JOURNAL OF EXPERIMENTAL ZOOLOGY 298A:44–52 (2003)
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 LEEn Department of Biology, National Changhua University of Education, Changhua, Taiwan 50058, Republic of China
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 crustaceans is an important endocrine center that produces and releases a host of regulatory neuropeptides (Cooke and Sullivan, ’82; Keller ’92; Soyez, ’97). Among these neuropeptides, crustacean 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 regulatory 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 r 2003 WILEY-LISS, INC.
the XO-SG appears to be the major site of CHH synthesis and release, recent studies have reported convincing evidence showing that CHH is synthesized 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: [email protected]
Received 8 January 2002; Accepted 12 March 2003 Published online in Wiley InterScience (www.interscience.wiley. com) DOI:10.1002/jez.a.10273
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On the other hand, several neurotransmitters or neuromodulators are implicated in the regulation of CHH release. It has been reported that injection of serotonin, dopamine, or enkephalins altered hemolymph glucose levels, suggesting that the glycemic response was due to increased or decreased CHH release from the XO-SG (Keller and Beyer, ’68; Fingerman et al., ’81; Lu ¨ schen et al., ’91; Rothe et al., ’91; Lu ¨ schen et al., ’93; Kuo et al., ’95; Sarojini et al., ’95; Lee et al., 2000). Leuenkephalin and serotonin have been shown to inhibit and enhance, respectively, release of hyperglycemic factor(s) from isolated eyestalk ganglia, as evidenced by bioassays (Rothe et al., ’91; Lee et al., 2001); identification of the serotonin-released hyperglycemic factor as CHH was confirmed by the result showing that it was immunoprecipitible by an anti-CHH antiserum (Lee et al., 2001). As to dopamine, studies on different species reported conflicting results. In the crayfish Procambarus clarkii, it was shown that injection of dopamine decreased hemolymph glucose levels and suggested that it inhibited CHH release via activation of enkephalinergic neurons (Sarojini et al., ’95); in the crab Carcinus maenas and shrimp Penaeus monodon, however, similar experimental approach demonstrated that dopamine increased hemolymph glucose levels implying that CHH release was enhanced (Lu ¨ schen et al., ’93; Kuo et al., ’95). To investigate the role of dopamine in CHH release we developed, using antibodies specific for Prc CHH (Lee et al., 2001), a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) that is sensitive enough to directly measure hemolymph CHH levels in P. clarkii. Using this ELISA, it was demonstrated that dopamine increased hemolymph CHH level in a time- and dose-dependent manner with corresponding changes in glucose levels, and that the dopamine-induced increases in CHH and glucose levels were absent in the eyestalk-ablated animals. Furthermore, dopamine significantly stimulated the release of CHH from in vitro incubated eyestalk ganglia. These results provide the first demonstration that dopamine treatment increases hemolymph levels of CHH, which in turn evoke a hyperglycemic response. The source of dopamine-released CHH appears to be the XO-SG in the eyestalk. It is expected that the ELISA will be useful for future studies of neural regulation of CHH release.
MATERIALS AND METHODS
Animals and experimental procedures Animals (Procambarus clarkii) used in the present study were obtained and reared as described previously (Lee et al., 2001). Unless stated otherwise, experimental animals were kept in water tanks with temperature held at 24711C using a temperature controller. The experimental procedures described below were always carried out in the morning in order to reduce possible interference due to circadian changes in CHH and hemolymph glucose levels (Gorgels-Kallen and Voorter, ’85; Kallen et al., ’90). For the injection experiments, animals received an injection (10 ml) of HEPES-buffered saline (Van Harreveld, ’36) or HEPES-buffered saline containing dopamine (3–hydroxytyramine, Sigma) at the doses indicated. Except where noted otherwise, hemolymph was withdrawn (approximately 200 ml/ animal) 1 h after injection for determination of glucose and CHH levels. Injection and bleeding procedures were as described previously (Lee et al., 2001). For the stress experiment, animals held in 241C were bled (zero time), transferred to tanks with water temperature kept at either 241C (control) or 341C (stressed), and bled (approximately 100 ml/animal) at designated times after transfer. Hemolymph samples were used for determination of CHH levels.
Glucose assay Four parts of the hemolymph withdrawn from animals were immediately diluted with 1 part of 0.01 M EDTA solution and the mixture was centrifuged (2000g, 20 min, 41C). The supernatant was collected and analyzed for glucose levels using the Trinder Glucose Assay Kit (Sigma) as described previously (Lee et al., 2000).
