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First published online July 2, 2004
Journal of Experimental Biology 207, 2845-2857 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01117

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A crustacean nitric oxide synthase expressed in nerve ganglia, Y-organ, gill and gonad of the tropical land crab, Gecarcinus lateralis

Hyun-Woo Kim, Luisa A. Batista, Jodi L. Hoppes, Kara J. Lee and Donald L. Mykles^*

Department of Biology, Colorado State University, Fort Collins, CO 80523, USA

^* Author for correspondence (e-mail: don{at}lamar.colostate.edu)

Accepted 27 May 2004

NO signaling is involved in many physiological processes in invertebrates. In crustaceans, it plays a role in the regulation of the nervous system and muscle contraction. Nested reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE) PCR generated a full-length cDNA sequence (3982 bp) of land crab NO synthase (Gl-NOS) from molting gland (Y-organ) and thoracic ganglion mRNA. The open reading frame encoded a protein of 1199 amino acids with an estimated mass of 135 624 Da. Gl-NOS had the highest sequence identity with insect NOS. The amino acid sequences for binding heme and tetrahydrobiopterin in the oxygenase domain, binding calmodulin and binding FMN, FAD and NADPH in the reductase domain were highly conserved. Gl-NOS had single amino acid differences in all three highly conserved FAD-binding sequences, which distinguished it from other NOS sequences. RT-PCR showed that the Gl-NOS mRNA was present in testis, ovary, gill, eyestalk neural ganglia, thoracic ganglion and Y-organ. NOS mRNA varied between preparations of Y-organ, thoracic ganglion and gill, while NOS mRNA was at consistently high levels in the ovary, testis and eyestalk ganglia. Immunohistochemistry confirmed that the Gl-NOS protein was expressed in Y-organ, ovary and gill. These results suggest that NOS has functions in addition to neuromodulation in adults, such as regulating or modulating ecdysteroid synthesis in the Y-organ.

Key words: Crustacea, Arthropoda, nitric oxide synthase, Y-organ, gonad, ovary, gill, eyestalk ganglion, molting gland, nervous tissue, tissue distribution, cDNA cloning, DNA sequence, amino acid sequence, gene expression, mRNA

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