The crustacean hyperglycemic hormone (CHH) family of peptides includes CHH,moult-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). In the crab Cancer pagurus, isoforms of these peptides, as well as CHH precursor-related peptide (CPRP), have been identified in the X-organ-sinus gland (XO-SG) system. Using peptides isolated from the C. pagurus SG, antibodies to each family member and CPRP were generated. These sera were then used to map the distributions and co-localization patterns of these peptides in the neuroendocrine organs of seven Cancer species: Cancer antennarius, Cancer anthonyi, Cancer borealis, Cancer gracilis, Cancer irroratus, Cancer magister and Cancer productus. In addition to the XO-SG, the pericardial organ (PO) and two other neuroendocrine sites contained within the stomatogastric nervous system, the anterior cardiac plexus (ACP) and the anterior commissural organ (ACO), were studied. In all species, the peptides were found to be differentially distributed between the neuroendocrine sites in conserved patterns: i.e. CHH, CPRP, MIH and MOIH in the XO-SG, CHH, CPRP and MOIH in the PO, and MOIH in the ACP (no immunolabeling was found in the ACO). Moreover, in C. productus (and probably in all species), the peptides present in the XO-SG and PO were differentially distributed between the neurons within each of these neuroendocrine organs (e.g. CHH and CPRP in one set of XO somata with MIH and MOIH co-localized in a different set of cell bodies). Taken collectively, the differential distributions of CHH family members and CPRP both between and within the neuroendocrine organs of crabs of the genus Cancersuggests that each of these peptides may be released into the circulatory system in response to varied, tissue-specific cues and that the PO- and/or ACP-derived isoforms may possess functions distinct from those classically ascribed to their release from the SG.

Research on crustaceans has played a major role in advancing our understanding of neuroendocrine structure and control. Nearly 80 years ago,Koller (Koller, 1925; Koller, 1927) demonstrated that hemolymph-borne agents were responsible for color change in shrimp. In the decade that followed, Hanström(Hanström, 1931; Hanström, 1933; Hanström, 1935; Hanström, 1937)identified a structure located in the eyestalk, the sinus gland (SG) as the source of these substances. Later, Bliss(Bliss, 1951) and Passano(Passano, 1951) independently demonstrated the neurohemal nature of the SG, a first for any neural structure in the animal kingdom. In recent years, work from many laboratories has shown that the SG is one of the major neuroendocrine organs in crustaceans and a number of hormones contained within it have been identified, including a group of structurally related peptides that have collectively been termed the crustacean hyperglycemic hormone (CHH) family.

In crustaceans, the CHH family includes CHHs, moult-inhibiting hormones(MIHs), gonad-inhibiting hormones (GIHs), vitellogenesis-inhibiting hormones(VIHs) and mandibular organ-inhibiting hormones (MOIHs), all of which are large peptides (72-78 amino acids) that share considerable amino acid sequence and structural homology, including six conserved cysteine residues and three disulfide bridges (Keller,1992; de Kleijn and van Herp,1995; Soyez, 1997; van Herp, 1998; Webster, 1998; Chan et al., 2003; Chen et al., 2005; Fanjul-Moles, 2006). As their diverse names imply, members of the CHH family have been shown to influence a broad array of physiological processes including, but not limited to, the regulation of metabolism, somatic growth and reproductive maturation(Sedlmeier and Keller, 1981; Sedlmeier, 1982; Sedlmeier, 1988; Keller, 1992; Charmantier-Daures et al.,1994; Wainwright et al.,1996; Santos et al.,1997; Khayat et al.,1998; van Herp,1998; Webster,1998; Chung et al.,1999; Spanings-Pierrot et al.,2000; Sonobe et al.,2001; Chan et al.,2003; Serrano et al.,2003).