Purification and biotinylation of IgG The rabbit anti-CHH antiserum used in the present study was raised against HPLC-purified Prc CHH I (Lee et al., 2001). Previous characterization of the antiserum against HPLC-fractionated sinus gland extract revealed that it recognized both Prc CHH I and II (Lee et al., 2001), which share a common amino acid sequence and differ from each other only in the configuration (L or D) of the third residue (Yasuda et al., ’94). In addition, a small pair of UV-absorbant peaks eluted immediately before CHH I and II was also recognized by the antiserum (Lee et al., 2001).
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These two UV-absorbant peaks probably contain N-terminally unblocked forms of Prc CHH I and II, respectively, as have been demonstrated in Orconectes limosus (Soyez et al., 2000). To purify IgG from the antiserum, an aliquot (0.5 F 1.0 ml) of the serum was buffer-exchanged to binding buffer (20 mM Na-phosphate, pH 7.0) using a PD-10 column (Amersham Pharmacia Biotech) and the collected eluate was injected onto a protein G column (Amersham Pharmacia Biotech). The column was washed and eluted according to the manufacture’s protocol. The collected purified IgG fractions were pooled and dialyzed three times using a dialysis cassette (Pierce) against 0.01 M phosphate-buffered saline (PBS, 8 mM Na2HPO4, 2 mM KH2PO4, 0.14 M NaCl, 0.01 M KCl, pH 7.4). Aliquots of the resulting IgG solution were used for protein assay using a commercially available assay kit and bovine gamma globulin (Bio-Rad) as standard. For biotinylation, the purified IgG was dialyzed against bicarbonate-buffered saline (0.1 M NaHCO3, 0.1 M NaCl, pH 7.4) three times and aliquots of the resulting IgG solution were used for protein assay as described above. Biotinamidocaproate N-hydroxysuccinimide ester (Sigma) dissolved in dimethyl sulfoxide was added to the IgG in bicarbonate-buffered saline in the ratio of 100 mg biotin/1 mg IgG. The mixture was incubated at room temperature for 2 h and dialyzed three times against 0.15 M NaCl to remove free biotin, and subsequently three times against 0.01 M PBS containing 0.1% sodium azide (Delves, ’95). Aliquots of the biotinylated IgG in PBS were used for protein assay as mentioned above.
Sandwich ELISA A sandwich ELISA was developed in order to quantify hemolymph CHH levels. The unlabeled IgG solution was diluted to 10 mg/ml with coating buffer (0.1 M sodium carbonate, pH 9.6) and 100 ml of the diluted solution was added to wells of an ELISA plate (Corning). The plate was incubated at 41C overnight. All subsequent incubations were performed at room temperature. After three washes with washing buffer A (0.01 M PBS containing 0.1% Tween 20 and 0.02% sodium azide, pH 7.4), 100 ml of blocking buffer (0.01 M PBS containing 2% BSA [bovine serum albumin]) was added and the plate was incubated for 1 h. After three washes with washing buffer A, 100 ml of HPLC-purified Prc CHH I (1.5F50.0 fmol in blocking buffer, Lee et al., 2001) or hemolymph
sample (80 ml of hemolymph diluted with 20 ml of 0.01 M PBS containing 2% BSA and 2% glycine ethyl ester) was added and the plate was incubated for 2 h. Except where noted otherwise, each hemolymph sample was analyzed once. After three washes with washing buffer A, 100 ml of biotinylated IgG (diluted to 2.5 mg/ml with blocking buffer) was added and the plate was incubated for 2 h. After three washes with washing buffer B (0.01 M PBS containing 0.1% Tween 20, pH 7.4), 100 ml of avidin-biotinylated peroxidase complex (Pierce) was added and the plate was incubated for 0.5 h. After three washes with washing buffer B, 100 ml of a substrate solution of peroxidase, 2,2’–azino-bis(3–ethylbenzthiazoline–6–sulfonic acid (ABTS, Sigma), was added and the plate was incubated for 0.5 h. Optical density was read at 405 nm using an ELISA reader (Bio-Tek). CHH levels in hemolymph samples were inferred from the standard curve constructed from a series of simultaneously assayed Prc CHH I and corrected for the dilution factor using KC4 software (Bio-Tek), and reported as fmol of CHH I equivalents per ml of hemolymph.
