Functional studies of crustacean hyperglycemic hormones (CHHs) of the blue crab, Callinectes sapidus – the expression and release of CHH in eyestalk and pericardial organ in response to environmental stress J Sook Chung and N Zmora Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, USA Keywords hypoxia; pericardial organ and sinus gland crustacean hyperglycemic hormones; temperature stress Correspondence J S Chung, Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 East Pratt Street, Columbus Center, Suite 236, Baltimore, MD 21202, USA Fax: +1 410 234 8896 Tel: +1 410 234 8841 E-mail: chung@comb.umbi.umd.edu (Received October 2007, revised December 2007, accepted 11 December 2007) doi:10.1111/j.1742-4658.2007.06231.x The rapid increase in the number of putative cDNA sequences encoding crustacean hyperglycemic hormone (CHH) family in various tissues [either from the eyestalk (ES) or elsewhere] underscores a need to identify the corresponding neuropeptides in relevant tissues Moreover, the presence of provided structural CHH implies the level of the complexity of physiological regulation in crustaceans Much less is known of the functions of nonES CHH than of those of its counterpart present in ESs In the blue crab, Callinectes sapidus, we know little of CHH involvement in response to the stressful conditions that naturally occur in Chesapeake Bay We have identified two isoforms of CHH neuropeptide in the sinus gland of the ES and isolated a full-length cDNA encoding CHH from the pericardial organ (PO) The functions of ES-CHH and PO-CHH in this species were studied with regard to expression and release in response to stressful episodes: hypoxia, emersion, and temperatures Animals exposed to hypoxic conditions responded with concomitant release of both CHHs In contrast, the mRNA transcripts encoding two CHHs were differentially regulated: PO-CHH increased, whereas ES-CHH decreased This result suggests a possible differential regulation of transcription of these CHHs In recent years, crustacean hyperglycemic hormones (CHHs), traditionally identified in the medulla terminalis X-organ and sinus gland (SG) in the eyestalk (ES), have been found in non-ES tissues Reported sites for the synthesis of CHH-like neuropeptides in non-ES tissues include the gut [1,2], subesophageal ganglion (SOG) [3], pericardial organs (POs) [4,5], and cells in the abdominal segments of embryos [6] In Carcinus maenas, the expression and translation of gut CHH occurs exclusively during premolt [1,2], whereas ES-CHH is molt stage independent [7] The amino acid sequence of gut CHH is identical to that in the ES [1,8], but only 66% homologous to PO-CHH [4,5,8] Thus, it seems that C maenas [1,4,8], Homarus americanus [3,9,10], Pachygrapsus marmoratus [5] and Machrobrachium rosenbergii [11] exhibit multiple isoforms and synthesis sites for CHH, suggesting that this may be a common feature among crustaceans Many putative CHH sequences have been identified with the aid of cDNA cloning, but there is little information about the localization or the physiological function of active CHH neuropeptides in corresponding tissues Prototypical actions attributed to ES-CHH include induction of hyperglycemia, suppression of Abbreviations AK, arginine kinase; CHH, crustacean hyperglycemic hormone; CPRP, crustacean hyperglycemic hormone precursor-related peptide; eIF4A, eukaryotic translation initiation factor 4A; ES, eyestalk; PO, pericardial organ; SALDI, surface-assisted laser desorption ⁄ ionization; SG, sinus gland; SOG, subesophageal ganglion; TD-PCR, touchdown PCR; TG, thoracic ganglia FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS 693 Expression and release of CHH in the blue crab J S Chung and N Zmora 694 Results Identification and bioactivity of CHHs from ES sinus glands and PO Two RP-HPLC peaks (1 and 2) as presented in Fig showed strong cross-reactivity with anti-ES-CHH (Fig 1) The result of a glucose bioassay with these peaks confirmed that peak is the major type of Cal sapidus ES-CHH-II, as it is for Car maneas and Cancer pagurus [29,30] The molecular masses of peaks and 2, determined by ESI MS, were 8494.20 Da and 8478.01 Da, respectively The typical separation of PO extract (50 PO equivalents) by RP-HPLC is presented in Fig Fractions Absorbance at 405 nm Absorbance at 210 nm 0.2 0.0 10 20 30 Retention time, 40 Fig CHH neuropeptide profile of single SG extract of Cal sapidus on an RP-HPLC C18 column The gradient condition was 30– 80% solution B over 45 (solution A, 0.11% trifluoroacetic acid in water; solution B, 0.1% trifluoroacetic acid in 40% water and 60% acetonitrile) Absorbance and flow rate were monitored at 210 nm at a flow rate of mLỈmin)1 Peak 1, CHH-I; peak 2, CHHII The mass of each neuropeptide determined by ESI MS was as follows; CHH-I, 8494.2 Da; CHH-II, 