Based on preprohormone organization, the CHH family can be divided into two sub-groups: (1) CHH and (2) MIH/GIH/VIH/MOIH(de Kleijn and van Herp, 1995; Lacombe et al., 1999; Chan et al., 2003; Chen et al., 2005; Fanjul-Moles, 2006). The preprohormones of the CHH subgroup always contain a second peptide, commonly referred to as CHH precursor-related peptide or CPRP, which is C-terminally flanked by the CHH (Weidemann et al.,1989; Tensen et al.,1991; de Kleijn et al.,1994a; de Kleijn et al.,1995; Ohira et al.,1997a; Davey et al.,2000; Toullec et al.,2002; Marco et al.,2003; Chen et al.,2004; Mettulio et al.,2004; de la Iglesia et al.,2005; Hsu et al.,2005a; Toullec et al.,2006). The preprohormones of the MIH/GIH/VIH/MOIH sub-group lack precursor-related peptides (Klein et al.,1993; de Kleijn et al.,1994b; Sun, 1994; Lee et al., 1995; Ohira et al., 1997b; Chan et al., 1998; Gu and Chan, 1998; Umphrey et al., 1998; Tang et al., 1999; Lu et al., 2001; Yang and Rao, 2001; Edomi et al., 2002; Krungkasem et al., 2002; Ohira et al., 2005). Although CPRP has been shown to circulate in the hemolymph, the physiological role(s)it plays in crustaceans remains unknown(Wilcockson et al., 2002).

In brachyuran crabs, particularly those of the genus Cancer, much work has been done in an effort to identify and characterize the actions of the native CHH-family members and CPRPs in the X-organ (XO)-SG system. In this genus, four distinct CHH isoforms have been identified [two from Cancer pagurus and two from Cancer productus(Chung et al., 1998; de la Iglesia et al., 2005; Hsu et al., 2005a); Table 1] as have two isoforms of MIH [one from Cancer magister and the other from C. pagurus (Chung et al.,1996; Umphrey et al.,1998); Table 1] and two isoforms of MOIH [both from C. pagurus(Wainwright et al., 1996); Table 1]. Similarly, nine isoforms of CPRP have been characterized from the SG of Cancerspecies [one from C. pagurus and four each from C. productusand Cancer borealis (Chung et al., 1998; de la Iglesia et al., 2005; Fu et al.,2005a; Fu et al.,2005b; Hsu et al.,2005a); Table 1]. As Table 1 shows, the isoforms of any given group are nearly identical in amino acid sequence, even when comparing isoforms from different species.

Although most studies concerning CHH-family members and CPRPs have focused on the SG, there is evidence that some of these peptides are also present in other regions of the crustacean nervous system(de Kleijn et al., 1995; Sun, 1995; Chang et al., 1999; Dircksen et al., 2001; Lu et al., 2001; Gu et al., 2002; Wilcockson et al., 2002; Basu and Kravitz, 2003; Chen et al., 2004; Fu et al., 2005b; Ohira et al., 2005; Toullec et al., 2006). CHH-and CPRP-like immunoreactivities have been found in the neuroendocrine pericardial organ (PO) of C. pagurus(Wilcockson et al., 2002) and putative CPRP fragments have been sequenced from this structure in C. productus (Fu et al.,2005b). The functional relevance of this dual XO-SG/PO distribution of CHH in these crabs is currently unknown. However, it has been postulated that the signals governing its release from each site are distinct. Moreover, the isoforms present in one versus the other structure may also be distinct (Dircksen et al.,2001; Toullec et al.,2006) and subserving different physiological roles, as has been shown for the CHHs isolated from the SG and PO of the crab Carcinus maenas (Dircksen et al.,2001).

Recently, we began a study to identify and compare the hormone complements present in the neuroendocrine organs of several Cancer species(Christie et al., 2004; Christie and Messinger, 2005; de la Iglesia et al., 2005; Fu et al., 2005a; Fu et al., 2005c; Hsu et al., 2005a; Hsu et al., 2005b; Messinger et al., 2005a; Messinger et al., 2005b; Cruz-Bermúdez et al.,2006). As a first step toward determining the identity, tissue distributions and physiological roles played by each CHH-family member and CPRP in the neuroendocrine systems of these species, we undertook the immunohistochemical mapping studies presented in this report. Specifically,antisera previously generated against native C. pagurus peptides(Webster, 1996; Wilcockson et al., 2002) were used to map the distributions and co-localization patterns of the CHH family and CPRP in the neuroendocrine organs of Cancer antennarius, Cancer anthonyi, C. borealis, Cancer gracilis, Cancer irroratus, C. magister and C. productus. As the data that follow show, most members of the CHH family and CPRP were immunologically detected in multiple neuroendocrine organs, with the complement of peptides present in each site conserved across species. In at least C. productus, the peptides detected in the XO-SG and PO were also differentially distributed among the neurons comprising each site. Collectively, the data presented here represent the most extensive and complete immunohistochemical survey of CHH family members and CPRP in crustacean neuroendocrine organs to date. Moreover, our findings support the hypotheses that: (1) members of CHH family and CPRP are released in response to varied, tissue-specific cues and (2) the isoforms released from non-XO-SG sites probably possess functions distinct from those ascribed to them when secreted from the SG. Some of these data have appeared previously in abstract form (Hsu et al., 2004).

