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.

CRISPR/Cas9 Ribo nucleoprotein complex‐mediated editing of the OBP13 gene affected the response of male Bactrocera dorsalis (Diptera: Tephritidae) to methyl eugenol

“…The G0 stage of B. dorsalis eggs was collected from gravid females as standardized earlier (Ashok, Bhargava, Asokan, Pradeep, Kennedy, Rai, et al., 2023). The injection mixture/RNP complex (sgRNA: Cas9) was set assembling; 500 ng/μL NEBEnGen Spy Cas9‐NLS protein, 500 ng/μL of sgRNA and 5 mM KCl,1X NEB r3.1 Buffer made a reaction volume using autoclaved MilliQ water and incubating at 25°C for 45 min.…”

Developing genetic tools to control the Oriental fruit fly: Potential approaches and target genes

“…In B. dorsalis, pesticide resistance has been reported in several countries (Azhar et al, 2018;Jin et al, 2011;Liu et al, 2019;Scott, 2008). Researchers are urged by the crucial need for a novel, sustainable, and environmentally safe means of controlling this pest (Ashok et al, 2023;Legros et al, 2021;Mutamiswa et al, 2021). Biological means of control, such as the sterile insect technique (SIT) (Vreysen et al, 2007), entomopathogenic fungi, parasitoids (Muriithi et al, 2016), and predatory ants , have been implemented in area-wide integrated pest management programmes (AW-IPM) against B. dorsalis (Heve et al, 2021).…”