Table 1.
Oligonucleotide sequences.
Figure 1.
CHH gene map based on GenBank sequence AF474409.
1. The position of the exons, introns and the PCR primers within the sequence are marked. The expected amplicon expands between exon 2 and 4. B. The primer position is shown (underlined base pairs) over the eyestalk CHH mRNA precursor sequence (Gene Bank acc. no. AB027291.1). Bold letters represent the coding region of the CHH protein. C. Base pair sequence alignment between the eyestalk CHH mRNA precursor (Eyestalk-CHH) and the amplicon sequences obtained in this work: the optic lobe sequence, PcOL-CHH, the brain sequence, PcB-CHH and the brain CHH-like sequence, CG-CHHL (GenBank acc. no. AY256877.1). D. The amino acid sequence alignments between Eyestalk-CHH, the deduced amino acid sequence from the optic lobe and brain amplicons obtained in this work, PcOL-CHH and PcB-CHH, the CHH-like sequence from the thoracic ganglia (1–5 TG-CHHLs; GenBank acc. nos. AAL79193.1, AF474408.1, JF311403, AY256876.1, JX856149) and the deduced amino acid sequence from CG-CHHL.
Figure 2.
CHH mRNA levels daily and circadian oscillations in eyestalk and brain of P. clarkii.
Chronograms showing rhythmic CHH mRNA expression in the optic lobe (A, B) and brain (C, D) under 12∶12 light–dark cycles and continuous darkness. Data are the mean ± standard error (SE) (n = 6). The results are expressed relative to the highest level of expression in each graph. The upper black and white bars in each graph denote the light and dark phases. Different letters above some time points represent statistically significant differences (post hoc analysis, LSD); please see the text for the explanation.
Table 2.
Cosinor Analysis.
Figure 3.
Confocal images showing brain CHH-positive cells and fibers.
Immunohistochemical localization of CHH-IR cells in the crayfish brain. The drawing represents a dorsal view of the brain as they relate to the representative confocal 1.6 µm optical sections stacked in 10 µm images of CHH-IR in the neuronal clusters (Cl 6, Cl 14,15 and 17). CHH is marked in red, and the cell nucleus marked in blue (DAPI). PR, protocerebrum; DE, deuterocerebrum; TR, tritocerebrum; PT, protocerebral tract. The white calibration bars equal 40 µm. The inset shows three horizontal serial sections of PR from the dorsal (upper panel) to central (lower panel) brain (CHH is shown in red). Immunopositivity is visible in all of the sections and is brighter in the central section. White calibration bar = 100 µm.
Figure 4.
Imunohistochemical localization of CHH in perikarya and nuclei in the central brain of crayfish.
Stacked confocal images of cluster 6 of the protocerebrum (A) and tritocerebral clusters 14 and 15 (B) and cluster 17 (C) of P. clarkii with small CHH-immunoreactive somata and some fibers (red). Nuclei are counterstained with DAPI (blue). Arrowheads show CHH-IR in the perikarya and the nuclei. Each image shows the blue (DAPI) and red (anti-CHH) channels as well as the merged image. Horizontal white bars = 40 µm.