Animals

Brown rock crabs Cancer antennarius Stimpson and yellow rock crabs Cancer anthonyi Rathbun were purchased from Coastal Catch (Santa Barbara, California, USA). Jonah crabs Cancer borealis Stimpson were purchased from the Marine Biological Laboratory (Woods Hole, Massachusetts,USA). Graceful crabs Cancer gracilis Dana were collected by hand at False Bay, San Juan Island, Washington, USA. Atlantic rock crabs Cancer irroratus Say were purchased from J&A Seafood (Brunswick, Maine,USA). Dungeness crabs Cancer magister Dana and red rock crabs Cancer productus Randall were collected by hand, trap or trawl at multiple locations throughout the San Juan Archipelago and greater Puget Sound areas of Washington State, USA. Regardless of species, animals were maintained in either aerated natural seawater aquaria chilled to 10°C (Department of Biology, University of Washington, Seattle, Washington, USA) or in flow-through natural seawater tables [Friday Harbor Laboratories, Friday Harbor, Washington, USA (locally caught animals only); ambient water temperature was approximately 8-12°C, depending on season].

Tissue collection

For the collection of tissue, crabs were anesthetized by packing in ice for 30-60 min. After anesthesia, the eyestalks were isolated, then the dorsal carapace was removed and the foregut and the lateral walls of the pericardial chamber were dissected free in chilled (approximately 10°C) physiological saline [composition: 440 mmol l-1 NaCl; 11 mmol l-1 KCl;13 mmol l-1 CaCl2; 26 mmol l-1MgCl2; 10 mmol l-1 Hepes; pH 7.4 (adjusted with NaOH)]. To obtain the XO-SG system, the carapace encasing an eyestalk was split both dorsally and ventrally and one half of the split shell was gently teased away from the other half. The remaining half of the eyestalk was then pinned in a wax-lined Pyrex dish filled with chilled physiological saline and the optic ganglia containing the XO-SG system were subsequently isolated. POs were obtained by pinning the isolated walls of the pericardial chamber in a wax-lined Pyrex dish containing chilled physiological saline. The nerve roots forming each PO were then dissected free from the muscles and connective tissues of the pericardial wall. For isolating the stomatogastric nervous system (STNS), which contains two recently identified neuroendocrine organs(Christie et al., 2004; Messinger et al., 2005a), the anterior cardiac plexus (ACP) and the anterior commissural organ (ACO), the foregut was flattened by making a longitudinal cut from the esophagus to the pylorus on its ventral side followed by a pair of medial cuts directed along the ossicles of the cardiac sac/gastric mill. After its opening, the teeth of the gastric mill were removed and the flattened foregut was pinned, inside down, in a wax-lined Pyrex dish containing chilled physiological saline. The STNS was then dissected free from the muscles and connective tissues of the foregut.

Antibodies and antibody production

Primary antibodies

All of the primary antibodies used in our study were rabbit polyclonal antibodies generated against native C. pagurus peptides(Table 1). These peptides were purified from a common pool of approximately 2000 SGs as described in several previous publications (Chung et al.,1996; Wainwright et al.,1996; Webster,1996; Chung et al.,1998). For production of the CHH antibody, native C. pagurus (Capa)-CHH II was used. The development of this antibody is described elsewhere (Webster,1996). For the production of the CPRP antibody, the antigen was C. pagurus (Capa)-CPRP conjugated to bovine thyroglobulin using either glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The production of this antibody is also described elsewhere(Wilcockson et al., 2002). For the production of the MIH and MOIH antibodies, methods similar to those employed for the production of the CHH antibody were used. Specifically, New Zealand white rabbits (one for each peptide) were injected subcutaneously at multiple sites with 5 nmol of native C. pagurus (Capa)-MIH or native C. pagurus (Capa)-MOIH I, each dissolved in 0.3 ml of phosphate-buffered saline and emulsified with an equal volume of complete Freund's adjuvant. Four weeks after the initial immunization, a booster of 5 nmol of peptide (Capa-MIH or Capa-MOIH I) in Freund's incomplete adjuvant was injected into each rabbit. Similarly, 8 weeks after the initial immunization,rabbits were boosted again, this time with 3 nmol of peptide (Capa-MIH or Capa-MOIH I) in Freund's incomplete adjuvant. Twelve weeks after the initial immunization, rabbits were terminally exsanguinated under anesthesia and serum was collected from the retracted clots. Production of all antisera used in this study was done at the University of Wales Bangor (Bangor, UK).

