Window to the Fluorescence: TheWhiteEye-Color Gene of Western Corn Rootworm,Diabrotica virgifera virgifera

Abstract

Eye-color mutations have proven useful in multiple insect species to help facilitate the development and use of transgenic tools for functional genomics. While there is species-specific variation in the pigments used to color insect eyes, every species studied thus far requires an ortholog of the ABC transporter gene white for proper pigmentation of the eyes. Previously, we generated transgenic western corn rootworm, Diabrotica virgifera virgifera, and found that their wild-type eye color obscured our ability to visualize a fluorescent marker driven by the widely used 3xP3 eye-specific promoter. Therefore, we sought to identify the D. v. virgifera ortholog of white (Dvvw). Here we report the discovery, cloning, and analysis of Dvvw cDNA and promoter.We also utilize RNA interference to knock down Dvvw mRNA in a transgenic strain, thereby demonstrating the utility of eye-color mutations when developing transgenic technologies.

Structural and functional insights into the Diabrotica virgifera virgifera ATP-binding cassette transporter gene family

“…Studies have revealed that some ABCG proteins perform other crucial physiological roles in the transport of lipids, sterols, and drugs [73]. In the current study, RNAimediated knockdown of Dvvw resulted in a white-eyed phenotype consistent with prior observations in T. castaneum [30], and with our own previous findings in D. v. virgifera [46]. Our findings support a prediction that Dvvw is part of the ommochrome pathway, where it is likely acting within a heterodimeric complex to import ommochrome pigments into the pigment granules of the compound eye.…”

“…Prior to death, it was noted that the body mass of treated individuals was less than that of similarly-aged larvae treated with buffer alone ( Specifically, injection of dsRNA targeting Dvvw, gave the expected white-eye phenotype (Fig. 4E); indeed, we had identified this white ortholog previously [46]. Injection of DvvABCG_3712 dsRNA into pre-pupae caused developmental defects that resulted in 80% mortality (Table 3; Fig.…”

“…Analogously, we were unable to identify a direct ortholog to TcABCG-8A, so we targeted DvvABCG_14042, the closest identifiable homolog according to BLASTp results. Finally, while the ABCG genes TcABCG-9A and TcABCG-9B represent the orthologs of the T. castaneum eye-color genes scarlet and white, respectively [32], results of BLASTx searches of the DNASTAR assembly resulted only in the identification of an ortholog of white [46]. Semiquantitative PCR of these eight D. v. virgifera ABC transcripts showed that all are expressed across all of the developmental stages examined (Fig.…”

Structural and functional insights into the Diabrotica virgifera virgifera ATP-binding cassette transporter gene family

“…virgifera [47]. Our findings support a prediction that DvvABCG_13829 is part of the ommochrome pathway, where it is likely acting within a heterodimeric complex to import ommochrome pigments into the pigment granules of the compound eye.…”

“…Specifically, injection of dsRNA targeting DvvABCG_13829, which is most closely related to eye-color transporters, gave a white-eye phenotype (Fig. 4E); indeed, DvvABCG_13829 corresponds to the ortholog of white we had identified previously [47]. Injection of DvvABCG_3712 dsRNA into pre-pupae caused developmental defects that resulted in 80% mortality (Table 3; Fig.…”