8478.1 Da Fractions that positively cross-reacted with Car maenas CHH antiserum are shown as bars Absorbance at 405 nm 0.5 Absorbance at 210 nm ecdysteroid and methyl farnesoate synthesis, inhibition of ovarian protein synthesis and osmoregulation [12– 19] Hyperglycemia, the commonly observed adaptive response, is caused by the release of CHH from the ES in response to changes in environmental conditions such as oxygen, temperature, and salinity [19–24] CHHs from the gut and the cells in embryonic abdominal segments seem to be particularly involved in the process of water uptake during ecdysis and hatching, respectively [1,6] The physiological roles of SOGCHH and PO-CHH have not been defined [3–5,11] Although CHH secretion from the ES in response to stress is well documented [19–24], the effect of stresses on CHH transcription in the ES remains unanswered Thus, despite > 90 putative cDNA structures for CHH being deposited in GenBank, it is apparent that much work is still required to address the physiological roles and the localization of the corresponding neuropeptides in the tissues from which many CHH cDNAs were derived The blue crab, Callinectes sapidus, an economically valuable euryhaline species in Chesapeake Bay, experiences migration and seasonal changes in environmental conditions, including temperature, salinity and dissolved oxygen (http://www.dnr.state.md.us/bay/ monitoring/water/index.html) In particular, it is noted that in the Bay, low temperatures during winter and anoxia during summer, in combination with the changes in salinity, are associated with high mortality in this species [25–27] In view of CHH involvement in response to stress in other crustacean species, it is reasonable to think that Cal sapidus CHH may also play an important regulatory role in adaptation to naturally occurring stressful conditions Thus, we were interested in isolating the cDNA of PO-CHH and identifying the native CHH neuropeptide in the ES, after the recent report of CHH cDNA from the ES [28] Also, we examined the physiological responses of the release and expression of these two CHHs under stressful conditions, especially severe hypoxia, hypothermia and hyperthermia, in an attempt to define their functions To address these questions, we cloned the fulllength cDNA of PO-CHH and identified the presence of the neuropeptide forms in the ES and PO For the first time in crustaceans, the expression profiles of these two CHHs in response to oxygen and temperature changes were documented using quantitative real-time RT-PCR To further define the physiological role of PO-CHH, the levels of this CHH were measured from the same animals, along with ES-CHH, using RIAs under control normoxic and hypoxic conditions 0.0 10 20 30 Retention time, 40 Fig Separation of the extract of PO (50 equivalents) on an RPHPLC C18 column Elution conditions were the same as described for Fig The cross-reacted fractions initially tested with Car maenas CHH antiserum are shown as bars FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS Glucose (ug/1 mL hemolymph) J S Chung and N Zmora Expression and release of CHH in the blue crab 1200 C *** 900 D 600 A 300 Saline E ES-CHH PO-CHH Fig In vivo bioassay of PO-CHH and ES-CHH (A) Glucose assay: open bars, controls at time = 0; closed bar, t = h Results are presented as mean ± SE (n = 6–8) ES-CHH showed a significant increase in hemolymph glucose (***P < 0.001, paired t-test) analyzed by ELISA are marked on a bar graph that shows positive cross-reactivity with PO-CHH antiserum Immunopositive fractions (numbers 30–32) were further analyzed by surface-assisted laser desorption ⁄ ionization (SALDI) to localize fraction 31 containing PO-CHH PO-CHH retained in fraction 31 showed two masses with a 16 Da difference: 8373 Da and 8356 Da This fraction was subjected to further purification, and the final peak was collected for bioassay Native PO-CHH (20 pmol) or ES-CHH (10 pmol) of the blue crab was injected into intact blue crabs ES-CHH increased hemolymph glucose five-fold, as compared to controls, whereas the injection of PO-CHH did not elevate hemolymph glucose (Fig 3) Animals injected with 20 pmol of oxidized ES-CHH, in which one of the Met residues was oxidized to give a 16 Da higher molecular mass than that of non-oxidized ES-CHH (peak 2, CHH-II), showed only a modest two-fold increase in glucose level from 77.79 ± 7.3 to 151.58 ± 13.4 lgỈmL)1 hemolymph (n = 10, P < 0.01, paired t-test) Interestingly, the injection of 20 pmol of Car maenas ES-CHH also induced a significant increase (P < 0.05, paired t-test) in hemolymph glucose from the blue crab (257 ± 34.6 to 633 ± 229 lgỈmL)1 hemolymph, n = 10) PO immunohistochemistry Figure 4A shows the intrinsic multipolar cells in the posterior bar of the PO;  50 cells, 30–40 lm in length, were positive