Secondary and tertiary antibodies

The secondary and tertiary antibodies used in our study were kind gifts from either Jackson ImmunoResearch Laboratories, Inc. (West Grove,Pennsylvania, USA) or Molecular Probes (Eugene, Oregon, USA). These included donkey anti-rabbit IgG labeled with Alexa Fluor 488 (Molecular Probes; catalog#A-21206), Alexa Fluor 594 (Molecular Probes; catalog #A-21207), FITC (Jackson ImmunoResearch; catalog # 711-095-152) or Rhodamine Red X (Jackson ImmunoResearch; catalog # 711-295-152); goat anti-rabbit IgG labeled with Alexa Fluor 488 (Molecular Probes; catalog # A11008) or Texas Red (Jackson ImmunoResearch; catalog #111-075-144); unlabeled monovalent goat anti-rabbit IgG antigen-binding (Fab) fragments (Jackson ImmunoResearch; catalog #111-007-003) or donkey anti-goat IgG labeled with FITC (Jackson ImmunoResearch, catalog # 705-095-147).

Whole-mount immunohistochemistry

Single labeling

For single labeling, tissue was fixed overnight in a freshly made solution of 4% paraformaldehyde (EM grade; Electron Microscopy Sciences, Hatfield,Pennsylvania, USA; catalog #15710) in 0.1 mol l-1 sodium phosphate buffer (pH 7.4) followed by five rinses (at 1 h intervals) in a solution of sodium phosphate buffer containing 0.3% Triton X-100 (P-Triton). After rinsing, tissue was incubated with gentle agitation in a 1:5000 dilution of primary antibody for approximately 72 h. Primary antiserum was diluted in P-Triton, with 10% normal donkey serum (NDS; Jackson ImmunoResearch; catalog#017-000-121) or normal goat serum (NGS; Jackson ImmunoResearch; catalog#005-000-121) added to diminish nonspecific binding. Following incubation in primary antibody, tissue was again rinsed five times at 1 h intervals in P-Triton and then incubated overnight in a 1:300 dilution of secondary antibody. As with the primary antibody, secondary antibody incubation was done in P-Triton with 10% NDS or NGS, using gentle agitation. After secondary antibody incubation, each preparation was rinsed five times over approximately 5 h in sodium phosphate buffer and then mounted between a glass microscope slide and coverslip using Vectashield mounting medium (Vector Laboratories,Inc., Burlingame, California, USA; catalog # H-1000). Fixation and incubation in both primary and secondary antibody was done at 4°C. All rinses were done at room temperature (approximately 20°C) without agitation. Secondary antibody incubation, and all subsequent processing, was conducted in the dark. Likewise, slides were stored in the dark at 4°C until examined.

To increase our confidence that the immunoreactivities reported here were specific for peptides related to the antigen used for the production of each antiserum, we conducted a series of adsorption controls. In these experiments,native Capa-CHH II, Capa-CPRP, Capa-MIH and Capa-MOIH I isolated from the SG of C. pagurus (Table 1) were used as blocking agents. It should be noted that due to a limited supply of these peptides, adsorption controls were only conducted on C. productus tissues and that the adsorptions used for labeling one preparation were sometimes recycled for use in blocking studies on other samples. In each adsorption experiment, the antibody was incubated with a blocking peptide (approximately 10-5 mol l-1) for 2 h at room temperature prior to applying the solution to the tissue. For each tissue in which an antibody produced labeling, a complete block of staining was achieved only when the antiserum was adsorbed with the peptide used for its production (three preparations for each peptide and antibody in each tissue;data not shown). Adsorption of an antibody with the other peptides had no appreciable effect on immunolabeling (three preparations for each peptide and antibody in each tissue; data not shown).

