DIPA-CRISPR Mediated Knockout of Vermilion Generates a Visible Eye Color Marker for The Band-Legged Ground Cricket...

The molecular basis of photoperiodism, by which insects use photoperiodic cues to anticipate seasonal changes and regulate key life-history events such as development, diapause, and reproduction, remains poorly understood. Studies on the molecular mechanisms of photoperiodism in hemimetabolous insects are limited compared with those in holometabolous insects, largely due to the lack of appropriate model organisms. The band-legged ground cricket Dianemobius nigrofasciatus represents a valuable model system because it exhibits clear photoperiodic responses in the maternal induction of embryonic diapause, the wing morph, and the rate of nymphal development. With the recent availability of the D. nigrofasciatus genome sequence, the establishment of effective genome-editing methods and reliable marker genes is expected to promote functional genomic analyses. In this study, we aimed to establish a direct parental (DIPA)-CRISPR genome-editing approach and evaluate the utility of vermilion (Dn-v), a gene involved in ommochrome synthesis, as a visible eye color marker for mutant screening. Cas9 ribonucleoprotein complexes were injected into females 3-5 days after adult emergence, during the vitellogenic stage, successfully yielding Dn-v knockout mutants. These mutants had white compound eyes throughout development, with pigmentation reaching a vermilion color about 20 days after adult emergence. We further examined the photoperiodic response associated with maternal diapause induction in knockout mutants. Similar to the wild-type, knockout mutants exhibited low and high diapause incidence under long-day and short-day conditions, respectively. Our results demonstrate that DIPA-CRISPR is an effective genome-editing method in D. nigrofasciatus and that Dn-v serves as a practical and reliable marker gene. The establishment of these genomic tools provides a foundation for future functional analyses aimed at elucidating the molecular basis of photoperiodism in hemimetabolous insects.