Studies On Infection Mechanism Of HcNPV And Its Combination With Bt In Larvae Of Fall Webworm, And Sustainable Control In Host Population

Hyphantria cunea nucleopolyhedrovirus (HcNPV), an effective baculoviral insecticide to control population of the severe intrusive insect pest the Fall webworm, has been used on a large scale in China. The application of HcNPV will present very important value in economy, ecology and environment and will boost biological control in forestry insect pest management. Investigation of mechanism of its infection, transmission among its host and combination infectious mechanisms with Bt will contribute practical and theoretical references to the bio-insecticide application in control of the fall webworm.Bioassay experiments of the virus and Bt showed that the larvae of H. cunea exhibited either symptom of virus infection or that of Bt infection when the inoculating concentration approached their respective LC50 of the two pathogens, and the respective peaks of incidence of diseases also occurred under the same concentrations. Compared with inoculation of single pathogen, peak stage of combined pathogenesis was 12-24h earlier. The results indicated that combination of the two pathogens could enhance their respective pathogenesis. Enhancement of HcNPV virulence was observed when concentration of Bt was at 10 and 25 mg/L, while antagonism occurred at 5 mg/L. Virulence of Bt was enhanced when concentration of HcNPV was at 1.6×105,1.6×106,1.6×107 PIBs/mL, respectively. The LT50 was 0.5-2.1d shorter at HcNPV and Bt co-infection illustrated that application of the two pathogens could significantly effect their pathogenesis on the host insect. Allotment of the two pathogens concentrations was the key factor to enhance the efficiency of infection.The H. cunea larvae were sampled at different time after the two pathogens inoculation at early 4th instar and stained with H.E. after wax sectioned. Pathologies of the larval midgut epithelia, fat body cells and testis cells were observed microscopically. During 6 to 48 h co-infection of the virus and Bt, top of midgut epithelium showed swell and cystoid, where cytoplasm accumulated. Virus infectious symptom occurred during 72-144 h co-infection, which was earlier than that of virus single infection. The pathogenic changes and cellular dissociation also accelerated comparing to single infection, which led to rapid death of the larvae.Ultrastructural observation of the larvae infected with Bt showed that cytoplasm of the midgut epithelia and goblet cells appeared vacuoles 48 h after inoculation. At 72 h, pathological changes aggravated that most epithelia shed from the basal membrane. For the co-infected larvae, disease changes occurred in midgut epithelia at 24 h and virion amplified at 96 h, while no disease changes were observed in fat body at 24 h. Disease changes occurred in fat body at 48 h and virion amplified at 120 h. During 144 h to 168 h after co-infection, virion amplified actively in the nuclei of the epithelia and fat body cells. At the same time the nuclei swelled that were full of the viral polyhedra. In the nuclei of testis cells infected with HcNPV at 144 h virogenic stroma appeared. The results showed that infection of the virus occurred in midgut epithelia 24 h earlier than that in fat body cells.Location of HcNPV in the host tissue was studied by immunohistochemistry. Some positive signals were detected in midgut 48 h after infection. During infection of 72-96 h viral antigen was detected in the tissues of trachea, fat body and epidermis, while positive signal increased in midgut, At 120 h nuclei of tracheal epidermis, fat body cells and dermal cells swelled where positive signals increased. At 144 h positive signals were detected in the tissues mentioned above. The results indicated that positive signals appeared earliest in the midgut and increased slowly, while they appeared later in other tissues and proceeded faster. Occurrence of the signals in HcNPV and Bt co-infection were earlier than that in single infection of HcNPV indicated that co-infection enhanced amplification of the virus in the host. No signals were detected in muscle tissue showed no infection of the virus occurred in the tissue.Concentrations of the infected host hemolymph proteins were detected and SDS-PAGE was used to analyze. Concentrations of the infected hemolymph proteins were higher than those of no-infected controls except at 72 h and 96 h after infection. The results implied that infection of the virus probably suppressed synthesis of hemolymph protein via destroying fat body tissue. There were no significant differences in hemolymph protein patterns (including total proteins, glycoproteins and lipoproteins) between infected and no-infected host.Some impact indices, such as larval mortalities, pupal weight, oviposition, percentage of egg hatch of respective parental, first offspring, second offspring generations after infection, were studied by bioassay. Significant differences existed between treatments and controls. The results indicated that HcNPV could pass through different host's generations and provided a sustainable control to the host population. Amplified PCR product of HcNPV polyh gene was detected in total DNA of the first and second generations of host's eggs showed HcNPV could vertically pass through parent host to its offspring.There were no significant differences of the viral virulence after 7 passages through the host analyzing with the techniques of bioassay, ultrastructure, SDS-PAGE, genomic DNA restriction endonuclease analysis. However, a 42.0 kD band was observed in SDS-PAGE of virion proteins of the third and 7th viral generation. These results showed that genetic characteristics of the virus remained stable after 7 generation passages. The newly occurred 42.0 kD protein was probably the change of interaction between the virus and its host.This paper also compared biology of 5 HcNPV strains. One of a strain was more virulent than other 4 strains expressed in LC50 and LT50. There were some differences in viral proteins and restriction endonuclease patterns of the 5 viral strains.