Double labeling

For preparations that were double-labeled (C. productus tissues only), the staining protocol for single labeling was modified in order to permit the use of primary antibody pairs that were both developed in the same host species (rabbit in this case). In brief, POs and SGs were dissected,fixed and incubated in the first primary antibody as described above for 1 or 2 days, respectively. Following incubation in the first primary antibody,tissues were rinsed with P-Triton as described for single labeling, then the SGs were incubated overnight in a 1:25 dilution of monovalent goat anti-rabbit IgG Fab fragments that also contained 10% NDS whereas the POs were incubated for 2 days in a 1:10 dilution of goat anti-rabbit IgG Fab fragments containing 10% NDS. For both tissues, this incubation with Fab fragments was done to sterically cover and immunologically convert the first primary antibody from rabbit to goat. After incubation with the Fab fragments, tissues were rinsed every hour for 5 h and then incubated overnight in a 1:300 dilution of FITC-conjugated donkey anti-goat IgG containing 10% NDS. Following this overnight incubation, tissues were rinsed as before then incubated in the second primary antibody for 1 (PO) or 2 (SG) days. After incubation in the second primary antibody, tissues were rinsed and then incubated overnight in a 1:300 dilution of Rhodamine Red X-conjugated donkey anti-rabbit IgG containing 10% NDS. All subsequent processing was the same as for the single labeling described above.

To assess the veracity of our double-labeling protocol, we conducted controls to determine the extent to which the Fab fragments were capable of sterically covering, and hence masking, the first primary antibody from detection via the second secondary antibody. Specifically, a single primary was applied to a tissue, sterically converted to goat viaincubation in Fab fragments and then incubated in the Rhodamine Red X-conjugated donkey anti-rabbit IgG. For all antibodies and all tissues in which double labeling was undertaken (3 preparations for each antibody in each tissue), a complete masking of the primary antibody was achieved, and hence little or no cross-talk between the first and second primary antibody sets should have occurred in our double-labeled preparations (data not shown).

Confocal and epifluorescence microscopy

After immunoprocessing, preparations were viewed and data were collected using one of two Bio-Rad MRC 600 laser scanning confocal microscopes (Bio-Rad Microscience Ltd., Hemel Hempstead, UK), a Bio-Rad Radiance 2000 laser scanning confocal microscope or a Nikon Eclipse E600 epifluorescence microscope. Descriptions of the hardware and software used for imaging on these systems are extensively documented in previous publications(Christie et al., 1997; Christie et al., 2003; Christie et al., 2004; Messinger et al., 2005a).

Figure production

For the production of figures, Bio-Rad.pic files collected using the MRC 600 or Radiance 2000 systems were converted to tif files using ImageJ (available free of charge from the National Institutes of Health at http://rsb.info.nih.gov/ij/). Individual micrographs and composite figures were produced from the tif files using a combination of ImageJ and Photoshop (version 7.0;Adobe Systems Inc., San Jose, California, USA). Schematic diagrams were produced using Canvas (version 8.0; Deneba Systems Inc., Miami, Florida, USA). It should be noted that the contrast and brightness of final figures were adjusted as needed to optimize the clarity of printed images.

The X-organ-sinus gland system

As in other brachyurans, the XO-SG system of Cancer species is located within the eyestalk (Figs 1, 2), with the XO (a loosely associated cluster of somata) situated in the proximoventral corner of the medulla terminalis and the SG (the neurosecretory terminals derived from the XO somata) present at the junction of the medulla interna and medulla externa(Cooke and Sullivan, 1982; Beltz, 1988; Fingerman, 1992). As stated earlier, isoforms of all members of the CHH family (i.e. CHH, MIH and MOIH)have been isolated from the XO-SG system of C. pagurus, as has an isoform of CPRP (Chung et al.,1996; Wainwright et al.,1996; Chung et al.,1998). Using antibodies generated against C. paguruspeptides (Webster et al., 1996; Wilcockson et al., 2002; this study), we immunolabeled eyestalks from C. antennarius, C. anthonyi, C. borealis, C. gracilis, C. irroratus, C. magister and C. productus to determine if the XO-SG system of these species also contains members of these peptide groups.