with anti-PO-CHH serum Most cells (35–40) were located in the anterior (Fig 4A) and posterior (Fig 4B) bars, whereas the rest were located in trunks Two types of cells were observed: the majority of cells showed homogeneous CHH staining in the cytoplasm (Fig 4C) with a visibly large nucleus, whereas others had less intensive but punctuated and B Fig Immunohistochemistry of PO staining with PO-CHH antiserum (A, B) Intrinsic multipolar cells, located in anterior (A) and posterior (B) bars (C, D) Cells at · 1200 magnification (E) PO-CHH staining shown in nerve fibers located in trunk Scale bars = 50 lm granulated cytoplasm (Fig 4D) Figure 4E shows the possible release sites on the surface of the PO and many nerve fiber tracts and varicosities in trunks of the PO Cloning and sequencing of PO-CHH cDNA The first PCR of PO cDNA with a combination of a set of primers LF1 and LR1 (Table 1) produced an amplicon of 500 bp On the basis of this sequence, genespecific primers for 5¢-RACE (LR and LR2) and 3¢-RACE (LF2) were generated A nested PCR with a combination of LF2 and 3¢ nested primer (Invitrogen, Carlsbad, CA, USA), using the template from a touchdown product, generated an amplicon of  1.7 kbp The sequencing of the cloned vector of this amplicon as an insert was completed using M13 F and R and a walking primer (PWF1, ATGGGATATGTTCTCAGT), revealing the presence of a long 3¢-UTR ( 1.5 kbp) The amplicon (132 bp) produced from the nested PCR of 5¢-RACE cDNA contained a 5¢-UTR and the remaining sequence of the 5¢-end of PO-CHH The complete cDNA sequence of PO-CHH, shown in Fig 5, contained a 5¢-UTR, a signal peptide (MQS IKTVCQITLLVTCMMATLSYTHA), a crustacean hyperglycemic precursor-related peptide (RSAEG LGRMGRLLASLKSDTVTPLRGFEGETGHPLE), a CHH, a non-amidated C-terminus (QIYDSSCK GVYDRAIFNELEHVCDDCYNLYRNSRVASGCR ENCFDNMMFETCVQELFYPEDMLLVRDAIRG) and a 3¢-UTR of  1.5 kbp FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS 695 Expression and release of CHH in the blue crab J S Chung and N Zmora Table List of primers used for cloning of 5¢-3¢-RACE of PO-CHH LF and LR primers were used for standard cRNA production for quantitative RT-PCR, whereas SF and SR primers were for quantitative RT-PCR A combination of CHH-SF and ES-SR was used for ES-CHH, and for PO-CHH, PO-SR primer was used Cal sapidus AK, Q9NH49; Cal sapidus CHH, AY536012; Cal sapidus eIF4A, DQ667140; Cal sapidus PO-CHH, DQ667141 Primers Sequence (5¢- to 3¢) LF1 LF2 LR1 LR2 LR3 PWF1 CHH-SF ES-SR PO-SR AK LF AK SF AK LR AK SR eIF4A LF eIF4A SF eIF4A LR eIF4A SR CAATCCATCAAAACCGTGTG TGCTACAGCAACTGGTGATCAGAAGGG CCCTTCCTGATCACCATGTTGCTGT GGCCATCATACAGGTGACTAGGAGGGT GGGTGATTTGACACACGGTTTTGATGGA ATGGGATATGTTCTCAGT ACAGATTTACGACTCCTCCTG CATGTTGCTGTAGCAGTTTGAT ATATAAGCTTATCCTCTGATAGC GACCCATCATCGAGGACTA ACCACAAGGGTTTCAAGCAG CCACACCAGGAAGGTCTTGT GGTGGAGGAAACCTTGGACT ACGTCAACATGTCCGACAAA CGGTGGAGACAACAAGGACT TGCGTTTCGTTTGACTTCAC GGCTGATGGCTTCTCAAAAC Hemolymph titers of PO-CHH and ES-CHH in response to changes in dissolved oxygen Hypoxia induced the release of CHHs from the PO and ES of the juvenile crabs (Fig 6) At the initial control normoxic condition, the amount of ES-CHH (376 ± 67 fmolỈmL)1) in hemolymph was  9-fold higher than that of PO-CHH (45.6 ± 8.6 fmolỈmL)1) One hour of exposure of hypoxia induced CHH secretion both from the ES and PO ES-CHH doubled in level from 376 ± 67 to 762 ± 143 fmolỈmL)1 hemolymph (n = 9, P < 0.05) The level of PO-CHH increased from 45.6 ± 8.6 to 74.5 ± 19.8 fmolỈmL)1 hemolymph (n = 9) but there was no statistical difference Interestingly, ES-CHH levels in the hemolymph of animals 10 after they were returned to control normoxic seawater, after previous exposure to hypoxia for h, significantly decreased to 156 ± 50 and 44.5 ± 12.8 fmolỈmL)1 hemolymph (n = 9, P < 0.05), respectively Effects of stresses on the levels of glucose and lactate in hemolymph and gene expression in the ES and PO Hypoxia and emersion Hypoxia and emersion caused hyperglycemia and hyperlactemia (Fig 7A,B) Crabs exposed to hypoxia 696 and emersion showed a  3-fold increase in glucose and a more than 30-fold increase in lactate, whereas the levels in controls remained constant The arginine kinase gene (AK) and the eukaryotic translation initiation factor 4A gene (eIF4A, DQ667140) were initially selected as control genes, but the expression levels in both tissues were significantly increased in response to hypoxia and emersion Thus, all the expression levels were presented as total copy number ⁄ tissue, as in Fig The ES and PO from the animals that experienced h of hypoxia and emersion showed significant changes in CHH gene expression As shown in Fig 7C, hypoxia and emersion greatly reduced ES-CHH gene expression ( 10-fold and five-fold) from 1.36 ± 0.42 · 108 to 1.06 ± 0.16 · 107 and 2.49 ± 0.35 · 