melanization by Heliothis virescens ascovirus 3h is helpful for HvAV-3h viral replication. In this study, the serpin-2.1 like gene from Spodoptera exigua ( Seserpin-2.1 like ) which is a specific serpin after HvAV-3h infection was identified. Seserpin-2.1 like has the typical characteristics of serpin proteins family. Seserpin-2.1 like widely distributed in various tissues (including hemolymph, fat body, midgut, cuticle and head), and expressed at a higher level in fat body, hemolymph and head. At different developmental stages, it tends to express at eggs and 5th instar larvae. Seserpin-2.1 like shows special phenomenon for HvAV-3h infection. On the one hand, the transcription level of Seserpin-2.1 like in hemolymph was significantly upregulated after HvAV-3h compared to other immune challenges Gram-positive bacteria ( Bacillus thuringiensis ), Gram-negative bacteria ( Escherichia coli ), and Baculovirus ( Autographa californica nucleopolyhedrovirus ). On the other hand, its expression disappeared during 72 to 120 h post infection. In summary, our results suggest that Seserpin-2.1 like may be involved in innate immunity of S. exigua after infection with HvAV-3h. And HvAV-3h may inhibit the expression of Seserpin-2.1 like to promote the melanization of host larvae for viral replication.

Melanization induced by Heliothis virescens ascovirus 3h promotes viral replication.

Melanization is an important innate immune defense mechanism of insects, which can kill invading pathogens. Most pathogens, for their survival and reproduction, inhibit the melanization of the host. Interestingly, our results suggested that after infection with Heliothis virescens ascovirus 3h (HvAV-3h), the speed of melanization in infected Spodoptera exigua larval hemolymph was accelerated and that the phenoloxidase (PO) activity of hemolymph in larvae infected with HvAV-3h increased significantly (1.20-fold at 96 hpi, 1.52-fold at 120 hpi, 1.23-fold at 144 hpi, 1.12-fold at 168 hpi). The transcription level of the gene encoding S. exigua prophenoloxidase-1 (SePPO-1 gene) was upregulated dramatically in the fat body during the middle stage of infection. In addition, when melanization was inhibited or promoted, the replication of HvAV-3h was inhibited or promoted, respectively. In conclusion, infection with HvAV-3h can markedly induce melanization in the middle stage of infection, and melanization is helpful for HvAV-3h viral replication.

Characterization of Heliothis Virescens Ascovirus 3h orf21 that Encodes a Virion Protein

Homologues of Heliothis virescens ascovirus 3h (HvAV-3h) orf21 are found in 9 completely sequenced members of the ascoviruses, but so far their functions are unknown. Here, orf21 (3h-21) was cloned in-frame into a pET-28a bacterial expression vector. The fusion protein produced by this construct was used for the preparation of a polyclonal antiserum. RT-PCR analysis showed a single transcript of 3h-21 of approximately 0.7kb was transcribed beginning at 24h post-infection in infected Helicoverpa armigera larvae. Western blot analysis of extracts from HvAv-3h-infected Helicoverpa armigera larvae detected a 25.6 kDa protein late in infection. This antiserum also reacted with a 25.6 kDa protein in purified virions of HvAv-3h. The protein was not extensively modified post-translation. Immunoelectron microscopy confirmed that the 3H-21 is associated with the structure of HvAV-3h virions.

Regulation of Chitinase in Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) During Infection by Heliothis virescens ascovirus 3h (HvAV-3h).

