Plasmids containing a modified genome of the Junonia coenia lepidopteran densovirus (JcDNV) integrate into the genome of a host insect cell rather than becoming infectious. These plasmids have been utilized to achieve somatic transformation in Drosophila that is stable from embryo to adult (Royer et al, 2001, Insect Mol. Biol. 10, 275). We have assessed the effect of additional modifications including different expression cassettes on the efficiency of JcDNV plasmid somatic transformation activities in three orders of insects. The plasmid pJDsRed[3xP3EGFP]H, which contains the Densovirus P9 promoter controlling expression of DsRed fluorescent protein and the 3xP3EGFP expression cassette (Berghammer et al., 1999. Nature 402, p370), was injected into syncytial embryos of the fruitfly Drosophila melanogaster wm, the moth Plodia interpunctella, and the beetle, Tribolium castaneum. Somatic transformation was observed on the basis of either DsRed and/or GFP fluorescence in G0 embryos and larvae of all four species at rates from 40-95%. Where comparisons could be made, the expression patterns were consistent with germ line transformed insects. Removal of the JcDNV coding sequences for nonstructural proteins or the right inverted terminal repeat had no effect on the rate of somatic transformation. Inclusion of the P9DsRed expression cassette in a piggyBac transformation vector demonstrated the wide utility of this cassette as a maker for transformation in various insect orders. These experiments demonstrate the utility of JcDNV vectors in assessing promoter activity in insects.