CRISPR/Cas9-induced vitellogenin knockout lead to incomplete embryonic development in Plutella xylostella

Vitellogenin (Vg) is important for insect egg maturation and embryo development . In the present study, we characterized the molecular structure and expression profile of Vg gene, and analyzed its reproductive functions in diamondback moth , Plutella xylostella (L.), a destructive pest of cruciferous crops, using CRISPR/Cas9 system. The P. xylostella Vg ( Px Vg) included all conserved domains and motifs that were commonly found in most insect Vgs except for the polyserine tract. PxVg gene was highly expressed in female pupae and adults. Px Vg protein was detected in eggs and female adults. PxVg was mainly expressed in the fat body and its protein was detected in most tissues, except in the midgut. CRISPR/Cas9-induced PxVg knockout successfully constructed a homozygous mutant strain with a 5-base pair nucleotide deletion. No Px Vg protein was found in the mutant individuals and in their ovaries. There were no significant differences between wild (WT) and mutant (Mut-5) types of P. xylostella in terms of ovariole length and the number of fully developed oocytes in newly emerged females. No significant difference was observed in the number of eggs laid within two days, but there was a lower egg hatchability (84% for WT vs. 47% for Mut-5). This is the first study presenting the functions of Vg in ovary development , egg maturation, oviposition and embryonic development of P. xylostella . Our results suggest that the reproductive functions of Vg may be species-specific in insects. It is possible that Vg may not be the major egg yolk protein precursor in P. xylostella . Other “functional Vgs” closely involved in the yolk formation and oogenesis would need to be further explored in P. xylostella . • PxVg is mainly expressed in female adults, specifically expressed in the fat body. • A homozygous mutant of P. xylostella with 5-bp-nucleotide deletion in the PxVg was developed using CRISPR/Cas9 system. • Knockout of PxVg showed completely inhibition of Vg deposition in the ovaries of mutant individuals. • No significant difference was observed in ovarian development after knockout of PxVg. • No significant difference was observed in oviposition , but there was a lower egg hatchability after knockout of PxVg.