Immunohistochemical detection of CHH, MIH, MOIH and CPRP in the XO-SG system

For each antibody, intense labeling was evident in nerve terminals in the SG (Figs 3, 4) as well as in approximately three-dozen somata in the XO (Fig. 3; MIH labeling shown) in each species (N≥4 preparations for each antibody in each species). Within the SG, the nerve terminals labeled by the CHH and CPRP antisera(Fig. 4A,B) had a tendril-like appearance, whereas the terminals labeled by the MIH and MOIH antisera(Fig. 4C,D) appeared more oval and blob-like. Regardless of antibody or species, all labeling in the SG could be traced back to the immunopositive XO somata via labeled axons in the sinus gland tract (sgt), suggesting that these somata are the sole source of the SG staining.

Fig. 1.

Schematic representation of the nervous system of a brachyuran crab illustrating the relative locations of the known neuroendocrine organs of Cancer species. The central nervous system (CNS) of brachyurans is generally considered to consist of the supraesophageal (SoG) and fused thoracic ganglia, which are connected via the circumesophageal connectives (cocs). The optic nerves (optns) link the SoG with the ganglia of the eyestalks, the location of the neuroendocrine sinus gland (SG). Another well-known neuroendocrine site is the pericardial organ(PO), which is located in the pericardial chamber surrounding the heart. The POs consist of elaborations of the segmental nerves (sns), which project from the fused thoracic ganglia. Two additional neuroendocrine sites,the anterior cardiac plexus (ACP) and the anterior commissural organ (ACO),are contained within the stomatogastric nervous system (STNS), an offshoot from the CNS that overlies the foregut. The ACPs are located on the anterior cardiac nerves (acns) and the ACOs are located within the commissural ganglia (CoGs). For the sake of future discussion, the stomatogastric ganglion(STG) is also shown in this schematic. It should be noted that this illustration is not drawn to scale and that other portions of the nervous system have been excluded for the sake of simplicity.

Fig. 1.

Schematic representation of the nervous system of a brachyuran crab illustrating the relative locations of the known neuroendocrine organs of Cancer species. The central nervous system (CNS) of brachyurans is generally considered to consist of the supraesophageal (SoG) and fused thoracic ganglia, which are connected via the circumesophageal connectives (cocs). The optic nerves (optns) link the SoG with the ganglia of the eyestalks, the location of the neuroendocrine sinus gland (SG). Another well-known neuroendocrine site is the pericardial organ(PO), which is located in the pericardial chamber surrounding the heart. The POs consist of elaborations of the segmental nerves (sns), which project from the fused thoracic ganglia. Two additional neuroendocrine sites,the anterior cardiac plexus (ACP) and the anterior commissural organ (ACO),are contained within the stomatogastric nervous system (STNS), an offshoot from the CNS that overlies the foregut. The ACPs are located on the anterior cardiac nerves (acns) and the ACOs are located within the commissural ganglia (CoGs). For the sake of future discussion, the stomatogastric ganglion(STG) is also shown in this schematic. It should be noted that this illustration is not drawn to scale and that other portions of the nervous system have been excluded for the sake of simplicity.

Fig. 2.

Schematic diagram of the optic ganglia of Cancer species highlighting the location and organization of the X-organ-sinus gland (XO-SG)system. The nervous system contained within the eyestalk consists of several distinct regions, including the medulla terminalis (MT), the medulla interna(MI), the medulla externa (ME), the lamina ganglionaris (LG) and the retina. This system of ganglia is connected to the supraesophageal ganglion (SoG) via the optic nerve (optn). Located in the MT is a loosely associated collection of neurosecretory somata that are collectively termed the X-organ (XO). The release site of hormones produced by these somata is the sinus gland (SG), which is located at the junction of the MI and ME. The sinus gland tract (sgt) links the XO and SG.

Fig. 2.