107 (copy number ⁄ tissue, one-way anova, P < 0.05), respectively In contrast, the expression level of the PO-CHH gene was dramatically increased from 1.95 ± 0.5 · 107 to 1.45 ± 0.45 · 109 and 2.21 ± 0.79 · 109 (copy number ⁄ tissue, one-way anova, P < 0.05) Emersion caused significantly higher expression of eIF4A (5.85 ± 2.38 · 107) than in controls (2.86 ± 0.73 · 106) in the ES, at P < 0.05 (one-way anova), whereas hypoxia induced slightly higher expression of eIF4A (3.81 ± 2.49 · 106) than in controls (2.86 ± 0.73 · 106), which was not statistically significant Hypoxia and emersion, on the other hand, greatly increased eIF4A expression in the PO (1.34 ± 0.637 · 108, 9.99 ± 2.68 · 107, respectively), as compared with controls (1.31 ± 0.163 · 107) Levels of increase in AK expression in the ES and PO were approximately 3–5-fold, as compared with controls Temperature A h exposure to hyperthermic (29 °C) conditions caused a 2.5-fold increase in glucose levels in hemolymph, as shown in Fig 8A, whereas in controls and under hypothermic conditions (4 °C) there was a slightly higher glucose level In contrast to the modest increase in glucose, the levels of lactate from all three groups were significantly elevated after h, as compared with those at the beginning of the experiment The increase in lactate levels was pronounced, in that both thermal stresses caused increases from seven-fold (hypothermal) to 13-fold (hyperthermal), whereas a 3.5-fold increase was observed in animals maintained at 22 °C The effect of different temperatures on gene expression in the ES and PO is shown in Fig 8C The expression of ES-CHH was modestly increased at 29 °C (P = 0.08, one-way anova) as compared with FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS J S Chung and N Zmora Expression and release of CHH in the blue crab Fig Sequence alignments of nucleotide and deduced amino acids of full-length PO-CHH (DQ667141) and ES-CHH (AY536012) PO-CPRP and ES-CPRP are in bold italic, and both CHHs are in bold The 5¢-UTR and a signal peptide are italicized A dibasic cleavage site (KR) is underlined The stop codon (TAA) is marked as *, and a putative polyadenylation site (AATAAA) is underlined that in controls at 22 °C; whereas the level of POCHH expression was only slightly higher, at 29 °C In contrast to the results obtained with hypoxia and emersion, as shown in Fig 7, there was little change in AK and eIF4A expression in the ES and PO at 22 °C and 29 °C, except that animals exposed to °C showed higher AK expression in the PO, as compared with those exposed to 22 °C and 29 °C Discussion In this article, we describe studies on the identification, localization and bioactivity of CHH neuropeptides of the blue crab, Cal sapidus, using biochemical, molecular and immunological methods Blue crabs produce a CHH neuropeptide in the PO that shows a 66% deduced amino acid sequence identity with ES-CHH [28] We have also demonstrated, for the first time in crustaceans, the differential expression of these two CHHs in response to changes in the following environmental conditions: hypoxia, emersion, and temperature More important with respect to physiology, we measured the hemolymph titers of these two CHHs under different dissolved oxygen levels in seawater Our results indicate that the regulatory mechanisms governing the expression of ES-CHH and PO-CHH are different Yet, the release of both CHHs seems to be sensitive to dissolved oxygen in seawater, suggesting an adaptive role A single SG in the ES of Cal sapidus contains two isoforms of CHH The molecular mass difference suggests that the major CHH may have pyroglutamate at the N-terminus via post-translational cyclization of Glu of peak This feature appears to be common in CHHs of brachyuran crab species, including Car maenas [29] and Cancer pagurus [30], and differs from those in astacuran species [10,31–33] The relative abundance of these CHH isoforms in the SG among FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS 697 J S Chung and N Zmora Control Emersion Hypoxia B *** 450 *** 300 150 Control Hypoxia Emersion C 1010 b b 10 b a 10 bb b b a b b a 10 a b a b a a 10 H H PO PO AK -e IF 4A these species varies as a ratio of two isoforms (peaks and 2), ranging from : for Cal sapidus, to : in Car maenas, to : 10 in Can pagurus [29,30], yielding peak as the major CHH The immunohistochemistry studies show that the intrinsically staining cells are responsible for PO-CHH synthesis The staining patterns are similar to those of Car maenas, in that most cells (approximately 35–40) were localized in the anterior and posterior bars [4] However, PO-CHH staining appears to be somewhat different from that of crustacean cardioactive peptide (CCAP) in the PO, where it is known mainly at its release site [34] Overall, the immunohistochemisty