Functional Conservation of Clock Output Signaling between Flies and Intertidal Crabs

Intertidal species have both circadian and circatidal clocks. Although the behavioral evidence for these oscillators is more than 5 decades old, virtually nothing is known about their molecular clockwork. Pigment-dispersing hormones (PDHs) were originally described in crustaceans. Their insect homologs, pigment-dispersing factors (PDFs), have a prominent role as clock output and synchronizing signals released from clock neurons. We show that gene duplication in crabs has led to two PDH genes (beta-pdh-I and beta-pdh-II). Phylogenetically, beta-pdh-I is more closely related to insect pdf than to beta-pdh-II, and we hypothesized that beta-PDH-I may represent a canonical clock output signal. Accordingly, beta-PDH-I expression in the brain of the intertidal crab Cancer productus is similar to that of PDF in Drosophila melanogaster, and neurons that express PDH-I also show CYCLE-like immunoreactivity. Using D. melanogaster pdf-null mutants (pdf(01)) as a heterologous system, we show that beta-pdh-I is indistinguishable from pdf in its ability to rescue the mutant arrhythmic phenotype, but beta-pdh-II fails to restore the wild-type phenotype. Application of the three peptides to explanted brains shows that PDF and beta-PDH-I are equally effective in inducing the signal transduction cascade of the PDF receptor, but beta-PDH-II fails to induce a normal cascade. Our results represent the first functional characterization of a putative molecular clock output in an intertidal species and may provide a critical step towards the characterization of molecular components of biological clocks in intertidal organisms.