Schematic diagram of the optic ganglia of Cancer species highlighting the location and organization of the X-organ-sinus gland (XO-SG)system. The nervous system contained within the eyestalk consists of several distinct regions, including the medulla terminalis (MT), the medulla interna(MI), the medulla externa (ME), the lamina ganglionaris (LG) and the retina. This system of ganglia is connected to the supraesophageal ganglion (SoG) via the optic nerve (optn). Located in the MT is a loosely associated collection of neurosecretory somata that are collectively termed the X-organ (XO). The release site of hormones produced by these somata is the sinus gland (SG), which is located at the junction of the MI and ME. The sinus gland tract (sgt) links the XO and SG.

Patterns of co-localization in the XO-SG system of C. productus

As just described, the SG terminals labeled by the CHH and CPRP antibodies were tendril-like whereas those labeled by the MIH and MOIH antibodies were distinctly blob-like in appearance in all species(Fig. 4). For C. productus, we quantified and compared the number of somata labeled by each antibody. In this species, the numbers of XO somata labeled by the CHH and CPRP antibodies were essentially identical [anti-CHH: mean=40±1.90 somata/XO, range=36-49, 6 preparations; anti-CPRP: mean=41±2.12 somata/XO, range= 37-47, 4 preparations (Student's t-test, P>0.05)] as were the number of somata labeled by the MIH and MOIH sera [anti-MIH: mean=32±1.38 somata/XO, range=25-36, 8 preparations;anti-MOIH: mean=29±1.92 somata/XO, range= 22-35, 6 preparations(Student's t-test, P>0.05)]. By contrast, the number of somata stained by either the CHH or CPRP antibody was statistically different from those labeled by either the MIH or MOIH antibody (ANOVA, P<0.0005; Tukey's test, P<0.05). Collectively, these findings suggested the potential for CHH/CPRP and MIH/MOIH co-localization in the XO-SG system, and simultaneously implied that the former two peptides were contained in subpopulations of neurons distinct from those producing the latter two peptides. To directly assess the patterns of co-localization present among the CHH family members and CPRP in C. productus, we developed a double-immunohistochemistry method utilizing primary antisera generated in a common host species and conducted double-immunolabeling for all possible combinations of the four antisera using this method.

Fig. 3.

Moult-inhibiting hormone (MIH)-like immunoreactivity in the X-organ-sinus gland (XO-SG) system of Cancer productus. (A) MIH-like labeling in the SG. Whole-mount immunolabeling of the nervous system contained within the eyestalk with antibody to MIH consistently stained nerve terminals in the SG. This labeling could be unambiguously traced via immunopositive axons in the sinus gland tract (sgt) to somata in the XO. This micrograph is a brightest pixel projection of 29 optical sections taken at 1.95 μm intervals. (B) MIH-like labeling in the XO. Immunoprocessing using anti-MIH consistently labeled 30 or so somata in the XO. This micrograph is a brightest pixel projection of 19 optical sections taken at 1.95 μm intervals. A and B are taken from the same preparation and are shown at the same magnification. Scale bar in B, 150 μm.

Fig. 3.

Moult-inhibiting hormone (MIH)-like immunoreactivity in the X-organ-sinus gland (XO-SG) system of Cancer productus. (A) MIH-like labeling in the SG. Whole-mount immunolabeling of the nervous system contained within the eyestalk with antibody to MIH consistently stained nerve terminals in the SG. This labeling could be unambiguously traced via immunopositive axons in the sinus gland tract (sgt) to somata in the XO. This micrograph is a brightest pixel projection of 29 optical sections taken at 1.95 μm intervals. (B) MIH-like labeling in the XO. Immunoprocessing using anti-MIH consistently labeled 30 or so somata in the XO. This micrograph is a brightest pixel projection of 19 optical sections taken at 1.95 μm intervals. A and B are taken from the same preparation and are shown at the same magnification. Scale bar in B, 150 μm.

As Fig. 5 shows,co-localization of CHH and CPRP as well as of MIH and MOIH (shown in Fig. 5A) was seen in XO somata in each of the C. productus eyestalks labeled using these antibody combinations (6 preparations for each combination). In none of the eyestalks labeled with any other combination of primary antisera was any co-localization evident in XO somata (Fig. 5B;6 preparations for each antibody combination). All somata that expressed CHH-like immunoreactivity were co-labeled by the CPRP antibody, a finding that is not surprising given their production from the same preprohormone in C. productus (de la Iglesia et al.,2005; Hsu et al.,2005a). Interestingly, complete co-localization was also seen in the double-labels pairing of the MIH and MOIH antibodies(Fig. 5A), suggesting that all XO somata that produce MIH also produce MOIH.

