The Masc gene product controls masculinization in the black cutworm, Agrotis ipsilon

Sex determination has been studied in the model lepidopteran species Bombyx mori, but it remains poorly understood in lepidopteran pests. In the present study, we identified and characterized the Masculinizer (Masc) gene in a Noctuidae pest species, Agrotis ipsilon. Sequence analysis revealed that AiMasc encodes a protein of 658 amino acids that has two CCCH-type zinc finger domains and two conserved cysteine residues (Cys-277 and Cys-280). We assessed the masculinizing activity AiMasc in BmN cells and found that/z'Masc induced expression of the male-specific doublesex isoform. Disruption of Masc via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in A. ipsilon caused abnormalities in abdominal segments and external genitalia, resulting in male-specific sterility. These results suggest that Masc participates in the process of sex determination in A. ipsilon. Successful identification of sex-determination gene in a pest species may enable the development of novel genetic approaches for pest control.

CRISPR/Cas9-mediated Tyrosine hydroxylase knockout resulting in larval lethality in Agrotis ipsilon

Tyrosine hydroxylase (TH)is involved in insect melanin and the catecholamine biosynthesis pathway.TH as an enzyme catalyzing the conversion of tyrosine to 3,4- dihydroxyphenylalanine is the first step reaction in the pathway.Although TH has been proven to affect the pigmentation of the epidermis and development in many insects,there is no report about physiological function of the THgene inAgrotis ipsilon.Here we cloned the TH gene from A.ipsilon.Semi-quantitative real-time polymerase chain reaction (PCR) analysis showed that AiTH was expressed at all development stages.Moreover,its high expression levels in the head and epidermis suggest that it is mainly related to pigment deposition and insect development.Then,we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9system to target the AiTH gene:deletion events were detected at the target sites.Compared with the control group,a few mutants with the phenomenon of narrowing in the egg shell and embryos can develop but cannot hatch;the other hatched embryos were seriously dehydrated after hatching and died within the first day.Quantitative real-time PCR analysis revealed that THwas down-regulated inAiTHmutants.Here,our work demonstrated thatAiTHplays an important role in growth and development of newly hatched larvae;meanwhile,it would be a promising target to explore a control strategy for A.ipsilon.

CRISPR/Cas9-based mutation reveals Argonaute 1 is essential for pigmentation in Ostrinia fumacalis

Ostrinia fumacalis (Lepidoptera: Pyralidae) is one of the most destructive agricultural pests in Asia. Traditional pest-management methods include sex pheromone capture, transgenic crops that produce Bacillus thuringiensis toxin, and pesticides. Although these strategies control pest populations effectively, they also cause negative side effects, including dramatically increased pesticide resistance, severe pollution, and hazards for human health. Recently developed genome editing tools provide new prospects for pest management and have been successfully used in several species. However, few examples have been reported in the agricultural pest O. fumacalis due to a lack in genomic information. In this report, we identified only one transcript of O. fumacalis Argonaute 1 (OfAgo 1) gene from the genome and cloned the open reading frame. OfAgol presented the maximum expression at the embryo stage or in the fat body during the larval stages. To understand its function, an OfAgol mutant was constructed using the Clustered Regularly Interspaced Short Palindromic Repeat/RNA-guided Cas9 nuclease (CRISPR/Cas9). Mutagenesis of OfAgol disrupted cuticle pigmentation by dowregulating micro RNAs and pigmentation-related genes. This is the first report for the cloning and functional analysis of OfAgol, revealing a role of OfAgol in cuticle pigmentation. The current report also established a CRISPR/Cas9 system in O. fumacalisy providing a new insight for pest management.