Insect chitinases play essential roles in the molting and metamorphosis of insects. The virus Heliothis virescens ascovirus 3h (HvAV-3h) can prolong the total duration of the larval stage in its host larvae. In this study, the molecular character and function of chitinase and chitin-binding domain (CBD) were analyzed in larvae of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). In detecting the chitinase activity of mock-infected and HvAV-3h-infected larval whole bodies and four different larval tissues, the results showed that larval chitinase activity was significantly decreased at 48 h post infection (hpi) and that the chitinase activity of HvAV-3h-infected larval fat body and cuticle was notably decreased at 144 and 168 hpi. The transcription level of S. exigua chitinase 7 (SeCHIT7) was down-regulated at the 6, 9, 12, 48, 72, and 96 hpi sample times, the S. exigua chitinase 11 (SeCHIT11) was down-regulated at 3–96 hpi, while both S. exigua chitinases (SeCHITs) were up-regulated at 120–168 hpi. Further tissue-specific detection of SeCHIT7 and SeCHIT11 transcription showed that SeCHIT7 was down-regulated at 144 and 168 hpi in the fat body and cuticle. SeCHIT11 was down-regulated at 168 hpi in the fat body, midgut, and cuticle. Additionally, the transcription and expression of S. exigua chitin-binding domain (SeCBD) could not be detected in HvAV-3h-infected larvae. The in vitro analyses of SeCHIT7N, SeCHIT11, and SeCBD showed that SeCHIT7N and SeCHIT11 were typical chitinases. Conversely, no chitinase activity was detected with SeCBD. SeCBD, however, could significantly increase the activity of SeCHIT7N and SeCHIT11. In conclusion, HvAV-3h not only interfered with the transcription and expression of SeCHITs but also affected the normal transcription and expression of SeCBD and, in doing so, influenced the host larval chitinase activity. These results will aid in providing a foundation for further studies on the pathogenesis of HvAV-3h.

3H-117, a structural protein of Heliothis virescens ascovirus 3h (HvAV-3h).

The open reading frame 117 (3h-117) of Heliothis virescens ascovirus 3h (HvAV-3h), which is a conserved coding region present in all completely sequenced ascovirus members, was characterized in this study. By RT-PCR detection, 3h-117 transcription began at 6-h post-infection (hpi) and remained stable until 168 hpi in HvAV-3h-infected Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae. In addition, 3h-117 putatively encodes a 21.5-kDa protein (3H-117) predicted to be a CTD-like phosphatase. Western blot analysis using a prepared rabbit polyclonal antibody specific to 3H-117 showed that the product could be detected at 24 hpi, which remained stably detectable until 168 hpi. The same analysis also demonstrated that the 3H-117 protein localized in the virions of HvAV-3h. Immunofluorescence analysis showed that at 24 hpi, 3H-117 was mainly located in the nuclei of H. armigera larval fat body cells and later spread into the cytoplasm. In summary, our results indicate that 3H-117 is a structural protein of HvAV-3h.

Changes in lipid, protein and carbohydrate metabolism in Spodoptera exigua larvae associated with infection by Heliothis virescens ascovirus 3h.

Spodoptera exigua (Lepidoptera: Noctuidae) larvae were used to analyze the regulation of lipid, protein and carbohydrate metabolism in host larvae infected with the Heliothis virescens ascovirus 3h (HvAV-3h). Using histological sections, significant pathological changes were found in the fat bodies of infected larvae from 24 h to 72 h post-infection (hpi). The lipid and protein contents of the infected larvae were significantly higher than those of the uninfected larvae, while the carbohydrate content of the infected larvae was significantly lower than that of the mock-infected larvae. The selected primary metabolite metabolism-associated genes showed different expression patterns. Further co-relationship analysis of the gene expression level and content changes of primary metabolites indicated the following: the acyl-CoA dehydrogenase and fatty acid synthase genes were closely associated with lipid metabolism, and the hexokinase and the glycogen synthase gene expression levels were related to carbohydrate metabolism, while the aminopeptidase N and the protein disulfide isomerase gene expression levels were not correlated with protein metabolism. These results indicate that the HvAV-3h virus stimulates host larval lipid and protein syntheses and inhibits carbohydrate synthesis.

Response analysis of host Spodoptera exigua larvae to infection by Heliothis virescens ascovirus 3h (HvAV-3h) via transcriptome

Heliothis virescens ascovirus 3 h (HvAV-3h), a dsDNA insect virus, belonging to the family Ascoviridae, can infect caterpillars of several Noctuidae species by ovipositing parasitoid wasps. In order to provide a comprehensive overview of the interactive responses of host larvae after infection by the ascovirus, a transcriptome analysis of Spodoptera exigua to HvAV-3h was conducted from 6 to 168 hours post infection (hpi). Approximately 101.64 Gb of RNA sequencing (RNA-seq) data obtained from infected and uninfected S. exigua larvae were used to perform a de novo transcriptome assembly, which generated approximately 62,258 S. exigua unigenes. Using differential gene expression analysis, it was determined that the majority of host transcripts were down-regulated beginning at 6 hpi and continuing throughout the infection period, although there was an increase in up-regulated unigene number during the 12 to 72 hpi stage. It is noteworthy that the most abundantly enriched pathways in KEGG annotation were Metabolism terms, indicating that the host larval metabolic mechanisms were highly influenced post HvAV-3h infection. In addition, the host cuticle protein encoding unigenes were highly down-regulated in most of the situations, suggesting that the host larval cuticle synthesis were inhibited by the viral infection.

