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Keywords:

  • Indexing terms: pigment-dispersing hormone;
  • circadian rhythms;
  • isopod;
  • Eurydice pulchra;
  • gene expression;
  • in situ hybridization;
  • immunohistochemistry;
  • quantitative RT-PCR

Abstract

Pigment-dispersing factor (PDF) is well known as a circadian clock output factor, which drives daily activity rhythms in many insects. The role of its homologue, pigment-dispersing hormone (PDH), in the regulation of circadian and/or circatidal rhythmicity in crustaceans is, however, poorly understood. The intertidal isopod crustacean, Eurydice pulchra has well-defined circatidal (12.4-hour) activity rhythms. In this study we show that this runs parallel to a circadian (24-hour) cycle of chromatophore dispersion. As a first step in determining the potential role of PDH in these rhythms, we have identified a novel form of PDH expressed in this species. Because conventional homology cloning was unsuccessful, we employed immuno-identification and Edman microsequencing to determine the primary structure of this peptide. From this, cDNA cloning identified the nucleotide encoding sequence and thus facilitated description of PDH neurons by in situ hybridization and immunohistochemistry. We show them to be morphologically similar to those that co-ordinate circadian activity rhythms in insects. In animals expressing both tidal (activity) and circadian (chromatophore) rhythms, however, there was no evidence for a corresponding periodicity in the expression of pdh transcript, as determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in Eurydice heads. It is therefore suggested that any role for PDH in daily/tidal timing in Eurydice is not mediated at the transcriptional level, rather rhythms in neurohemal release may be important in such co-ordination. J. Comp. Neurol. 519:562–575, 2011. © 2010 Wiley-Liss, Inc.