Studies of crustacean hyperglycaemic hormone in the Norway lobster 'Nephrops norvegicus' (Linnaeus, 1758)

Abstract

Nephrops norvegicus is a deep water marine decapod crustacean which burrows in fine muddy substrata. It is a commercially important as a fisheries species, but knowledge of its biology, particularly concerning its endocrinology, is limited. This thesis describes the endocrinology of Nephrops norvegicus with particular reference to crustacean hyperglycaemic hormone. Histological investigations of the eyestalk of Nephrops norvegicus enabled the identification d" the X-organ sinus gland complex. The development of a microbioassay allowed the determination of increases of glycaemia following the injection of crude sinus gland extracts into a host animal. The optimal dose for induced haemolymph glycaemia was determined. HPLC separation was used to isolate and purify several neuropeptides from crude sinus gland extracts, which had varying degrees of hyperglycaemic activities. The use of ELISA (enzyme linked immunosorbent assay) showed that the peptides reacted with polyclonal rabbit antiserum raised against the CHH of the crayfish Orconectes limosus and with polyclonal guinea pig antiserum raised against the CHH of the lobster, Homarus americanus. SDS-PAGE of these peptides enabled an estimation of their molecular weights and the purity of one of the active peptides was determined using capillary electrophoresis. The effects of photoperiod and severe hypoxia on the CHH-induced hyperglycaemia of Nephrops norvegicus were also investigated. The responses of N. norvegicus appeared to differ in some respects from other decapods species. Pairs of oligonucleotide primers, based on the sequence of the lobster, Homarus americanus, complimentary to regions of the CHH sequence, were used in the polymerase chain reactions (PCR). Complimentary first strand DNA (cDNA) was synthesised from total Nephrops eyestalk RNA. 35 rounds and 40 rounds of PCR amplification produced a l00bp and 230bp double stranded DNA product respectively, which were resolved by electrophoresis on a tris-borate-EDTA gel. The product size was compared with known standards. Finally, the identification of CHH and GIH synthesis and storage in the eggs of Nephrops norvegicus at various stages of development was investigated. By the use of PCR, it was not possible to determine if synthesis of CHH was occurring in 50% developed eggs. The use of ELISA, however, demonstrated that in 90% developed eggs there was a significant increase of both CHH and GIH immunoactivity.