HPLC fractionation of hemolymph Two animals were stressed for 2 h according to the procedure described above. Hemolymph was then withdrawn from these animals and pooled. Two ml of the hemolymph sample were diluted with 500 ml of 0.01 M EDTA solution and the mixture was centrifuged (2000g, 20 min, 41C). The supernatant was mixed with an equal volume of 4.1 M ammonium sulfate, kept at 41C for 2 h to precipitate the proteins, and centrifuged at 14,000g for 20 min. The pellet was suspended in 1 ml of 0.1 M PBS containing 0.1 M NaCl and 0.01% EDTA (pH 7.7) and dialyzed against the same PBS solution three times. Solution was recovered from the dialysis cassette, lyophilized, and reconstituted in 1 ml of 0.1N HCl. The resulting solution was subjected to chromatographic fractionation using a HPLC system as previously described (Lee et al., 2001). Briefly, a reversedphase column (Nucleosil, C–18, 5 mgr;m, 250.0 4.6 mm, MetaChem Technologies Inc.) was eluted at a flow rate of 1 ml/min. The elution gradient was 10% to 80% B over 170 min. Solvent A was 0.1% TFA in water; solvent B was 0.08% TFA in acetonitrile. The eluate was collected every 1 min and lyophilized. Dried residue in each fraction was reconstituted in 100 ml of ELISA
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blocking buffer and the reconstituted solution (100 ml /fraction) was assayed using the ELISA described above. In order to determine the retention time of Prc CHH I and II, HPLC-purified CHH I and II (Lee et al., 2001) were chromatographed immediately after the hemolymph sample using the same elution gradient.
In vitro incubation Eyestalk ganglia with intact X-organ-sinus gland complex, dissected from ice-anaesthetized animals, were incubated as described previously (Lee et al., 2001) with minor modifications. Briefly, eyestalk ganglia were pre-incubated in Van Harreveld saline buffered with 20 mM HEPES (400 ml/ganglia/incubation) for 30 min at 251C. After pre-incubation, each eyestalk ganglia was transferred to a new well containing 400 ml of buffered saline without or with various concentrations of dopamine and the incubation was performed for 1 additional h. The incubation media were collected and diluted (usually 10 or 15 folds) with ELISA blocking buffer. An aliquot (100 ml) of the diluted solution obtained from each incubation was assayed using the CHH ELISA described above.
Statistical analysis Effects of experimental treatments on CHH and glucose levels were analyzed by analysis of variance (ANOVA) and Fisher’s protected LSD test using StatView 4.01 obtained from Abacus Concepts. RESULTS A sandwich ELISA was developed for measuring hemolymph CHH levels. Typical standard (Prc CHH I) and sample titration curves are shown in Figure 1. The standard curve is linear over the range from 1.5 fmol to 50 fmol (correlation coefficient ¼ 0.99; slope ¼ 1.01). The assay sensitivity (i.e., the lowest amounts of CHH giving an optical density significantly greater than background) was about 1 fmol per well. A hemolymph sample was serially diluted and the diluted samples were assayed by ELISA. The resulting sample titration curve (correlation coefficient ¼ 0.98; slope ¼ 0.98) was parallel to the standard curve. Furthermore, analysis of HPLCfractionated hemolymph samples by the ELISA indicated that the majority of CHH-immunoreactivties was eluted at the same times (80 and 83
Fig. 1. Parallelism between ELISA standard curve and sample titration curve. Standard (Prc CHH I, a) and a hemolymph sample (b) were serially diluted and assayed by the ELISA as described in Materials and Methods. Each data point represents a mean7SEM (n ¼ 3).
