Fig. 2.

Factors required for the reciprocal cry2 and Pdp1_iso1 regulation in the gut. (A) Reproductive females that naturally produce JH were injected with dsRNA to silence the indicated genes, and the levels of cry2 and Pdp1_iso1 mRNAs in their guts were determined 4 d later, together with ovarian morphology. lacZ dsRNA served as a control. Depletion of Met, Clk, and Cyc (individually or in combinations) altered both transcripts toward their diapause mode; depletion of Met or Tai prevented oogenesis. (B) RNAi depletion of Met, Clk, or Cyc in diapause females partially reduced cry2 and increased Pdp1_iso1 mRNAs, equalizing their levels. (C) Met, Clk, and Cyc are necessary for the JH mimic methoprene to revert the levels of cry2 and Pdp1_iso1 mRNAs to the reproductive mode, as both transcripts became approximately equalized in the guts of Met, Clk, or cyc RNAi females that were given methoprene. (D and E) Pdp1_iso1 and cry2 form a feedback loop of mutual repressors in the gut. dsRNAs were injected to females 1 d after adult ecdysis, and transcript levels in their guts were monitored 1, 2, 3, 5, 7, 14, and 21 d later. Knockdown of either gene remained effective throughout this period. Removal of Cry2 in diapause females caused Pdp1_iso1 mRNA to increase near levels normally occurring in guts of reproductive females (D). Conversely, expression of cry2 was enhanced when reproductive females were subjected to Pdp1_iso1, although to a lesser extent than in diapause controls. Values are mean ± SEM from three independent experiments. (*P < 0.05 and **P < 0.001 vs. lacZ controls as assessed, Tukey honestly significant difference test).