of PO-CHH in the PO indicates its role as a neurotransmitter, as it may be directly targeted into the specific sites in which the nerve fibers of the PO innervate the anterior ramifications On the other hand, as a neurohormone, PO-CHH may be released from the surface of the PO and into branchiocardiac veins through the direct openings of anterior and posterior bars [35] Furthermore, considering the localization of PO-CHH-producing cells in the pericardial chamber, it may be pertinent to suggest that these intrinsic multipolar PO-CHH cells may be sensitive to homeostasis of hemolymph Cloning of cDNA of PO-CHH of Cal sapidus produced only one size (2004 bp) that is translated into PO-CHH neuropeptide The cDNA sequence of PO-CHH encodes a preprohormone containing a signal peptide (26 amino acid residue), one crustacean hyperglycemic hormone precursor-related peptide (CPRP, 36 amino acid residue), and PO-CHH, not 600 4A Fig Changes in hemolymph titers of PO-CHH and ES-CHH in response to dissolved oxygen CHH control values were obtained from animals in control normoxic water The closed bar shows a significantly increased level of ES-CHH (P < 0.05, one-way ANOVA, Krustal–Wallis test, INSTAT); the open bar is PO-CHH Bars represent mean ± SE (n = 9) Note the difference in the y-axis scales between ES-CHH and PO-CHH 698 PO C ia xia ox po orm Hy ln tro on C K or l n tro n Co xia mo 100 -A 20 -e IF c 200 200 ES 40 H a 400 ** ES 60 *** 300 -C H 600 400 ES 80 Glcuose (ug/mL hemolymph) 800 Lactate (ug/mL hemolymph) b A Copy number of genes (tissue) 100 1000 PO-CHH (fmol/mL hemolymph) ES-CHH (fmol/mL hemolymph) Expression and release of CHH in the blue crab Fig Effect of hypoxia and emersion on glucose (A), lactate (B) and gene expression (C) (A, B) Open bars: controls at t = Closed bars: h after exposure Paired t-test showed the statistical significance at **P < 0.01 and ***P < 0.001 (C) The profiles of CHH, AK and eIF4A expression in the ES and PO in response to hypoxia and emersion Closed bar: controls Hatched bars: hypoxia Crossed bars: emersion Bars represent mean ± SE (n = 6) Statistical analysis was performed using one-way ANOVA (P < 0.05, Krustal– Wallis test, INSTAT) amidated at the C-terminus (71 amino acids) This cDNA of Cal sapidus PO-CHH contains a much longer long 3¢-UTR ( 1.5 kbp) than that of Car maenas (518–759 bp) [4] Interestingly, a putative cDNA encoding a PO-CHH type has been cloned in thoracic ganglia (TG) of Cal sapidus and other tissues [28] However, the immunohistochemisty study using FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS J S Chung and N Zmora Expression and release of CHH in the blue crab Glucose (ug/mL hemolymph) A 300 ** 200 100 22 to 22 22 to 29 22 to Temperature (°C) Lactate (ug/ mL hemolymph) B 180 *** 120 ** 60 * 22 to 22 22 to 29 22 to Temperature (°C) 1e+9 1e+8 b 1e+7 aa H PO AK -e IF 4A PO -C H PO -e IF ES ES 4A 1e+6 -C H H ES -A K Copy no of genes (ES/PO) C Fig Effect of hypothermia and hyperthermia on glucose (A), lactate (B), and gene expression (C) (A, B) Open bars: controls at t = Closed bars: h after exposure The results were analyzed for statistical significance using a paired t-test (*P < 0.05, **P < 0.01, ****P < 0.001) (C) The profiles of CHH, AK and eIF4A expression in the ES and PO in response to hypothermia and hyperthermia Closed bar: controls Hatched bar: hypothermia Crossed bar: hyperthermia Bars represent mean ± SE (n = 6) Statistical analysis was performed using one-way ANOVA (P < 0.05, Krustal–Wallis test, INSTAT) anti-PO-CHH revealed exclusive positive staining in the PO but not in TG (our unpublished observation), suggesting that the putative cDNA encoding the PO-CHH type cloned in TG is not translated into a protein Similarly, for Car maenas, nine putative cDNA sequences of PO-CHH are listed in GenBank, despite the fact that only one of these encodes the conceptual neuropeptide sequence of PO-CHH [4] The results of homologous and heterologous bioassays of Cal sapidus ES-CHHs reflect high sequence identity (> 75%) of ES-CHHs between two crab species Our finding is in agreement with a previous report that the injection of Cal maenas ES-CHH triggered hyperglycemia in Can pagurus [30] Overall, such results of heterologous bioassays indicate that CHH receptors among these crab species may share some degree of similarity Cal sapidus PO-CHH injection (20 pmol) did not cause hyperglycemia in the blue crab This finding is not unexpected, as a similar result was observed in Car maenas [4] The close sequence analysis of ESCHH and PO-CHH shows that the greatest homology is in the first 40 amino acids, with much more difference in the latter half of the sequence Such differences are common in all CHH sequences currently available in GenBank, indicating that functionality inducing hyperglycemia may