Evaluation Of The Predation Of Natural Enemies In Rice Ecosystem With Monoclonal Antibodies

Using the monoclonal antibodies developed respectively against rice planthoppers, brown planthopper, Nilaparvata lugens (St?l), white-backed planthopper, Sogatella furcifera (Horváth) and collembola, field predation of the major natural enemies of rice planthoppers and collembola were evaluated. The results were as follows: 1) Four monoclonal antibodies (McAbs), namely 3B2, 3B11, 3H3 and 4B8, were developed against N. lugens using hybridoma technique. All McAbs had high absorption values even they were diluted over 4.096×106 times. They reacted with all the stages of N. lugens while not cross-reacted with other insect pests and predators in the rice paddy. The 3H3 and 4B8 were the most specific. SDS-PAGE and Western blot analysis indicated that 3B2, 3B11 bound specifically to one polypeptide with molecular weight of 287 kDa and 3H3, 4B8 reacted with the polypeptides with molecular weights of 215, 199 and 167 kDa. At the temperature of 25℃, the 3B2, 3B11, 3H3 and 4B8 could detect the epitopes after one female of N. lugens had been ingested by Pardosa pseudoannulata for about 35.04h, 11.45h, 8.81h and 45.91h, respectively. Immunodiffusion showed that all McAbs belong to IgG3 subclass. Based on the results as above, 4B8 was chosen to study the interactions between N. lugens and their predators in the field. 2) Five McAbs, namely 1B5, 2F10, 2G12, 3D8 and 4F8, were developed against the collembola using hybridoma technique. They had high absorption values even they were diluted over 1.024×108 times. All of the McAbs only reacted with the collembola while not cross-reacted with other insect pests and predators. Immunodiffusion showed that the 1B5, 2F10, 3D8 and 4F8 belong to IgG3 subclass, while the 2G12 belong to IgG1 subclass. 3) Three variations of ELISA, direct, indirect and double antibody sandwich methods of ELISA were examined for their abilities to detect N. lugens antigens in the predator guts. The results showed that the direct and indirect ELISA were affected more strongly by the not-target antigens than on the double antibody sandwich ELISA. When the negative effects of the non-target were about 5%, the concentration of the non-target antigens was 0.042mg/ml with the direct and indirect ELISA methods and 0.214mg/ml with the double antibody sandwich ELISA methods respectively. The double antibody sandwich ELISA methods were the most sensitive when the concentration of the non-target antigens was 0.214mg/ml. They could detect about 16.91 ng/ml (1/17509 individuals of the female/ml) of N. lugens antigens compared with 67.59ng/ml (1/4378 female/ml) for the indirect ELISA method and 135.18ng/ml (1/2189 female/ml) for the direct method. The double antibody sandwich method of ELISA was established with the 4B8 diluted 1000 times (28.130μg/ml), enzyme-linked antibody HRP-4B8 diluted 4000 times (1.045μg/ml) and sample diluted 100 times (100ml/individual) for detection of N. lugens antigens. The negative effect of the non-target antigens was lower than 5% and about 1.691 μg prey antigens (1/175 female) in a predator gut could be detected under this detecting system. 4) Indirect methods and double antibody sandwich methods of ELISA with 1B5, 2F10, 2G12, 3D8 and 4F8 were examined for their abilities to detect the collembola antigens in predator guts. The results showed that the double antibody sandwich ELISA with 2G12 was too insensitive to be used. The concentrations of the predator antigens in a sample had a greater effect on indirect ELISA method with 1B5, 2F10, 3D8 and 4F8 than on the double antibody sandwich ELISA method with the same monoclonal antibodies. The negative effects of the non-target antigens on the double antibody ELISA methods and on the indirect ELISA methods were about 20% and 75% when the concentration of the non-target antigens was 0.428mg/ml. The double antibody sandwich ELISA methods were the most sensitive under this concentration of the non-target antigens, by which the lowest concentration of the prey antigens that could be detected was 89.73ng/ml (1/104 individuals of the collembola/ml). They were about 179.45ng/ml (1/52 collembola/ml) for the indirect ELISA method with 1B5, 3D8 and 4F8 and 89.73ng/ml (1/104 collembola/ml) with 2F10. The double antibody sandwich method of ELISA was established with the 2F10 diluted 4000 times (34.193 μg/ml), enzyme-linked antibody HRP-2F10 diluted 1500 times (2.463 μg/ml) and sample diluted 50 times (50ml/individual) for detection of the collembola antigens. The negative effect of the non-target antigens was lower than 20% and 4.49μg prey antigens (1/2 individuals of the collembola) remained in a predator guts could be detect under this system. 