The role of nuclear receptor E75 in regulating the molt cycle of Daphnia magna and consequences of its disruption.

Biological rhythms regulate innumerable physiological processes, yet little is known of factors that regulate many of these rhythms. Disruption in the timing of these rhythms can have devastating impacts on population sustainability. We hypothesized that the timing of the molt infradian rhythm in the crustacean Daphnia magna is regulated by the joint action of the protein E75 and nitric oxide. Further, we hypothesized that disruption of the function of E75 would adversely impact several physiological processes related to growth and reproduction. Analysis of mRNA levels of several genes, involved in regulating the molt cycle in insects, revealed the sequential accumulation of E75, its dimer partner HR3, FTZ-F1, and CYP18a1 during the molt cycle. Exposure to the nitric oxide donor sodium nitroprusside early in the molt cycle had no effect on E75 or HR3 mRNA levels, but delayed the peak accumulation of FTZ-F1 and CYP18a1 mRNA. The subsequent exuviation was also delayed consistent with the delay in peak accumulation of FTZ-F1 and CYP18a1. These results supported our assertion that nitric oxide binds E75 rendering it incapable of binding HR3. Excess HR3 protein then enhanced the accumulation of the downstream products FTZ-F1 and CYP18a1. Similarly, suppression of E75 mRNA levels, using siRNA, had no effect on mRNA levels of HR3 but elevated mRNA levels of FTZ-F1. Consistent with these molecular responses, the suppression of E75 using siRNA increased the duration of the molt cycle and reduced the number of offspring produced. We conclude that the molt cycle of daphnids is regulated in a manner similar to insects and disruption of E75 results in a lengthening of the molt cycle and a reduction the release of viable offspring.