lie in the first 40 amino acid residues [36–40] Therefore, it is suggested that these two CHHs may have separate receptors in their target tissues, where PO-CHH may be mobilizing glucose but not be directly involved in hyperglycemia in hemolymph PO-CHHs among Cal sapidus, Car maenas and P marmoratus share overall 67% sequence identity, of which 85% and 68% are contributed by the first 40 residues and by the C-terminus, respectively [4,5] Thus, on the basis of the sequence identity among PO-CHHs, it is proposed that the physiological function of this CHH may be conserved in at least these crab species, although it has not yet been fully defined and understood To define the physiological function of PO-CHH, the release pattern was evaluated along with that of ES-CHH in response to hypoxia The basal level in the PO was surprisingly high at 10)11 m, although it was 10-fold less than that of ES-CHH This difference in the concentrations of two CHHs seems to reflect the amount present in these tissues, approximately 2– pmol, at least 20–50-fold less than that of ES-CHH (100 pmolỈES)1) It is noteworthy that the basal level of ES-CHH is an order of magnitude greater in Cal sapidus (10)10 m) than in Car maenas (10)11 m) [24] or Can pagurus (< 10)11 m) [20,23] Such a high CHH concentration in hemolymph of Cal sapidus may explain the high basal level of hemolymph glucose, which may reflect the behavioral differences among these species Moreover, crabs under hypoxia and emersion have shown differential redistribution of hemolymph to increase the flow, especially to the FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS 699 Expression and release of CHH in the blue crab J S Chung and N Zmora sternal artery, and overall cardiac output [41–43] Our finding of the elevated ES-CHH level in hemolymph is congruent with previous reports on CHH secretion under hypoxia or emersion [20–24] Thus, our finding supports the suggestion that PO-CHH and ES-CHH have an adaptive role in the physiological response to hypoxia In crustaceans, it seems to be rather difficult to find an ideal control gene for quantitative RT-PCR, the level of which does not change during molting or the reproductive cycle [7,44] As eIF4A, a member of the DEAD box and ATP-dependent RNA helicase family, may be considered as a temporal translation indicator, because of its involvement in the assembly of active polysome by unwinding secondary structure in the 5¢-UTR of mRNAs [45], we reasoned that eIF4A would be a good control gene as an indicator of the level of translation Therefore, Cal sapidus cDNA of the eIF4A gene was initially isolated, but the expression level of this gene was unexpectedly changed, suggesting that it is sensitive to oxygen level One hour of acute hypoxia and emersion induced different expression levels of PO and ES-CHH Chronic hypoxia caused downregulation of the hemocyanin gene in the hepatopancreas in the same species [46] Moreover, the degree of decrease in CHH expression in the ES is in proportion to that of CCAP expression in the same tissue, as described in Chung et al [47], but there is no change in CCAP of TG, indicating the tissue-specific regulation of CCAP expression Likewise, the present results concerning PO-CHH and ES-CHH expression in response to oxygen level suggest that the regulation of CHH expression is different and tissue specific, perhaps via tissue-specific alternative splicing or tissue-specific gene expression through different regulatory arrangements in the upstream promoter regions [37,38] It is reasonable to suggest that the levels of transcription of PO-CHH are in proportion to the demand for its release, whereas the inhibition of ES-CHH transcription may be caused by high glucose levels in hemolymph On the other hand, the elevated hemolymph glucose might have inhibited ES-CHH expression, as a previous report indicated that CHH neurons are hyperpolarized in response to 25 mm glucose in the media [48] Hyperglycemia may also inhibit the further release of CHH, whereas the low glucose level in hemolymph may have a positive influence on the release of CHH from SG in the ES, as described in Chung & Webster [24] With these observations, it would be interesting to see if in vitro incubation of the ES in high-glucose media causes the inhibition of CHH gene expression 700 We have shown the presence of ES-CHH neuropeptides and the putative cDNA sequence of PO-CHH that is translated into a neuropeptide in the PO The location of intrinsic multipolar cells and structure of nerve branches in the PO indicate that these cells may be sensitive to changes in hemolymph homeostasis Changes in dissolved oxygen levels in seawater immediately affect the release of CHHs from the PO and SG, strongly suggesting that PO-CHH has an adaptive role, in particular, in response