First report on the utility of pupal case for early determination of CRISPR/Cas9 ribonucleoprotein mediated genomic edits in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Tephritidae: Diptera).

Abstract

The Oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly invasive pest of quarantine importance affecting the global fruit trade. In managing B. dorsalis, methods like cultural, biological, chemical, sterile insect technique (SIT), and semiochemical-mediated attract-and-kill are in use with varying success. The SIT approach is the method of choice for a chemical-free, long-term suppression of B. dorsalis, followed in many countries across the globe. The nonspecific mutations caused by irradiation affect the overall fitness of flies, thus requiring a more precise method for a heritable, fitness-not-compromising approach. In this regard, CRISPR/Cas9-mediated genome editing enables the creation of mutations at the precise genomic location/s through RNA-guided dsDNA cleavage. Of late, DNA-free editing employing ribonucleoprotein complex (RNP) is preferred to validate the target genes at G0 stage embryos in insects. It requires characterizing genomic edits from adults after completing their life cycle, which may entail a few days to months, depending on longevity. Additionally, edit characterization is required from each individual, as edits are unique. Therefore, all RNP-microinjected individuals must be maintained until the end of their life cycle, irrespective of editing. To overcome this impediment, we predetermine the genomic edits from the shed tissues, such as pupal cases, to maintain only edited individuals. In this study, we have shown the utility of pupal cases from five males and females of B. dorsalis to predetermine the genomic edits, which corroborated the edits from the respective adults.