Selection of reference genes for expression analysis using RT-qPCR in the dissemination system of Heliothis virescens ascovirus 3\u2009h (HvAV-3h)

Ascoviruses are double-stranded DNA viruses that mainly infect noctuid larvae, and are transmitted by the parasitoid wasp Microplitis similis Lyle. Ascovirus-parasitoids wasp-noctuid insects constitute the dissemination system. Selection of suitable reference genes for the dissemination system could play an important role in elucidating the pathogenic molecular mechanisms of ascovirus. Unfortunately, such studies on potential reference genes in the dissemination system of ascoviruses are lacking. In the present study, we evaluated 11 candidate reference genes: β-actin1 (ACT1), β-actin2 (ACT2), elongation factor 1 (EF1), elongation factor 2 (EF2), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (SOD), 28S ribosome (28S), Tubulin (TUB) and 18S ribosome (18S). The samples were originally from various virus concentrations and points-in-time of experimental treatments using RefFinder and four algorithms. The results showed that EF1 was the most stable internal gene in S. exigua and M. similis and that EF2 was the most stable in the IOZCAS-Spex-II-A cell line, and the stability of reference genes were confirmed via the expression levels of two inhibitor of apoptosis-like (iap-like) genes from Heliothis virescens ascovirus 3 h (HvAV-3h). This study provides a crucial basis for future research that explores the molecular mechanisms of the pathogenesis of ascoviruses.

Genome analysis of Heliothis virescens ascovirus 3h isolated from China.

No ascovirus isolated from China has been sequenced so far. Therefore, in this study, we aimed to sequence the genome of Heliothis virescens ascovirus 3h (HvAV-3h) using the 454 pyrosequencing technology. The genome was found to be 190,519-bp long with a G+C content of 45.5%. We also found that it encodes 185 hypothetical open reading frames (ORFs) along with at least 50 amino acids, including 181 ORFs found in other ascoviruses and 4 unique ORFs. Gene-parity plots and phylogenetic analysis revealed a close relationship between HvAV-3h and three other HvAV-3a strains and a distant relationship with Spodoptera frugiperda ascovirus 1a (SfAV-1a), Trichoplusia ni ascovirus 6a (TnAV-6a), and Diadromus pulchellus ascovirus 4a (DpAV-4a). Among the 185 potential genes encoded by the genome, 44 core genes were found in all the sequenced ascoviruses. In addition, 25 genes were found to be conserved in all ascoviruses except DpAV-4a. In the HvAV-3h genome, 24 baculovirus repeat ORFs (bros) were present, and the typical homologous repeat regions (hrs) were absent. This study supplies information important for understanding the conservation and functions of ascovirus genes as well as the variety of ascoviral genomes.

Selection and validation of reference genes for RT-qPCR analysis in Sf9 cell line infected by Heliothis virescens ascovirus 3h (HvAV-3h)

The real-time quantitative reverse transcription PCR (qRT-PCR) is an efficient and pervasive technique for analysis gene expression. Despite the fact that this technique has been extensively used to explore the gene functions, the stability of the reference genes still requires being validated at different experimental conditions. This research aims to confirm that the expression stability of seven candidates reference genes under different concentrations sterile hemolymph containing virus (2, 4, 6, 108-fold) infects the Spodoptera frugiperda cells line (Sf9). The appropriate reference genes as endogenous controls were determined through four computational algorithms (ΔCt, NormFinder, BestKeeper, and geNorm) and an integrated online software RefFinder. According to results of this study, TUB and ACT1 were proved to be the most stable reference genes in E2 fold and E4 fold respectively. PPI was the best reference gene in E6 and E8 fold, 28S and TUB were the most stable pair reference genes through all samples. These results can facilitate basic researches of the molecular mechanism of HvAV-3h and also prove to be helpful for the standard RT-PCR method.

Characterization and Growing Development of Spodoptera exigua (Lepidoptera: Noctuidae) Larvae Infected by Heliothis virescens ascovirus 3h (HvAV-3h).

