Changes Of The Extracellular Enzymes From Entomopathogenic Fungi And Immune Response Of Haemolymph Of Scale Insect Infected By Entomopathogenic Fungi

Scale insects belong to the superfamily Coccoidea of the order Hemiper in the class Insecta.7500 species,49 families were recorded worldwide, and 830 species,16 families in China. Many species are important pests in forest plants, fruit trees and ornamental plants. Chemical pesticides have been used to control scale insects, but the wax layer on the surface of scale insects keep the insect from harming of chemical pesticide. Entomopathogenic fungus as an agent of biological control is very promising. In recent years, the entomopathogenic fungi and pathology of scale insects have been reported, but the interaction and immune response when the entomopathogenic fungi infecting scale insects have not been reported.The experiment consists of three parts:1. The four strains of the entomopathogenic fungi, Lecanicillium lecanii V3.4505, V3.4504, L. fungicola HEB02, and Fusarium incarnatum-equiseti HEB01, were used. Two species of scale insects, Ceroplastes japonicus Green and Rhodococcus sariuoni Borchsenius as the target insects were tested. The cuticle of the two scale insects was used as the sole carbon source in the medium for fungal culture. The activities of four extracellular enzymes, including lipase, protease (Pr1), chitinase, and N-acetyl-D-glucosaminidase (NAGase), produced by the four strains were determined. The function of the extracellular enzymes during the strains penetrating the integument of scale insects was analyzed. Meanwhile, the cumulative mortalities of the two scale insects infected with the four strains for eight days were assayed to evaluate the virulence of the strains aginst scale insects.2. By using phase contrast, fluorescence and transmission electron microscopy (TEM) technology, the hemocytes of the female adult of C. japonicus were studied, including their types and proportions, cellular immune response after infected by Lecanicillium lecanii V3.4505.3. By applying cytochemical localization technology and transmission electron microscopy, the peroxidase (POD) and phenoloxidase (PO) in hemolymph of C. japonicus were studied to compare the distribution and contents in haemolymph of control and fungi-treated group.The results are as follows.1. The role of the four extracellular enzymes of the four entomopathogenic fungus strains during degrading the integument of scale insectsThe four strains were prepared into suspension of 1×108spore/mL and were used to infect the C. japonicus and R. sariuoni by spraying. The cuticle of the two scale insects was used as the substrate in the medium for fungal culture. The activities of lipase, protease (Prl), chitinase, and N-acetyl-D-glucosaminidase (NAGase) produced by the four strains were determined and assayed in the 1-8 day culture.The activities of the four enzymes all changed significantly during the scale insect integument-degrading process, their changing trend all exhibited rising in the first few days and falling in the later. The activity peak of lipase appeared earliest that was at two days after inoculation, this is due to the wax on the uppermost layer of the integument. The lipase activity of the strains cultured on the cuticular medium of C. japonicus was obviously higher than that on R. sariuoni, this is due to that the wax layer of C. japonicus was obviously higher than R. sariuoni. The activity peak of Prl came after 3-4d while the activity peak of chitinase and NAGase appeared at 6 d and 4-5d, respectively; the peak of enzyme activities was in correspondence to the arrangement of protein and chitin in integument of scale insect. Among the four strains, V3.4505 strain caused the highest mortality rate of 73% for C. japonicus and 81% for R. sariuoni, respectively, and, that showed significant difference with other two strains, HEB02 and HEB01. The average values of Prl activity of four strains were significantly related to the cumulative mortality of the two scale insects, and the Linear equations were y=0.082x+5.822 (R2=0.823) for C. japonicus and y=0.119x+14.75 (R2=0.764) for R. sariuoni. Similarly, the average chitinase activity of four strains are also significantly related to the cumulative mortality of the two scale insects, the Linear equations were y =0.148x+15.89 (R2=0.645) for C.japonicus and y=0.095x+10.46 (R2=0.762) for R. sariuoni. This indicated that the Prl and chitinase played important role in the scale insect integument-degrading process and they could be used as the virulence indicator of the entomopathogenic fungi.2. The hemocytes of C. japonicus and the cellular immune response aginsted Lecanicillium lecaniiIn order to study the immune response of the haemolymph of the female adult of C. japonicus, the hemocyte morphology was observed and the number of each type of hemocytes was counted under the light microscope and TEM. Four types of hemocytes were identified, including prohemocytes accounting for 24.6%, plasmatocytes accounting for 36.6%, granulocytes accounting for 13.2%, and oenocytoids accounting for 25.6%.The suspension of 1×108 spore/mL of the Lecanicillium lecanii V3.4505 was used to infect the C. japonicus by spraying. After two days, hyphae in a thin layer were observed on the surface of scale insect. As infecting, the insects gradually turned to black and died in 5 days. The samples of hemolymph of infected scale insects were observed under phase contrast and florescence microscope. It was found that the interaction between hemocytes and fungus was strongest at the third day. The adhesion and phagocytosis behaviors were obviously observed under phase contrast and fluorescence microscope. The phagocytosis behavior of oenocytoids was also detected under fluorescence microscope. The hemocytes of the scale insects after infected three days were observed under the TEM. The ultrastructure changes were observed inside the hemocytes, including hemocyte vacuolization, double nucleus membrane separation and organelle deformation. After infection, the hemocyte types were not altered but their ratios differed. The numbers of plasmatocytes, granulocytes and oenocytoids raised for 6.3%,1.8% and 5.6%, respectively, while the number of prohemocytes decreased by 13.7%. It means plasmatocytes and oenocytoids may play important roles in the immune of scale insects. 3. The cytochemistry localization of phenoloxidase (PO) and peroxidase (POD) in the hemocytes of C. japonicusLabeled with L-3,4-dihydroxyphenylalanine (L-DOPA), many strong-positive phenoloxidases (PO) particles in the hemocytes of the infected C. japonicus were observed under the TEM, while PO particle was not found inside the fungus. Similarly, labeled with 3,3-diaminobenzidine tetrahydrochloride (DAB), strong-positive peroxidases (POD) particles were observed not only inside the infected hemocytes, but also in the fungus. Compared to the non-infected hemocytes, the infected hemocytes had more PO and POD particles with higher intensity. It meaned that PO and POD in haemolymph of C. japonicus, could help to resist the invasion of foreign pathogen.