even-skipped acts as a pair-rule gene in germ band stages of Tribolium development

The pair-rule gene even-skipped (eve) is essential for insect segmentation, yet its function varies among insect clades. While loss of eve results in typical pair-rule phenotypes in Drosophila, knock-down of eve orthologs shows segmental, gap-like, or asegmental phenotypes in non-Drosophila insects. In Tribolium, knock-down of the eve ortholog (Tc-eve) resulted in a graded phenotypic series ranging from strong to weak, the most informative of which was intermediate phenotypes. The strong knock-down embryos displayed asegmental phenotypes and severely disorganized germ bands which have prevented determination of Tc-eve function in later stages. In order to understand the segmentation function of Tc-eve during later germ band elongation stages, we analyzed intermediate Tc-eve^RNAi embryos in which germ band elongation was less affected. Most intermediate Tc-eve^RNAi germ bands displayed segmentation defects with a double segmental periodicity in the abdomen. In these intermediate embryos, Tc-engrailed (Tc-en) stripes were ectopically expanded into large bands with a double segmental periodicity, while the remaining Tc-en stripes between the expanded Tc-en stripes were absent or barely formed. The expanded Tc-en stripes seemed to be activated by primary Tc-eve stripes and Tc-paired, both of which failed to resolve into secondary segmental stripes. The absence of Tc-en stripes appeared to be a consequence of the absence of the secondary stripes of Tc-runt that were required for the activation of Tc-en stripes. These results suggest that Tc-eve functions as a pair-rule gene at least in the germ band stages of Tribolium development.