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leer Bluedot   Establishment and Characterization of three New Embryonic Spodoptera littoralis Cell Lines and Testing their Susceptibility to SpliMNPV

Ibrahim Ahmed
Studium: im Ausland (Irak)
Tätigkeit: Doktorand

Baculoviruses have a significant potential as biological pesticides. Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) could thus be applied to protect plants against the African Cotton Leafworm. For the in vitro production of SpliMNPV a cellular system has to be established. For this purpose three new continuous cell lines were established from the embryonic tissue of the cotton leaf worm Spodoptera littoralis. The three cell lines were designated as Spli-C, Spli-S and Spli-B. They consist mostly of spherical cells as well as spindle and giant cells. The population doubling time for the three cell lines Spli-C, Spli-S and Spli-B were 30.5, 31 and 44.5 hrs, respectively, at passage 19, while the population doubling time at passage 120 was 26, 27 and 32 hrs, respectively. The DNA fingerprinting techniques RAPD and DAF confirmed that the cell lines originated from Spodoptera littoralis tissues. Lactate dehydrogenase (LDH) isozyme analysis showed a distinguishable difference between the three new Spodoptera littoralis cell lines and the other insect cell lines that are used in our laboratory. The susceptibility tests of the three new cell lines showed that all the cell lines were susceptible to SpliMNPV, whereby many viral occlusion bodies were observed inside the infected cells. The susceptibility of the three cell lines to Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) was investigated too. All three cell lines were non-permissive to AcMNPV, with the cells dying a few hours post-infection and forming apoptosis-like bodies. Nuclear DNA fragmentation was observed in all AcMNPV-infected cell lines by DNA gel electrophoresis analysis. The SpliMNPV OB productivity of the three cell lines showed significant differences. The cell line Spli-C had the highest susceptibility to SpliMNPV compared to the other two cell lines. OB production was about 1x107 OBs/ml in the Spli-C cell line at MOI 1, while the amounts of OBs produced by the Spli-S and Spli-B cell lines were 3.3x106 and 2x106 OBs/ml, respectively. SpliMNPV polyhedrin gene expression and DNA replication in the three cell lines were also investigated over time using quantitative PCR (qPCR). In order to reach high cell densities the Spodoptera littoralis cells were immobilized using sodium cellulose sulfate (NaCS) and polydiallyldimethylammonium chloride (PDADMAC) capsules to protect cells from shear stress as a result of agitation and gas sparging in order to supply sufficient oxygen. The cell densities increased from 4-5x106 cells/ml in suspension culture to 1.3x107 cells/ml in capsules. Our results suggest that large-scale production of SpliMNPV as a biopesticide is possible with these cell lines.