Characterization, localization and temporal expression of crustacean hyperglycemic hormone (CHH) in the behaviorally rhythmic peracarid crustaceans, Eurydice pulchra (Leach) and Talitrus saltator (Montagu)

Abstract

Crustacean hyperglycemic hormone (CHH) has been extensively studied in decapod crustaceans where it is known to exert pleiotropic effects, including regulation of blood glucose levels. Hyperglycemia in decapods seems to be temporally gated to coincide with periods of activity, under circadian clock control. Here, we used gene cloning, in situ hybridization and immunohistochemistry to describe the characterization and localization of CHH in two peracarid crustaceans, Eurydice pulchra and Talitrus saltator. We also exploited the robust behavioral rhythmicity of these species to test the hypothesis that CHH mRNA expression would resonate with their circatidal (12.4h) and circadian (24h) behavioral phenotypes. We show that both species express a single CHH transcript in the cerebral ganglia, encoding peptides featuring all expected, conserved characteristics of other CHHs. E. pulchra preproCHH is an amidated 73 amino acid peptide N-terminally flanked by a short, 18 amino acid precursor related peptide (CPRP) whilst the T. saltator prohormone is also amidated but 72 amino acids in length and has a 56 residue CPRP. The localization of both was mapped by immunohistochemistry to the protocerebrum with axon tracts leading to the sinus gland and into the tritocerebrum, with striking similarities to terrestrial isopod species. We substantiated the cellular position of CHH immunoreactive cells by in situ hybridization. Although both species showed robust activity rhythms, neither exhibited rhythmic transcriptional activity indicating that CHH transcription is not likely to be under clock control. These data make a contribution to the inventory of CHHs that is currently lacking for non-decapod species.

Functional Characterization and Signaling Systems of Corazonin and Red Pigment Concentrating Hormone in the Green Shore Crab, Carcinus maenas

“…Gene expression was performed using Taqman MGB hydrolysis probes as described previously (Hoelters et al, 2016 ) using standard curves made from in vitro transcribed cRNA. In brief, standards were made from PCR products generated using gene-specific primers 5′ flanked by T7 phage promoter sequences.…”

Crustacean hyperglycemic hormone is synthesized in the eyestalk and brain of the crayfish Procambarus clarkii

“…Recently, in situ hybridization in two species of crustaceans, Eurydice pulchra (Leach) and Talitrus saltator (Montagu), showed that CHH mRNA was localized in protocerebral cells [32]. …”

New techniques, applications and perspectives in neuropeptide research

“…Neuropeptide localization in species and/or tissue(s) enables mapping in neuronal subtypes relative to structural components of the cell, tissue or whole organisms, which can then be used to inform the function of a target neuropeptide and to direct functional biology experiments (Hoelters et al, 2016). Overall, the rapid increase in 'omics'-derived neuropeptide sequence data has revolutionized our approach to the localization of neuropeptides and their signaling pathway components (Elphick and Mirabeau, 2014) and facilitated our ability to construct species-and neuropeptide-specific 'connectomes' (Shahidi et al, 2015).…”