Study On Physiological And Biochemical Mechanisms Of Pathogenesis Caused By Heterortiabditis Bacteirophora In White Grubs

Underground pest is a most important kind of agricultural pests. As one of thedominant population of underground pests, white grubs can cause a lot of economicloss to agricultural production in China or even all over the world every year.Entomopathogenic nematodes ("EPN or nematodes ", for short) have been one of themost widely used biological pesticide in the past few years. It’s indicated thatnematode was an ideal pesticide for underground pest control, especially for the whitegrubs. It’s the representative of biological pesticides. Many highly pathogenicnematodes species have been screened and identified for field application. However,pathogenesis caused by nematode in white grubs remains unknown at present. Inorder to obtain better application of entomopathogenic nematodes on controlling thewhite grubs, this paper focused on the pathogenic mechanisms of entomopathogenicnematodes. A series of physiological and biochemical researches were conducted, themain results were as follows:1. White grubs were infected with Heterorhabditis bacteriophora Cangzhoustrain. The difference of phenoloxidase (PO) and carboxylesterase (CarE) activitybetween infected grubs and control were observed. We find that that PO or CarEactivity of the infected grubs generally showed a "rise-peak-down" trend. In24,12,36h, the PO activity in the2nd-instar larvae of Hololtrichia oblita Faldermann,Holotrichia parallela Motschulsky and Anomala corpulenta Motschulsky reachedmaximum and was significantly higher than contro（lP<0.05）, respectively. Similarly,in48,36,48h, the CarE activity ran up to maximum and was significantly higher thancontrol（P<0.05）, respectively.2. TEM (transmission electron microscopy) techniques were used to observedmorphological changes in fat body and midgut cell,which came from2nd-instarlarvae of Hololtrichia oblita Faldermann and Holotrichia parallela Motschulsky(i.e.white grubs), repectively.200Ijs (infective juveniles, volume:5μL) nematodes wereinjected into white grubs. Of course, the nematode species was Heterorhabditisbacteriophora Cangzhou strain, too. There were gradually structure changes in fatbody and midgut cells after24h and48h of Ijs injection. After24h, fat droplet, located at fat body cells, became smaller. Its color became shallow. The endoplasmicreticulum, mitochondria became swells in both fat body and midgut cells. Microvillusof the midgut to began to fall off. After48h, the membrane structure that packageda lot of fat droplet was broken. Chromatin in fat body and midgut cell nucleusdissociated. Mitochondria and endoplasmic reticulum disintegrated completely inboth two kinds of cells. Microvillus of the midgut almost compeletely lost.3. The saturated phenol extraction method was screened out as the best whitegrub protein preparation method, taking protein yield, SDS-PAGE analysis and2-DE(two-dimensional electrophoresis) Gel mapping analysis into consideration. Thesaturated phenol extraction method integrated the proteome characteristics of whitegrubs, minimized the interference of other kinds of impurities. Its protein yield was14.18mg/g, and the protein sample could be easily redissolved. There were745spoton2-DE gel mapping, and26bands on SDS-PAGE gel. The number of spots andbands were both the most among three protein preparation method. What’s more,proteins from saturated phenol extraction method showed the best reproducibility in2-DE. Protein spots distribution was uniform, too.The method lay a foundation forthefurther use on differentially expressed proteome.4. Differentially expressed protein in white grubs under200Ijs/grub infectionwere analysed, using2-DE (two-dimensional electrophoresis) and MS (massspectrometry).The results showed that there were significant protein expressionchanges between control and treat. After24h of injection, there were9proteinsupregulated, while13proteins downregulated.8of these protein were successfullyidentified by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight)MS. These proteins are mainly haemolymph protein, immune related protein, actin,parasite relative proteins, etc.