Spodoptera exigua (Hübner) is a very serious worldwide pest capable of causing severe economic losses in numerous agricultural crops. The need for an effective, highly virulent, pathogenic microorganism for use as a biological control agent against S. exigua larvae is particularly important. Heliothis virescens ascovirus 3 h (HvAV-3h)-containing hemolymph with a titer of 9.58 × 10(12) genome copies per ml was used to inoculate S. exigua larvae per os with a 1.06 × 10(10) dosage per larva for the first- to second instar and 9.58 × 10(9) genome copies per larva for the third- to fifth instars. Intrahemocoelic injections were also used with a dosage of 1.53 × 10(9) genome copies per larva for third- to fifth instar. The postinjection mortality, body weight, and food intake of the S. exigua larvae were observed and recorded. The corrected mortality rates for the first- through fifth instar inoculated per os were 21.88 ± 0.98, 22.22 ± 4.00, 8.89 ± 4.01, 6.66 ± 3.33, and 8.89 ± 2.94%, respectively. The early instars were significantly easier to infect with virus compared to the later instar. The corrected mortality of the third, fourth, and fifth instars inoculated by injection was 96.58 ± 3.42, 98.83 ± 1.17, and 97.78 ± 2.22%, respectively. Compared to the healthy larval population, survival time of the diseased larval population was considerably extended. In addition, food intake was greatly reduced, and the body weight remained fairly constant in the third- and fourth instar. The body weight declined in the fifth instar corresponding to a reduction in food intake.

Imperfection works: Survival, transmission and persistence in the system of Heliothis virescens ascovirus 3h (HvAV-3h), Microplitis similis and Spodoptera exigua.

Ascoviruses are insect-specific large DNA viruses that mainly infect noctuid larvae, and are transmitted by parasitoids in the fields. Heliothis virescens ascovirus 3h (HvAV-3h) has been recently isolated from Spodoptera exigua, without parasitoid vector identified previously. Here we report that Microplitis similis, a solitary endoparasitoid wasp, could transmit HvAV-3h between S. exigua larvae in the laboratory. When the female parasitoid wasp acquired the virus and served as a vector, the period of virion viability on the ovipositor was 4.1 ± 1.4 days. Infected host larvae were still acceptable for egg laying by parasitoids, and the parasitoids thereafter transmitted virus to healthy hosts. Virus acquisition occurred only from donor hosts between 3 and 9 days post infection. The peak of virus acquisition (80.9 ± 6.3%) was found when M. similis wasps oviposited in larvae that had been inoculated with the virus 7 days previously. When virus infection of the host took place during the life cycle of the parasitoid wasp, it caused 1- to 4-day-old immature parasitoids death in the host, whilst a small proportion of 5- to 6-day-old and the majority of 7-day-old parasitoids larvae survived from the virus-infected hosts. Viral contamination did not reduce the life span or fecundity of female M. similis.

A Comparison of Growth and Development of Three Major Agricultural Insect Pests Infected with Heliothis virescens ascovirus 3h (HvAV-3h)

Ascoviruses are double-stranded DNA viruses that are pathogenic to lepidopteran hosts, particularly noctuid larvae. Infection of a larva is characterized by retarded growth, reduced feeding and yellowish body color. In this paper, we reported the growth and development of three major agricultural noctuid insect pests, Helicoverpa armigera (Hübner), Spodoptera exigua (Hübner) and Spodoptera litura (Fabricius), infected with Heliothis virescens ascovirus 3h (HvAV-3h). Using 10-fold serial dilutions (0 to 7) of HvAV-3h-containing hemolymph to infect S. litura larvae, we found no significant difference in larval mortalities from 0 to 103-fold dilutions; however, significant differences were observed at 104-fold dilution and above. Using a 10-fold dilution of HvAV-3h-containing hemolymph to infect H. armigera, S. exigua and S. litura larvae, we found that the growth and development were significantly affected. All infected larvae could not pupate; the survival times of treated H. armigera, S. litura and S. exigua larvae were significantly longer than untreated control larvae. Body weight showed significant difference between treated and untreated control group from day 1 after inoculation in H. armigera and S. exigua, but day 2 in S. litura. Additionally, food intake also showed significant difference between treated and untreated control group from day 2 after inoculation in H. armigera and S. litura, but day 3 in S. exigua.