An antibody to recombinant crustacean hyperglycaemic hormone of Nephrops norvegicus cross-reacts with neuroendocrine organs of several taxa of malacostracan Crustacea.
The crustacean hyperglycaemic hormones (cHHs) are multifunctional neuropeptides that play a central role in the physiology of crustaceans. A partial cDNA coding for cHH of the Norway lobster, Nephrops norvegicus, was cloned; this cDNA was fused to glutathione- S-transferase (GST) to obtain a recombinant fusion protein that was used to raise a rabbit antiserum and to perform a biological assay. The specificity of the purified antibody was demonstrated by means of Western blotting. To validate the specificity of the purified antibody to the cHH of N. norvegicus and its cross-reactivity with other species, we performed standard immunocytochemistry of the eyestalk on: (1) paraffin sections of the decapod species N. norvegicus, Munida rugosa and Astacus leptodactylus and of the stomatopod Squilla mantis; (2) semithin resin sections of N. norvegicus and Palaemon elegans; (3) ultrathin sections of N. norvegicus sinus gland (transmission electron microscopy studies). The pattern of immunoreactivity shown by N. norvegicus eyestalk sections conforms to distribution, relative amount and ultrastructural features of cHH-containing neurons and nerve endings as reported in the previous literature. In all the crustacean species examined, the antibody marks precisely the X organ-sinus gland complex and unspecific staining is completely lacking. In addition, its specific cross-reaction by immunoprecipitation depletes shrimp eyestalk extract of hyperglycaemic activity in an in vivo bioassay. The results obtained show a cHH-specific molecular recognition despite the fact that the species tested belong to systematic groups increasingly remote in the phylogenetic tree. The antibody could be used for advancing our knowledge on cHH activity in a variety of crustacean species, e.g. for monitoring reproductive and stress conditions.
Giulianini
P G
PG
BRAIN Centre for Neuroscience, Department of Biology, University of Trieste, via Licio Giorgieri 7, 34127 Trieste, Italy. giuliani@univ.trieste.it
Pandolfelli
N
N
Lorenzon
S
S
Ferrero
E A
EA
Edomi
P
P
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000918:Antibody Specificity; D060829:Arthropod Proteins; D003400:Astacoidea / Q000378:metabolism; D001681:Biological Assay; D003429:Cross Reactions; D003445:Crustacea / Q000378:metabolism*; D033364:Decapoda / Q000378:metabolism; D004702:Endocrine Glands / Q000502:physiology*; D005123:Eye / Q000737:chemistry / Q000648:ultrastructure; D005982:Glutathione Transferase / Q000235:genetics; D007447:Invertebrate Hormones / Q000276:immunology; D008121:Nephropidae / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000276:immunology*; D009474:Neurons / Q000648:ultrastructure; D011817:Rabbits / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D013045:Species Specificity
D000906:Antibodies; D060829:Arthropod Proteins; D007447:Invertebrate Hormones; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C015947:hyperglycemic hormone, crustacean; D005982:Glutathione Transferase