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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.

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