min) as the purified Prc CHH I and II (Fig. 2). Thus, results of these experiments demonstrate the specificity of the ELISA for CHH. As Prc CHH I was used as the standard in the ELISA, the total levels of CHHs measured using this assay are reported as CHH I equivalents per ml of hemolymph. This ELISA was also tested by monitoring changes in hemolymph CHH levels in the crayfish P. clarkii subjected to thermal stress, which has been shown to significantly increase
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creased CHH and glucose levels 2.4F3.6 and 1.8F7.0 folds, respectively. In order to examine whether dopamine stimulates CHH release from extra-eyestalk tissues, effects of dopamine on CHH and glucose levels were tested in eyestalk-ablated animals. Dopamine, at dose of 107 mol/animal, had no significant effect on CHH and glucose levels in eyestalk-ablated animals, whereas the same dose of dopamine significantly increased both CHH and glucose levels, 3.0 and 4.6 folds, respectively, in intact animals (Fig. 6). On the other hand, release of CHH from eyestalk ganglia was enhanced by dopamine. In vitro incubation of eyestalk ganglia in the presence of dopamine (106F103 M) increased the amounts of CHH released into the incubation media 1.3F2.3 folds (Fig. 7). Fig. 2. ELISA analysis of HPLC-fractionated hemolymph sample. Two ml of hemolymph withdrawn from stressed animals was processed and fractionated as described in Materials and Methods. HPLC fractions were lyophilized, reconstituted in ELISA blocking buffer, and assayed by ELISA. Arrows indicate the elution time of HPLC-purified Prc CHH I and II (80 and 83 min, respectively) that were chromatographed immediately after the hemolymph sample.
CHH levels in lobsters and crayfishes (Keller and Orth, ’90; Chang et al., ’98). The results showed that after animals were transferred from 241C to 341C, CHH levels rose significantly from 32.474.9 fmol/ml to 78.778.7 fmol/ml at 30 min, reached a peak (123.3721.1 fmol/ml) at 2 h, and remained significantly elevated (94.9712.5 fmol/ml) at 4 h (Fig. 3). On the other hand, CHH levels in control animals showed a slight but statistically insignificant increase during the sampling period, possibly due to handling stress (Fig. 3). Dopamine increased hemolymph CHH and glucose levels. After injection of dopamine (107 mol/animal), CHH levels increased significantly within 30 min from 28.577.2 fmol/ml to 66.67fmol/ml, peaked at 1 h (86.7720.0 fmol/ml), declined thereafter, and returned to the basal levels at 4 h (Fig. 4a); glucose levels fluctuated with a similar time course (Fig. 4b). Both parameters remained fairly stable during the 4 h period in saline-injected control animals (Fig. 4). Dose-dependent effects of dopamine on CHH and glucose levels are shown in Figure 5. At doses between 108F106 mol/animals, dopamine in-
DISCUSSION Results presented in this study show that dopamine increased hemolymph glucose levels in the crayfish P. clarkii (Figs. 4 and 5), which are in
Fig. 3. Effect of thermal stress on CHH levels in the crayfish P. clarkii. Hemolymph was withdrawn from animals acclimated to 241C before (time 0) and at designated times after being transferred to tanks with water temperature kept at either 241C (open circles) or 341C (filled circles). A separate group of animals was used for each time point. Hemolymph samples were used for CHH quantification. Each data point represents a mean7SEM (n¼8). n and nn represent significant difference from zero time controls at 5% and 1% levels, respectively.
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direct contrast to those of Sarojini et al. (’95) who also worked on the same species. The reason for such contradiction is unknown. However, it is
Fig. 4. Time courses of dopamine-induced changes in CHH and glucose levels. Hemolymph was withdrawn from animals before (time 0) and at designated times after receiving an injection of saline (open circles) or saline containing dopamine (107 mol/animal, filled circles). A separate group of animals was used for each treatment. Hemolymph samples were used for CHH (a) and glucose (b) quantification. Each data point represents a mean7SEM (n¼8). n and nn represent significant differences from corresponding zero time controls at 5% and 1% levels, respectively.
Fig. 5. Dose-dependent effects of dopamine on CHH and glucose levels. Animals received an injection of saline or saline containing dopamine (DA) at the doses indicated. One hour after injection, hemolymph was withdrawn from each animal and used for CHH (a) and glucose (b) quantification. Each column represents a mean7SEM (n¼8). n and nn represent significant difference from corresponding saline controls at 5% and 1% levels, respectively.
