# Functional Analysis of the Scarlet Gene in the Cricket Gryllus bimaculatus

## TL;DR

The study shows that the scarlet gene in crickets is responsible for eye pigmentation but does not affect eye structure or fertility.

## Contribution

The functional role of the scarlet gene in eye pigmentation of Gryllus bimaculatus is experimentally confirmed using CRISPR/Cas9.

## Key findings

- A yellow-eyed mutant line was created using CRISPR/Cas9, showing pigmentation defects from embryonic stages.
- The compound eye structure and reproductive capacity remained unaffected in the mutant line.
- Expression levels of white and brown genes were reduced in the mutant, suggesting a regulatory role.

## Abstract

Eye-color genes are widely used to study insect development and genetics. In this work, we focused on the scarlet gene in the cricket Gryllus bimaculatus. By applying CRISPR/Cas9 gene editing, we produced a stable yellow-eyed mutant line, in which the pigmentation defect was visible from the embryonic stage to adulthood. Despite the altered eye color, the microscopic structure of the compound eyes and the reproductive capacity of the Gbst−/− knockout strain were unaffected. These results show that this gene plays a specific role in eye pigmentation but not in eye development or fertility. Therefore, this gene can be considered a useful visible marker for genetic manipulation in crickets.

The scarlet gene encodes an ATP-binding cassette transporter involved in eye pigmentation across various insect species. In this study, we functionally characterized the scarlet homolog (Gbst) in the cricket Gryllus bimaculatus, a hemimetabolous model organism. Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9-mediated knockout of Gbst generated a stable yellow-eyed mutant line (Gbst−/−) with changed pigmentation evident from embryogenesis through adulthood. Quantitative real-time PCR analysis showed that scarlet expression was extremely low in Gbst−/−, and the transcript levels of white and brown were also reduced. Histological sections of the compound eyes showed that both WT and Gbst−/− mutant possessed complete and well-defined ommatidial structures, indicating that the scarlet gene does not affect compound eye structure. In addition, reproduction tests showed that knockout of the Gbst gene did not affect egg production or embryonic viability. These findings demonstrate that Gbst is a key factor involved in eye pigmentation in G. bimaculatus, and has potential for application as a visual transgenic marker gene.