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First published online March 22, 2004
Journal of Experimental Biology 207, 1453-1460 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00900

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The crayfish Procambarus clarkii CRY shows daily and circadian variation

María Luisa Fanjul-Moles^*, Elsa G. Escamilla-Chimal, Andrea Gloria-Soria and Gabriela Hernández-Herrera

Laboratorio de Neurofisiología Comparada, Departamento de Biología, Facultad de Ciencias, Universidad Nacional Autónoma de México, México DF 11000

View larger version (17K): [in a new window]   Fig. 1. (A) Schematic representation of the eyestalk of P. clarkii. R, retina; ME, medulla externa; MI, medulla interna; MT, medulla terminalis; HB, hemiellipsoid body; LG, lamina ganglionaris. The dark region represents cells expressing CRY immunoreactivity in MT-HB. Note the cluster of cells located in the inferior region of the hemiellipsoid body. (B) Schematic representation of the brain of P. clarkii. PR, protocerebrum; PT, protocerebral tract; AMC, protocerebral anterior median cluster; DC, deuterocerebrum; TC, tritocerebrum. Note the dark region representing the protocerebral median cluster immunoreactivity.

View larger version (58K): [in a new window]   Fig. 2. (A–D) Confocal image of the three-dimensional topography of the eyestalk and brain CRY immunoreactivity of P. clarkii at two times of day. All the images are optical sections of whole-mount preparations. (A) MT-HB complex cells expressing CRY at 03:00 h. The hemiellipsoid body shows a cluster of cells expressing a strong CRY immunoreaction; an immureactive cell apparently located in MT seems to branch towards the HB (small arrows). Note the protocebral tract (PT) expressing a dim immunoreaction. (B) MT-HB complex at 19:00 h, showing low immunoreaction. (C) Protocerebrum at 03:00 h; note the lack of immunoreactive signal at this hour. (D) Neurons of the protocebral anterior medial cluster (AMC) expressing strong CRY immunoreactivity at 19:00 h. Scale bars, 100 µm. (E) Fluorescence micrograph of a histological section showing the cytoplasm of some cells of the protocerebral anterior medial cluster (AMC) expressing CRY immunoreactivity at 11:00 h. Scale bar, 20 µm. See text for explanation.

View larger version (26K): [in a new window]   Fig. 3. Western blots showing the specificity of the the Drosophila CRY antibody for crayfish. (A) The anti-CRY antibody recognizes CRY in the brain of crayfish. The molecular mass of the crayfish protein matches that of the cryptochrome reported for Drosophila (approximately 60 kDa). At 07:00 h and 11:00 h, small bands of lower intensity appear below. (B) After incubation of the antibody with the control peptide the immunoreactive bands are not present, indicating the antibody specificity. Each lane represents a time point of sample collection from animals maintained in the dark for 72 h (N=3). The left side of the figure shows the position of molecular mass markers.

View larger version (27K): [in a new window]   Fig. 4. Chronograms illustrating the daily rhythmic pattern of CRY abundance in P. clarkii. Bars at the bottom of each panel indicate the illumination conditions (open, light; closed, dark). Values are means ± S.D. (N=3). The corresponding western blot is shown at the top of each panel. Each lane represents the time points of sample collection. D-CRY is the stained control peptide. (A–C) Changes in the daily pattern of CRY abundance in the eyestalk, showing no significant circadian rhythms. (D–F) Changes in the rhythmic pattern of CRY abundance in the brain of P. clarkii: when the organisms changed from LD to DD, a clear circadian pattern was observed. Note that CRY abundance rhythm mean (mesor) increases under continuous darkness, especially in the eyestalk. The data for B,C,E,F were obtained from tissues taken from animals maintained in the dark for 24 and 72 h, respectively. A and D show data obtained from tissues form animals maintained in LD. Relative CRY abundance is indicated by the relative average intensity of the immunoreactive area of the band.