Ectopic transgene expression in butterfly imaginal wing discs using vaccinia virus.

The rich diversity of wing color patterns within and between butterfly species makes butterflies an attractive system for studying the development and evolution of pattern formation. Study of the molecular and genetic basis of pattern formation in butterflies has revealed that a number of developmentally important genes possess expression patterns in the developing wing that correlate with the wing color patterns of the adult. For example, genes including engrailed/ invected, distal-less, cubitus inter-ruptis, hedgehog, Notch, and spalt have expression patterns in the fifth larval instar and pupal wing imaginal discs that correlate with the formation of the eyespot, a color pattern composed of a series of concentric rings (1–5). Although these data suggest that these genes play a role in eyespot formation, the evidence is indirect. One way to directly determine the role that these and other genes play would be to ectopically express genes in the developing wings. Currently, there are two viral vectors that enable transgene expression in vivo in arthropods, Sindbis virus (6) and baculovirus (7). In addition, transposon-based technology has also achieved ectopic expression in butterflies (8). Because baculoviruses naturally infect and can be potentially lethal to butterflies , their utility as a gene expression tool is limited. Furthermore, although Sindbis virus has been used as a gene expression vector in butterflies (6), this is an RNA virus and thus possesses a high mutation rate that could possibly impact expression of the gene of interest. In an effort to create an alternative viral system for ectopically expressing transgenes in butterfly pupal and larval wing tissue, we examined the use of vaccinia virus. It has been reported that vaccinia virus, a vertebrate poxvirus, is able to infect gypsy moth cell lines in vitro (9). The vaccinia virus enters the gypsy moth cells and carries out both early and late viral protein synthesis as well as viral DNA replication (9). However, processing of viral late proteins does not occur, and therefore infectious virions are not produced. In this study, we set out to determine whether vaccinia virus could be used as a gene expression vector to ectopically express transgenes on the wings of butterflies in vivo. To test this, we subcutaneously injected fifth instar Junonia coenia larva with vaccinia virus designed to express enhanced green fluorescent protein (EGFP) (10) through the larval cuticle in the area of the wing disc with 1–5 μL [approximately 10 5 plaque forming …