5) The effects of temperature, meal size and predator body size on detection period and antigen decay rate were studied using the 4B8 monoclonal antibody-based double sandwich ELISA. The prey antigens of N. lugens in the guts of P. pseudoannulata decayed exponentially (y=exp(a*t)). Prey detection periods decreased significantly with the increase of temperature. At the temperature of 16°C, the detection periods were 6.279d, 6.686d, 9.593d and 15.514d respectively for P. pseucoanlate predated 1, 2, 3, and 4 pregnant females of N. lugens. When temperature was up to 30°C, the detection periods were less than one day. At the temperature range of 16 to 37°C, the relationship between prey detection periods and temperature was exponentially (tDp =a+exp(b+c*T)). The meal sizes also affected the detection periods remarkably. At the temperature of 25℃, while meal size increased from one female to four females, the prey detection period lengthened from 1.890d to 5.980d. The predator body size had no significantly effects on the prey digested rate. The equation, R=Q0*d/f(0.1526+exp(3.9716-0.1347*T)),was established for the model system of P. pseucoanlate------N. lugens. 6) Predators collected from rice paddy were examined to identify qualitatively predator species of N. lugens, S. furcifera and collembola using the monoclonal antibodies 4B8, 2B6 and 2F10, respectively. The results showed that the positive rates of Cyrtorrhinus lividipennis, P. pseudoannulata, Pirata subpiraticus, Marpissa magister and Clubiona japonicola were the highest to 4B8, those of C. lividipennis, P. pseudoannulata, Coleosoma octomaculatum, Salticidae were the highest to 2B6, and those of Dolomedessp., Salticidae, P. pseudoannulata, M. magister and C. lividipennis were the highest to 2F10, while Tetragnatha spp. were not found positive response to any monoclonal antibody. There were some arthropods species predated all of the three insect prey species. The total positive rate of Dolomedes sp., P. pseudoannulata, C. lividipennis, P. subpiraticus, M. magister, Salticidae and C. japonicola were above 20%. 7) Field investigation showed that the densities of N. lugens, S. furcifera and P. pesucoanulate changed respectively within the ranges of 0~109 individuals/hill, 0~126individuals/hill, and 0~2 individuals/hill in the single rice cropping season in Hangzhou. Throughout the whole rice-growing season, the largest prey biomass that each P. pseudoannulata predated on N. lugens and S. furcifera were 0.449mg (0.33 female) and 1.198mg (1.32 female), respectively. The largest predation rates of them were 2.279% and 0.608%, respectively. This suggested that these two rice planthoppers can't be controlled by the predators. It was resulted from low population densities of P. pseudoannulata. 8) Correlation analysis indicated that the positive rates, the average prey captured biomass and the total prey captured biomass of P. pseudoannulata for N. lugens and S. furcifera increased with the increase of the field biomass of N. lugens and S. furcifera. There was significant relationship between the predation rate of P. pseudoannulata for N. lugens and the field biomass of N. lugens. The positive rates and the average prey captured biomass of P. pseudoannulata for S. furcifera, and the positive rate of P. pseudoannulata for N. lugens also increased with the increase of the occupying prey biomass of each predator, respectively. The relationship between the predation rates of P. pseudoannulata to collembola and the densities of collembola were not significant. The total positive rate of P. pseudoannulata to three prey species fluctuated at the range of 30%~60% throughout the whole rice growing season. The average and total prey biomass of P. pseudoannulata increased with the increase of total prey biomass in the field. The major prey species of P. pseudoannulata in the different rice growing periods were different. In the primarily periods after rice transplanted, the major prey species of P. pseudoannulata were collembola and S. furcifera, while S. furcifera and N. lugens were the major prey species in the middle and late rice growing periods.