Abstract
Crustacean hyperglycemic hormone (CHH) synthesizing cells in the optic lobe, one of the pacemakers of the circadian system, have been shown to be present in crayfish. However, the presence of CHH in the central brain, another putative pacemaker of the multi-oscillatory circadian system, of this decapod and its circadian transcription in the optic lobe and brain have yet to be explored. Therefore, using qualitative and quantitative PCR, we isolated and cloned a CHH mRNA fragment from two putative pacemakers of the multi-oscillatory circadian system of Procambarus clarkii, the optic lobe and the central brain. This CHH transcript synchronized to daily light-dark cycles and oscillated under dark, constant conditions demonstrating statistically significant daily and circadian rhythms in both structures. Furthermore, to investigate the presence of the peptide in the central brain of this decapod, we used immunohistochemical methods. Confocal microscopy revealed the presence of CHH-IR in fibers and cells of the protocerebral and tritocerebal clusters and neuropiles, particularly in some neurons located in clusters 6, 14, 15 and 17. The presence of CHH positive neurons in structures of P. clarkii where clock proteins have been reported suggests a relationship between the circadian clockwork and CHH. This work provides new insights into the circadian regulation of CHH, a pleiotropic hormone that regulates many physiological processes such as glucose metabolism and osmoregulatory responses to stress.
Highlights
The metabolic crustacean hyperglycemic hormone (CHH) is produced by neuroendocrine cells of the X-organ sinus gland (XO-SG) complex in the eyestalks of decapod crustaceans
CHH has been detected by immunocytochemistry in other regions of the nervous system of decapods,such as the subesophageal ganglion of Homarus [9] neurons in the so-called second thoracic roots of the ventral nerve cord in lobsters [10], the retina of the crayfish Procambarus clarkii (P. clarkii) [11] and the brain of other non-decapod crustacean species [12], [13], [14]
The Clustal W alignment of the optic lobe and brain amplicons revealed a 100% sequence identity with the CHH mRNA sequence obtained from the eyestalk of P. clarkii by Yasuda-Kamatani (GenBank accession number AB027291.1)
Summary
The metabolic crustacean hyperglycemic hormone (CHH) is produced by neuroendocrine cells of the X-organ sinus gland (XO-SG) complex in the eyestalks of decapod crustaceans. We isolated and cloned a similar CHH mRNA fragment from two of the putative pacemakers of crayfish, the eyestalk and the brain, and investigated whether the levels of this transcript showed circadian variations. At the end of the experimental period, the animals were sacrificed, and tissue samples from the brain and optic lobe were collected every 4 h beginning at 08:00 h; in total, samples were collected at six time points during the day.
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