Enhanced virulence of genetically engineered Autographa californica nucleopolyhedrovirus owing to accelerated viral DNA replication aided by inserted ascovirus genes.

Genetic engineering technology is an ideal method to improve insecticidal efficiency by combining the advantages of different pathogenic microorganisms. Thus, six ascovirus genes were introduced into the genomic DNA of Autographa californica nucleopolyhedrovirus (AcMNPV) to possibly transfer the intrinsically valuable insecticidal properties from ascovirus to baculovirus. The viral budded virus (BV) production and viral DNA replication ability of AcMNPV-111 and AcMNPV-165 were significantly stronger than that of AcMNPV-Egfp (used as the wild-type virus in this study), whereas AcMNPV-33 had reduced ones. AcMNPV-111 and AcMNPV-165 also exhibited excellent insecticidal efficiency in the in vivo bioassays: AcMNPV-111 showed a 24.1% decrease in the LT50 value and AcMNPV-165 exhibited a 56.3% decrease in the LD50 value compared with AcMNPV-Egfp against the 3rd instar of Spodoptera exigua larvae, respectively. Furthermore, the size of the occlusion bodies (OBs) of AcMNPV-33, AcMNPV-111, and AcMNPV-165 were significantly increased compared to that of AcMNPV-Egfp. AcMNPV-111 and AcMNPV-165 had stable virulence against the 2nd to 4th instars tested larvae and higher OB yield than AcMNPV-Egfp in the 3rd and 4th instar larvae. Correlation and regression analyses indicated that it is better to use 5 OBs/larva virus to infect the 2nd instar larvae to produce AcMNPV-111 and 50 OBs/larva virus to infect the 3rd instar larvae to produce AcMNPV-165. The results of this study obtained recombinant viruses with enhanced virulence and exhibited a diversity of ascovirus gene function based on the baculovirus platform, which provided a novel strategy for the improvement of baculovirus as a biological insecticide.

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 3 h 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.

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.

Altering of host larval (Spodoptera exigua) calcineurin activity in response to ascovirus infection.

BACKGROUND: Calcineurin (CaN) is involved in numerous cellular processes and Ca2+ -dependent signal transduction pathways. According to our previous transcriptome studies, thousands of host larval (Spodoptera exigua) transcripts were downregulated after the infection of Heliothis virescent ascovirus 3h (HvAV-3h), while the Spodoptera exigua calcineurin genes (SeCaNs) were significantly upregulated. To understand the regulation of SeCaNs in S. exigua larvae during the infection of HvAV-3h, the functions of CaN subunit A (SeCaN-SubA) and CaN binding protein (SeCaN-BP) were analysed. RESULTS: The in vitro assays indicated that the bacterial expressed SeCaN-SubA is an acid phosphatase, but no phosphatase activity was detected with the purified SeCaN-BP. The transcription level of SeCaN-SubA was upregulated after HvAV-3h infection and the CaN activity was significantly increased after HvAV-3h infection in S. exigua larvae. Interestingly, the SeCaN-BP transcripts were only detectable in the HvAV-3h infected larvae. Further immunoblotting results consistently agree with those obtained by qPCR, indicating that the infection of HvAV-3h causes the upregulated expression of SeCaN-SubA and the appearance of SeCaN-BP. An interaction between the cleaved SeCaN-SubA and SeCaN-BP was detected by co-immunoprecipitation assays, and the expression of SeCaN-BP in Spodoptera frugiperda-9 (Sf9) cells can help to increase the CaN activity of SeCaN-SubA. Further investigations with CaN inhibitors suggested that HvAV-3h. Further investigations with CaN inhibitors suggested that the inhibition on host larval CaN activity can also inhibit the viral replication of HvAV-3h. CONCLUSION: The increase in CaN activity caused by HvAV-3h infection might be due to the upregulation of SeCaN-SubA and the induced expression of SeCaN-BP, and increased CaN activity is essential for ascoviral replication. © 2019 Society of Chemical Industry.

Identification of four caspase genes from Spodoptera exigua (Lepidoptera: Noctuidae) and their regulations toward different apoptotic stimulations.

