Dopaminergic Regulation of Crustacean Hyperglycemic Hormone and Glucose Levels in the Hemolymph of the Crayfish Procambarus clarkii HONG-SHIN ZOU, CHI-CHIH JUAN, SHYH-CHI CHEN, HSIN-YUAN WANG, and CHI-YING LEE n Department of Biology, National Changhua University of Education, Changhua, Taiwan 50058, Republic of China ABSTRACT The effects of dopamine on crustacean hyperglycemic hormone (CHH) release and hemolymph glucose levels in the crayfish Procambarus clarkii were investigated. A quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using antibodies specific for Prc CHH was developed and characterized. The sensitivity of the ELISA was about 1 fmol/well. Specific measurement of CHH in hemolymph samples by the ELISA was demonstrated by the parallelism between CHH standard curve and sample (hemolymph) titration curve. Moreover, thermally stressed P. clarkii exhibited a characteristic change of hemolymph CHH levels as revealed by the ELISA. CHH and glucose levels increased significantly within 30 min of dopamine injection, peaked at 1 h, and returned to the basal levels at 4 h. Dose-dependent effects of dopamine on CHH and glucose levels were observed between 10 –8 to 10 –6 mol/animal. Dopamine-induced increases in CHH and glucose levels were absent in eyestalk-ablated animals. Finally, dopamine significantly stimulated the release of CHH from in vitro incubated eyestalk ganglia. These results suggest that dopamine enhances release of CHH into hemolymph that in turn evokes hyperglycemic responses and that the predominant site of dopamine-induced CHH release is the X-organ-sinus gland complex located within the eyestalk. J. Exp. Zool. 298A: 44–52, 2003. r 2003 Wiley-Liss, Inc. INTRODUCTION The X-organ-sinus gland complex (XO-SG) located within the eyestalks of decapod crusta- ceans is an important endocrine center that produces and releases a host of regulatory neuro- peptides (Cooke and Sullivan, ’82; Keller ’92; Soyez, ’97). Among these neuropeptides, crusta- cean hyperglycemic hormone (CHH) is involved in regulating blood glucose levels mainly through mobilization of glucose from glycogen depots (see Santos and Keller, ’93a), although other regula- tory functions have been proposed (e.g., Tensen et al., ’89; Chang et al., ’90; Charmantier-Daures et al., ’94; Yasuda et al., ’94; Liu et al., ’97; Santos et al., ’97). Sequence analysis of CHHs isolated from various decapods indicated that they are peptides of 72F73 amino acid residues with a considerable degree of similarity (Soyez, ’97). An intriguing feature of CHH is the existence of multiple molecular variants in a given species as has been reported in several species (Soyez et al., ’94, Yasuda et al., ’94; Aguilar et al., ’95; Yang et al., ’97; Chung et al., ’98). In addition, although the XO-SG appears to be the major site of CHH synthesis and release, recent studies have reported convincing evidence showing that CHH is synthe- sized and released outside the XO-SG (Chang et al., ’98, ’99; Chung et al., ’99; Webster et al., 2000). Regulation of CHH release has been the subject of many studies utilizing sensitive immunoassays. For example, release of CHH in response to electrical and elevated [K + ] stimuli has been characterized (Keller et al., ’94; Richmond et al., ’95); it has also been shown that CHH release was affected by various environmental stresses (Keller and Orth, ’90; Webster, ’96; Chang et al., ’98) and by hemolymph glucose and lactate levels (Santos and Keller, ’93b). Grant sponsor: National Science Council, Taiwan, Republic of China; Grant number: NSC 89–2311–B–018–005 and 90–2311–B–018– 001. n Correspondence to: Chi-Ying Lee, Department of Biology, National Changhua University of Education, Changhua, Taiwan 50058, Republic of China. E-mail: bicylee@cc.ncue.edu.tw Received 8 January 2002; Accepted 12 March 2003 Published online in Wiley InterScience (www.interscience.wiley. com) DOI:10.1002/jez.a.10273 r 2003 WILEY-LISS, INC. JOURNAL OF EXPERIMENTAL ZOOLOGY 298A:44–52 (2003)