Modifying oviposition behaviour of the Oriental fruit fly, Bactrocera dorsalis (Hendel) to obtain uniform G₀ stage eggs for microinjection

The CRISPR/Cas9 technology has opened up newer avenues in insect pest management like precision guided sterile insect technique (pgSIT) which achieves a highly specific mutation in the target genes such as spermatogenesis, sex determination related genes etc. In this regard, validating the loss-of-function of the target gene/s is a prerequisite before the final application. This is easily achieved through DNA-free editing by embryonic delivery of the cognate ribo nucleo protein complex (RNP) into G₀ stage eggs. Obtaining uniform G₀ stage eggs is necessary to offset the microinjection injury and have high heritability of the genomic edits. We to optimized a method to obtain intact eggs of Oriental fruit fly, Bactrocera dorsalis (Hendel) without injury for microinjection by modifying the oviposition behaviour of the gravid female of B. dorsalis to retrieve intact eggs and to obtain large number of G₀ eggs for genome editing. This paper describes a method to obtain required number of eggs for such studies. Out of two egg laying methods, the one with a small container with water covered with parafilm and topped with a thin banana pulp slice provided intact eggs. Maximum oviposition was observed between 20-60 days after eclosion. By the present finding we can obtain sufficient eggs for microinjection at 15-minute interval.