Apoptosis plays critical roles in multiple biological processes in multicellular organisms. Caspases are known as important participators and regulators of apoptosis. Here, four novel caspase genes of Spodoptera exigua were cloned and characterized, which were designated as SeCasp-1, SeCasp-6, SeCasp-7 and SeCasp-8. Analysis of the putative encoded protein sequences of these SeCasps indicated that SeCasp-1 and SeCasp-7 were possible homologs of executor caspases; SeCasp-8 was a possible homolog of initiator caspases; and SeCasp-6 was a unique caspase of S. exigua that shares low similarity with all the identified insect caspases. Based on baculovirus expression system analyses, SeCasp-1 exhibited similar caspase activity to human caspase-1, -3, -4, -6, -8 and -9; SeCasp-6 presented similar caspase activity to human caspase-2, -3, -4, -6, -8 and -9; SeCasp-7 exhibited similar caspase activity to human caspase-2, -3 and -6; and SeCasp-8 presented similar caspase activity only to human caspase-8. Induction with different chemicals revealed that SeCasp-1 showed extreme upregulation after 24 h in the treated fat body cell line (IOZCAS-Spex-II) of S. exigua. Developmental expression analysis revealed that SeCasp-1 was highly transcribed in the larval stages, while SeCasp-6, SeCasp-7, SeCasp-8 were down-regulated. The in vivo detection of the relative expression levels of SeCasps in S. eixgua larvae inoculated with different pathogens suggested that SeCasp-1 was sensitive to Bacillus thuringiensis infection and that SeCasp-6 was sensitive to baculovirus infection. SeCasp-7 and SeCasp-8 showed slight changes under either in vitro chemical apoptosis induction or in vivo pathogen infection.

In Vitro Infectious Risk Assessment of Heliothis virescens ascovirus 3j (HvAV-3j) toward Non-target Vertebrate Cells.

As specific pathogens of noctuid pests, including Spodoptera exigua, S. litura, Helicoverpa armigera, and Mythimna separata, ascoviruses are suitable for the development of bioinsecticides. In this study, the infectivity of Heliothis virescens ascovirus 3j (HvAV-3j) on insect and mammalian cells was evaluated. HvAV-3j infection induced drastic morphological changes in Sf9, HzAM1, SeFB, and HaFB cells, including swelling and detachment. Notably, the latter phenomena did not occur in HvAV-3j-inoculated mammalian cells (HEK293, 7402, HePG2, PK15, ST, and TM3). MTT assays indicated that HvAV-3j inhibited the growth of host insect cells from the 6th hpi, but no effects were detected in the HvAV-3j-inoculated mammalian cells. Furthermore, viral DNA replication, gene transcription, and protein expression were investigated, and the results consistently suggested that HvAV-3j viruses were not able to replicate their genomic DNA, transcribe, or express their proteins in the non-target vertebrate cells. The HvAV-3j genes were only transcribed and expressed in the four insect cell lines. These results indicated that HvAV-3j was infectious to cells derived from S. frugiperda, S. exigua, H. armigera, and H. zea but not to cells derived from human, pig, and mouse, suggesting that ascoviruses are safe to non-target vertebrate cells.

Parasitism of Two Spodoptera spp. by Microplitis prodeniae (Hymenoptera: Braconidae).

Early instar larvae of the tobacco cutworm Spodoptera litura (Lepidoptera: Noctuidae) and the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) are recognized hosts of the parasitic wasp Microplitis prodeniae Rao and Kurian (Hymenoptera: Braconidae), although M. prodeniae has previously been regarded as monophagous. In this study, we found the immature period and longevity of M. prodeniae developing in S. exigua was similar to that in S. litura. It was shown that the development time of M. prodeniae in S. exigua was 15.1 ± 0.3 d, not significantly different from 15.0 ± 0.2 d in S. litura. The parasitism rate of M. prodeniae attacking S. exigua was significantly lower than on S. litura (65.48 ± 2.29 and 43.83 ± 2.20%, respectively), whilst the female ratio of the wasp's offspring was not significantly different when developing on the two species. M. prodeniae females prefer to oviposit on the second- and third-instar host larvae of S. exigua, rather than other instars. The effects of development of M. prodeniae on two important lepidopterous pests are discussed.

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.