A somatic transformation gene vector that exploits the genomic integration properties of Junonia coenia lepidopteran densovirus (JcDNV) sequences in vivo has been developed. JcDNV somatic transformation vectors are derivatives of plasmids containing an interrupted genome of JcDNV that provide efficient, robust vectors that can be used to examine regulation of chromosomally integrated transgenes in insects. Microinjection of JcDNV plasmids into syncytial embryos of Drosophila melanogaster or the lepidopterans Plodia interpunctella, Ephestia kuehniella or Trichoplusia ni resulted in persistent transgene expression throughout development. Inclusion of transgenes with tissue-specific promoters resulted in expression patterns canonical with phenotypes of piggyBac germline transformants. Somatic transformation required the presence of the viral inverted terminal repeat in cis only and did not depend upon non-structural viral proteins.

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is an important pest of maize in the Americas and has recently been introduced into Africa. Fall armyworm populations have developed resistance to control strategies that depend on insecticides and transgenic plants expressing Bacillus thuringiensis toxins. The study of various resistance mechanisms at the molecular level and the development novel control strategies have been hampered by a lack of functional genomic tools such as gene editing in this pest. In the current study, we explored the possibility of using the CRISPR/Cas9 system to modify the genome of FAW. We first identified and characterized the abdominal-A (Sfabd-A) gene of FAW. Sfabd-A single guide RNA (sgRNA) and Cas9 protein were then injected into 244 embryos of FAW. Sixty-two embryos injected with Sfabd-A sgRNA hatched. Of these hatched embryos, twelve developed into larvae that displayed typical aba-A mutant phenotypes such as fused segments. Of the twelve mutant larvae, three and five eventually developed into female and male moths, respectively. Most mutant moths were sterile, and one female produced a few unviable eggs when it was outcrossed to a wild-type male. Genotyping of 20 unhatched Sfabd-A sgRNA-injected embryos and 42 moths that developed from Sfabd-A sgRNA-injected embryos showed that 100% of the unhatched embryos and 50% of the moths contained indel mutations at the Sfabd-A genomic locus near the guide RNA target site. These results suggest that the CRISPR/Cas9 system is highly efficient in editing FAW genome. Importantly, this gene editing technology can be used to validate gene function to facilitate an understanding of the resistance mechanism and lead to the development of novel pest management approaches.

Transcriptional activity of the Junonia coenia densovirus (JcDNV) P9 promoter depends on a 557-bp sequence located within the overlapping 3' sequences for viral capsid and nonstructural genes. Utilizing a somatic transformation assay to assess JcDNV promoter activity in Drosophila melanogaster and Plodia interpunctella, viral sequences were subjected to deletional analysis. Removal of a 685-bp fragment reduced P9-driven expression to background levels. Inclusion of a second expression cassette demonstrated vector persistence and confirmed somatic transformation. P9 promoter-driven expression was restored by insertion of a 557-bp JcDNV fragment or by inclusion of a heterologous baculovirus hr5 enhancer. Consensus polycomb transcriptional factor binding sites were identified within the 557-bp fragment, which suggests a potential role in regulating densoviral transcription.

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