| In this article, the isolate MA3 of Metarhizium anisopliae was used to study the pathogenesis in Brontispa longissima (Gestro), including the histopathology, defense response of prophenoloxidase-activating system and haemocytes, and the induction of cytochrome 450 family 4 (CYP4) gene. The purposes of this study were to provide the basis for the application of the pathogen in the integrated control of B. longissima, and new exemplum for the research of interaction between host and pathogens.The results of biological test revealed that MA3 had a good control against B. longissima under 1×107 conidia/mL concentration, with about 100% mortality to larvae and adults. The control effect was different under different temperature and humidity. Pathogenic effect could occurred remarkable under the condition of 30℃and 95% relative humidityA histopathological study on B. longissima larvae infected by M. anisopliae showed that the fungi infected body mainly by penetrating the cuticle. However, it also could infect through spiracle. The conidia could be found in haemocoel 24 h after infected, and a large number of haemocytes congregated just beneath the epidermis from where the fungi penetrated. Invasion of the hyphae to host tissues showed no selection, and the pathological changes occurred on all infected tissues and organs, including fat body, muscle, mid gut, salivary gland and trachea.Prophenoloxidase-activating system is one of the most important humoral immunity system in insects, which involves in defense reaction, cuticular hardening and wound healing. The lysate of M. anisopliae appear to be an elicitor of prophenoloxidase in hemocyte lysate solution and plasma, and it also has inhibitory effect to phenoloxidase. The activity of phenoloxidase in infected group was higher than that in uninfected group, but the activity of phenoloxidase induced by injection 10μg laminarin in infected group is significant lower than that in uninfected group. These results suggested that M. anisopliae infection activate the prophenoloxidase of B. Longissima, and also suppressed the prophenoloxidase system activation which induced by laminarin. The circulating hemocytes of B. longissima adults have been investigated by means of light and transmission electron microscopy (TEM). Five types of hemocytes were found in the hemolymph and they were identified as plasmatocytes, granulocytes, oenocytoids, spherulocytes and coagulocytes, respectively. Plasmatocytes are the most abundant cell type which represents more than 40% of the total cell population. As infected with M. anisopliae, two types of hemocytes which were plasmatocytes and oenocytoids involved in phagocytosis, and ultrastructure of all hemocytes cell types were changed obviously. However, phagocytosis, nodulation and encapsulation to hypha were not observed, which suggested that M. anisopliae has the ability to avoid the recognition of haemocytes. The total hemocytes counts decreased to very low level after a transient increase, and the relative percentage of hemocytes also changes obviously.Five partial cDNA fragment (GenBank accession numbers: DQ238840 ~ DQ238844) were amplified with degenerate primers designed with the conserved sequences of known CYP4, and the 3'end of BLWH4 was cloned by 3'-RACE. The deduced amino acid sequence of BLWH4 has CYP4 conserved domains of helix k and heme-binding motif. Semiquantitative RT-PCR analysis indicated that the BLWH4 mRNA expression in adults was more than larvae,and increased when adults and 5th instar larvae infected with M. anisopliae MA3, indicated that BLWH4 should plays a role in protection against pathogenic fungus.
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