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

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.

Bursicon and neuropeptide cascades during the ecdysis program of the shore crab, Carcinus maenas.

Very little is known regarding the release patterns of neuropeptides involved in ecdysis of crustaceans compared to insects. In particular, the dynamics of release of the insect cuticle hardening hormone bursicon, which has only recently been discovered in crustaceans, is unknown. Bursicon has not previously been identified as a circulating neurohormone in these animals. Since patterns of release were likely to be ephemeral, bursicon, as well as two other neurohormones involved in the ecdysis program in crustaceans, crustacean cardioactive peptide (CCAP) and crustacean hyperglycaemic hormone (CHH) were measured in single haemolymph samples in Carcinus maenas. For bursicon, an ultrasensitive time resolved-fluoroimmunoassay (TR-FIA) was developed, which firstly involved its characterisation by HPLC, bioassay and immunoassay. Simultaneous measurement of three neurohormones was performed at unparalleled levels of resolution, which has not previously been reported in any invertebrate. Additionally, expression patterns and architecture of neurones expressing both bursicon and CCAP were determined in the CNS during the moult cycle. Bursicon and CCAP are released in a massive surge, likely a single global exocytotic event on emergence, just after release of CHH. Despite co-localisation of CCAP and bursicon in neurones of the CNS, observations suggest that differential packaging of CCAP can occur in the pericardial organs in a small population of secretory boutons, thus accounting for observations showing release of some CCAP during the penultimate stages of the ecdysis program. The results obtained vividly illustrate the dynamism of neuropeptide cascades occurring during crustacean ecdysis, and also allow proposal of a hypothesis of its endocrine control.