noted that the injection volume used in that study was 100 ml (Sarojini et al., ’95), as opposed to 10 ml in ours. Prior testing by us indicated that high vehicle volume (450 ml) itself significantly increased hemolymph glucose levels. This may explain why the basal glucose levels of the control
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Keller, ’93b; Kuo et al., ’95; Webster ’96; Chang et al., ’98). Whether the opposite responses to dopamine are due to the different glycemic states of the animals awaits further investigation. Our results showing that dopamine exerted a hyperglycemic effect are consistent with those of other studies (Lu ¨ schen et al., ’93; Kuo et al., ’95) that also administered smaller injection volumes and reported lower basal glucose levels. It has been suggested that the hyperglycemic effect of dopamine is mediated by its stimulatory effect on the release of CHH (Lu ¨ schen et al., ’93; Kuo et al., ’95). However, this proposition has not yet been proven. A sensitive and specific immunoassay is required in order to provide direct evidence. Immunoassays have been successfully employed to study changes of CHH secretion in response to stresses, electrical stimuli, and glucose and lactate injection (Keller and Orth, ’90; Santos and Keller, ’93b; Keller et al., ’94; Richmond et al., ’95; Webster, ’96; Chang et al., ’98), and during the day/night cycle (Gorgels-Kallen and Voorter, ’85; Kallen et al., ’90). In the present study, a specific and sensitive sandwich ELISA was developed allowing us to directly measure hemolymph CHH
Fig. 6. Dependency of dopamine-induced increases in CHH and glucose levels on the presence of the eyestalk. Eyestalkablated (ablated) and intact animals received an injection of saline or saline containing dopamine (DA, 107 mol/animal). One hour after injection, hemolymph was withdrawn from each animal and used for CHH (a) and glucose (b) quantification. Each column represents a mean7SEM (n¼8). n and nn represent significant difference from corresponding saline controls at 5% and 1% levels, respectively.
(vehicle-injected) animals in the present study were about 10 mg/dl, whereas the corresponding values in the study of Sarojini et al. (’95) were approximately 35 mg/dl, which are also much higher than those reported for other crustaceans (Rothe et al., ’91; Lu ¨ schen et al., ’93; Santos and
Fig. 7. Effect of dopamine on the release of CHH from in vitro incubated eyestalk ganglia. Eyestalk ganglia were preincubated in saline for 30 min, transferred to saline in the absence or presence of various concentrations of dopamine (DA) as indicated, and further incubated for 1 h. Incubation media from the 1 h incubation were collected, diluted with ELISA blocking buffer, and assayed by ELISA. Each column represents a mean7SEM (n¼5). n and nn represent significant difference from saline controls at 5% and 1% levels, respectively.
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levels without pre-assay sample processing (Figs. 1 and 2). The time course and fold changes of the thermal stress-induced increase in CHH levels of the crayfish P. clarkii, as measured by this ELISA (Fig. 3), are comparable to those reported for other similarly stressed crustaceans (Keller and Orth, ’90; Chang et al., ’98). Using this ELISA, it was demonstrated that dopamine significantly increased hemolymph CHH levels with concomitant changes in glucose levels (Figs. 4 and 5). These results suggest that dopamine stimulates the release of CHH that in turn evokes the hyperglycemic responses. The predominant source of dopamine-induced release of CHH appears to reside within the eyestalk, i.e., the XO-SG. Expression of CHH gene in the ventral nerve cord of the lobster Homarus americanus has been reported by earlier studies (De Kleijn et al., ’95; Reddy et al., ’97). In fact, recent reports provided compelling evidence showing that CHHsecreting cells are present not only in lobster ventral nerve cord (Chang et al., ’99) but also in the gut of the crab (C. maenas) (Chung et al., ’99; Webster et al., 2000). The results showing that dopamine did not significantly increase CHH levels in the eyestalk-ablated animals (Fig. 6) suggest that contribution of these extra-eyestalk tissues to the dopamine-induced CHH release is at most minimal in P. clarkii. However, it should be mentioned that in C. maenas CHH is released from the gut during ecdysis (Chung et al., ’99). If a similar ecdysis-associated CHH release also occurs in crayfish, whether dopamine stimulates this CHH release is a question requiring further investigation (animals used in the present studies were intermolt crayfishes). On the other hand, dopamine had a dose-dependent effect on the release of CHH from eyestalk ganglia incubated in vitro (Fig. 7). However, it is not yet clear that dopamine is acting directly on the eyestalk ganglia when it elevates CHH levels in vivo. The combined results of the present and our previous studies (Lee et al., 2000, 2001) indicated that two biogenic amines, serotonin and dopamine, stimulate CHH release from the XO-SG. Physiological significance of biogenic amine-induced CHH release and hyperglycemia has not yet been addressed. It has been shown that various external and internal signals regulate CHH release. For example, environmental stresses are capable of rapidly increasing hemolymph CHH levels (Keller and Orth, ’90; Webster, ’96; Chang et al., ’98); hemolymph glucose and lactate levels exert their effects on CHH release in negative and
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