The orcokinins are a highly conserved family of crustacean
peptides that enhance hindgut contractions in the crayfish Orconectes limosus (Stangier et al. [1992]
Peptides 13:859-864). By combining immunocytochemical and mass spectrometrical analysis of the stomatogastric nervous system (STNS) in the crayfish Cherax destructor, we show that multiple orcokinins are synthesized in single neurons. Immunocytochemistry demonstrated
orcokinin-like immunoreactivity in all four ganglia of the STNS and in the pericardial organs, a major neurohaemal organ. Identified neurons in the STNS were stained, including a pair of modulatory interneurons (inferior ventricular nerve neuron, IVN), a neuron with its cell body in the stomatogastric
ganglion that innervates cardiac muscle c6 via the anterior median nerves (AM-c6), and a sensory neuron (anterior gastric receptor neuron). Five
orcokinin-related
peptides were identified by matrix-assisted
laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) post source decay fragmentation in samples of either the stomatogastric
ganglion or the pericardial organs. Four of these
peptides are identical to
peptides derived from the cloned Procambarus clarkii precursor (Yasuda-Kamatani and Yasuda [2000] Gen. Comp. Endocrinol. 118:161-172), including the original [Asn(13)]-
orcokinin (NFDEIDRSGFGFN, [M+H](+) = 1,517.7 Da), [Val(13)]-
orcokinin ([M+H](+) = 1,502.7 Da), [Thr(8)-His(13)]-
orcokinin ([M+H](+) = 1,554.8 Da), and FDAFTTGFGHS ([M+H](+) = 1,186.5 Da). The fifth
peptide is a hitherto unknown
orcokinin variant: [Ala(8)-Ala(13)]-
orcokinin ([M+H](+) = 1,458.7 Da). The masses of all five
peptides were also detected in the inferior ventricular nerve of C. destructor, which contains the cell bodies and axons of the IVNs as well as the axons of two other
orcokinin-like immunoreactive neurons. In the oesophageal nerve, in which all the
orcokinin-like immunoreactivity derives from the IVNs, at least two of the orcokinins were detected, indicating that multiple orcokinins are synthesized in these neurons. Similarly, all four
orcokinin masses were detected in the anterior median nerves, in which all the
orcokinin-like immunoreactivity derives from the AM-c6 neuron. This study therefore lays the groundwork to investigate the function of the
orcokinin peptide family using single identified neurons in a well-studied system.