The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.  In the construction of CRISPR/Cas9-mediated dsLmRNase2^–/–mutant locusts, we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned.  However, the heritable efficiency of the dsLmRNase2 deletion to the next generation G₁ progeny was similar between adults derived from the tanned or less tanned engineered eggs.  Further, the similar effective mutant ratios in the normally developed eggs and G₀ adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9.  Moreover, we found that the syncytial division period, which was longer than the time for tanning, conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs.  We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection.  Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure.  In summary, tanned eggs possess stronger defense responses and higher efficiency of genome editing, providing an improved model for developing Cas9-mediated gene editing procedures in locusts.

Cytochrome b₅ (Cyt-b₅) is a small heme protein and known to be involved in a wide range of biochemical transformations, including cytochrome P450 monooxygenase (CYP)-mediated metabolism of endogenous and exogenous compounds.  Studies on Cyt-b₅ are more concentrated in mammals, but are relatively rare in insects.  The characteristics and function of Cyt-b₅ from Locusta migratoria have not been described yet.  We sequenced the full-length cDNA sequence of Cyt-b₅ from L. migratoria (LmCyt-b₅) by reverse transcription-PCR (RT-PCR) based on locust transcriptome database.  The phylogenetic analysis showed that LmCyt-b₅ was closely related to the Cyt-b₅ from Blattodea.  LmCyt-b₅ was highly expressed in ovary, Malpighian tubules, midgut, gastric caeca, and fat bodies.  Silencing of LmCyt-b₅ had no effect on the susceptibility of L. migratoria to four different insecticides.  Suppression of LmCyt-b₅ or silencing of both LmCyt-b₅ and LmCPR did not significantly change the total CYP activity toward the substrate 7-ethoxycoumarin (7-EC).  However, coexpression of LmCYP6FD1 with LmCPR and LmCyt-b₅ together in Sf9 cells by using Bac-to-Bac baculovirus expression system significantly increased the catalytic activity of LmCYP6FD1 toward 7-EC as compared with the coexpression of LmCYP6FD1 with cytochrome P450 reductase (LmCPR) or LmCyt-b₅ separately.  These results suggest that LmCyt-b₅ plays an important role in the catalytic reaction of LmCYP6FD1 toward 7-EC in our in vitro experiments.  Further study is needed to clarify the role of LmCyt-b₅ in CYP-mediated catalytic reactions in L. migratoria.