to oxygen level Defining the physiological function of PO-CHH may seem to be a challenge, as the structural similarity of PO-CHH places it as ‘a tacit CHH’, despite the fact that it does not induce hyperglycemia in hemolymph However, we have taken a positive step towards identifying a physiological function of PO-CHH, as its release is recorded under the changes in dissolved oxygen levels in seawater For the future, binding studies are required to identify its target tissues and second messenger, as the next step towards defining the physiological function of PO-CHH Furthermore, given the high sequence similarity of PO-CHH among Cal sapidus, Car maenas and P marmoratus, the function of this neuropeptide may be similar in these crab species, as is the function of ES-CHH Experimental procedures Animals Juvenile Cal sapidus (carapace width: 45–80 mm) were obtained from the blue crab hatchery program [49], Aquaculture Research Center, Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD and were maintained in individual compartments in artificial seawater [15 parts per thousand (p.p.t.) salinity and 22 °C] under ambient daylight conditions Experimental animals were fed daily with chopped frozen squid and pelleted sea bream (EWOS, Surrey, Canada) All experimental animals were at intermolt stage [50] Identification, isolation and quantification of neuropeptides from SGs and POs Neuropeptides of SGs in the ES and PO were purified using RP-HPLC, as described in Chung & Webster [29] and Dircksen et al [4], respectively CHH peaks were initially identified using ELISA with a combination of Carcinus CHH antisera, the method described in Wilcockson et al [23] Amino acid analysis was carried out for the quantification of neuropeptides [29], and ESI MS of SG neuropeptides was used for mass determination Confirmation of the RPHPLC fraction containing PO-CHH and its mass determination were performed using SALDI FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS J S Chung and N Zmora RACE of PO-CHH Total RNA from POs was extracted in TRIzol reagent and quantified using RIBO green (Invitrogen) The GeneRacer protocol (Invitrogen) was employed for the synthesis of 5¢RACE and 3¢-RACE cDNAs from lg of total RNA For the first amplification of 3¢-RACE cDNA, a touchdown PCR (TD-PCR) was used with a forward gene-specific primer (LF1: 5¢-CAATCCATCAAAACCGTGTG-3¢) and 3¢ universal primer (Invitrogen) Conditions of TD-PCR were as follows: initial denaturation at 94 °C for min; three cycles at 94 °C for 30 s, 54 °C for 30 s, and 72 °C for min; three cycles at 94 °C for 30 s, 52 °C for 30 s, and 72 °C for min; three cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for min; 24 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for min, and a final extension at 72 °C for A nested PCR was carried out with a forward gene-specific primer (LF2: 5¢-TGCTACAG CAACTGGTGATCAGAAGGG-3¢) and 3¢ universal nested primer (Invitrogen), with the following conditions: after initial denaturation at 94 °C for min, 30 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for min, and a final extension at 72 °C for The PCR product was electrophoresed on 1% agarose gel, and the band was excised, extracted and cloned into a TOPO-TA vector for sequencing The sequence of the 3¢-UTR was obtained by primer walking using the gene-specific forward primer (5¢-ATATAAGCTTATCCTCTGATAGC-3¢) For 5¢-RACE, the same PCR conditions were employed as described above for 3¢-RACE, using a gene-specific reverse primer (LR1: 5¢-TTCCTGATCACCATGTT GCTGT-3¢) and a 5¢ universal primer for the first PCR Following this, the nested PCR was conducted using LR2 (5¢-GGGTGATTTGACACACGGTTTTGATGGA-3¢) and 5¢ nested universal primers (Invitrogen) The methods of PCR analysis and cloning were as described above Quantitative real-time RT-PCR The cRNA standards of quantitative RT-PCR, including PO-CHH and ES-CHH, AK and eIF4A, were initially PCR amplified using a combination of LF and LR primers (Table 1) Further cRNA synthesis and RNA quantification were performed as described in Chung & Webster [6,7], with a modification for purification of in vitro transcribed cRNAs (Ambion, Austin, TX, USA), eluting on a spin-column (BD Biosciences, Mountainview, CA, USA) Total RNA extracted from the tissues as described above was treated with DNase, and each of lg or 0.5 equivalent of PO RNAs were primed with random hexamers for cDNA synthesis using avian myeloblastosis virus or Moloney murine leukemia virus reverse transcriptase Final cDNA samples were diluted to 40 lL, and lL of each sample was analyzed for the expression of genes using SYBR gold (ABI, Foster City, CA, USA) on ABI Expression and release of CHH in the blue crab Prism with a pair of gene-specific primers (SFs and SRs; Table 1) PO-CHH and ES-CHH antisera production C-terminal fragments of PO-(FDNMMFETCVQELFY PEDMLLVRDAIRG; Proteintech Group Inc., Chicago, IL, USA) and ES-CHH (EDLLIMDNFEEYAR KIQVV-NH2; Invitrogen) were synthesized with the addition of Cys at the N-terminus These modified synthetic peptides, after being N-terminally conjugated with bovine thyroglobulin using m-maleimidobenzoyl-N-succinimide ester [47], were used for antisera production in rabbits (Proteintech Group Inc., Chicago) Whole-mount immunohistochemistry of PO Immediately after dissection, POs were fixed in a fixative containing 7% picric acid and 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4) overnight Procedures for washing and application of primary (· 1000 dilution) and secondary antiserum (Vector Laboratories, Burlingame, CA, USA) were as described in Chung & Webster [5] Z-stacked images of PO preparations were collected using a Zeiss Confocal Microscope with a BioRad program (COMB, UMBI) Bioassays of CHH neuropeptides Levels of glucose in hemolymph were estimated before and h after injection of 10 or 20 pmol of each of ES-CHH and PO-CHH, oxidized ES-CHH, and Car maenas ESCHH, using the glucose assay described in Webster [20] Iodination of neuropeptides and RIA The detailed procedures for iodination of ES-CHH and C-terminal synthetic peptide of PO-CHH, RIA and hemolymph sample preparation were as described in Chung & Webster [24,29] Standards of both RIAs ranged from 500 fmol per tube to 3.8 fmol per tube, and the detection limit was < fmol per tube for both CHHs ED50 values were 120 ± and 134 ± (fmol per tube) for SG-CHH and PO-CHH, respectively The results were analyzed using assayzap (Biosoft, Cambridge, UK) Effects of environmental factors on the levels of hemolymph and gene expression of CHH – dissolved oxygen, emersion and temperature Thirty minutes before the experiment, L of artificial seawater (15 p.p.t., 22° C) was continuously purged with nitrogen to reduce the level of dissolved oxygen to < 0.5% FEBS Journal 275 (2008) 693–704 ª 2008 The Authors Journal compilation ª 2008 FEBS 701 Expression and release of CHH in the blue crab J S Chung and N Zmora (YSI 58 Dissolved Oxygen Meter) Test juvenile animals (60–80 mm carapace width) were exposed for h to hypoxic seawater under continuous nitrogen purging, whereas controls remained in aerated normoxic seawater At the end of the h exposure, either to anoxia or the control, hemolymph samples were withdrawn from the hypobranchial sinus through the arthrodial membrane between the chelae and the first walking leg, using a mL syringe with a 23-gauge needle The ES and PO were dissected out, immediately frozen on dry ice, and kept at )80° C until further processing For emersion experiments, juvenile crabs were exposed to air for h at 22° C, whereas controls were maintained in 15 p.p.t artificial seawater at 22° C At the end of the experiment, the hemolymph and tissue samples were collected as described above Hemolymph CHHs were estimated using RIAs after the elution of hemolymphs on a Sep-Pak C18 column (Waters; 360 mg cartridges), as described in Chung & Webster [24,29] Temperatures Juvenile crabs (45–60 mm carapace width) were initially held at 22° C (15 p.p.t.) Once hemolymph samples were drawn as described above at time 0, animals (n = 6) were subjected for h to the following temperatures: 29° C, 22° C, and 4° C The second hemolymph and tissue samples were collected at the end of exposure Total RNA was extracted using the method described above Statistical analysis The data were tested for statistical significance using graphpad instat version 3.0 (GraphPad Software, San Diego, CA, USA) Acknowledgements The authors wish to thank Drs S G Webster (University of Wales, Bangor, UK) for amino acid analysis of neuropeptides, and M M Ford for comments on the manuscript We also wish to thank to Dr S Moore for SALDI (Ciphergen) and Mr M Prescott for ESI MS analyses (University of Liverpool) We are indebted to Mr O Zmora and hatchery personnel for the young juvenile crabs, and S Rogers and E Evans for maintaining the water quality of the recirculation system This article is contribution no 07-177 of the Center of Marine Biotechnology (University of 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Hemolymph titers of PO -CHH and ES -CHH in response to changes in dissolved oxygen Hypoxia induced the release of CHHs from the PO and ES of the juvenile crabs (Fig 6) At the initial control normoxic... of CHH cDNA from the ES [28] Also, we examined the physiological responses of the release and expression of these two CHHs under stressful conditions, especially severe hypoxia, hypothermia and. .. mortality in this species [2 5–2 7] In view of CHH involvement in response to stress in other crustacean species, it is reasonable to think that Cal sapidus CHH may also play an important regulatory