The pericardial organ

Another crustacean neuroendocrine system known to contain CHH-related peptides is the PO (Dircksen et al.,2001; Wilcockson et al.,2002; Fu et al.,2005b). In brachyuran species, including C. productus,the PO consists of two or more longitudinal nerve trunks that are connected by vertical nerve bars (Fig. 1). The trunks and bars that form each PO are elaborations of the segmental nerves(sns) that originate from the fused thoracic ganglia(Fig. 1). Both intrinsically and extrinsically located cell bodies contribute to the release terminals that lie under the epineurium that covers the PO(Cooke and Sullivan, 1982; Beltz, 1988; Fingerman, 1992). As stated earlier, both CHH and CPRP have been detected in the PO of C. pagurususing immunohistochemistry (Wilcockson et al., 2002). In this species, all labeling for both peptides originates from three intrinsic somata located in or near the anterior bar of the PO (Wilcockson et al.,2002). To assess if CHH-, CPRP-, MIH- or MOIH-like peptides were present in the POs of other species of Cancer and to determine their distributions and patterns of co-localization in C. productus, we immunoprocessed this tissue with the same set of antisera used in our mapping of these substances in the SG.

Immunohistochemical detection of CHH, CPRP and MOIH, but not MIH in the PO

Immunoprocessing of POs with the CHH, CPRP and MOIH antisera consistently produced labeling within this tissue in each of the Cancer species investigated (Fig. 6; N≥3 preparations for each antibody in each species). By contrast,no staining was seen when POs of any crab were processed with the MIH antiserum (N≥3 preparations for each species). For both the CHH and CPRP antibodies, labeling in the POs of each species consisted of up to three somata (usually two bipolar and one multipolar) in the anterior bar or in the sns just anterior to it, as well as fine processes and peripherally located nerve terminals covering extensive regions of the anterior and posterior bars and portions of all three nerve trunks(Fig. 6A,B). For both antibodies, the vast majority of the immunopositive fine fibers and nerve terminals appeared to originate from the arborizations of the intrinsic somata. In each of the CHH-immunopositive somata, labeling was cytoplasmic,uniformly diffuse and often extended for a considerable distance into the axons emanating from these cell bodies. By contrast, labeling in the CPRP-immunopositive somata in all species was cytoplasmic and granular, with little immunoreactivity evident in their projecting axons.

Fig. 4.

The distribution of crustacean hyperglycemic hormone (CHH)-like and CHH precursor-related peptide (CPRP)-like labels in the sinus gland (SG) differ from those of moult-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH) in Cancer productus. Within the SG, terminals labeled by the CHH and CPRP antibodies had the appearance of flocculent tendrils,whereas those revealed by the MIH and MOIH antibodies appeared more oval and blob-like. (A) CHH-like immunopositive terminals in the SG. (B) CPRP-like immunopositive terminals in the SG. (C) MIH-like immunopositive terminals in the SG. (D) MOIH-like immunopositive terminals in the SG. All micrographs are single optical sections shown at the same magnification. Scale bar in D, 50μm.

Fig. 4.

The distribution of crustacean hyperglycemic hormone (CHH)-like and CHH precursor-related peptide (CPRP)-like labels in the sinus gland (SG) differ from those of moult-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH) in Cancer productus. Within the SG, terminals labeled by the CHH and CPRP antibodies had the appearance of flocculent tendrils,whereas those revealed by the MIH and MOIH antibodies appeared more oval and blob-like. (A) CHH-like immunopositive terminals in the SG. (B) CPRP-like immunopositive terminals in the SG. (C) MIH-like immunopositive terminals in the SG. (D) MOIH-like immunopositive terminals in the SG. All micrographs are single optical sections shown at the same magnification. Scale bar in D, 50μm.

In all animals, MOIH-like staining was weak in the axons and in patches of superficially located nerve terminals in both the anterior and posterior bars,as well as in the nerve trunks (Fig. 6D). Whereas no MOIH-like staining was seen in any intrinsic PO somata in any of the species, approximately four MOIH-immunopositive cell bodies were present in the fused thoracic ganglia of C. productus (3 preparations; data not shown). As the thoracic ganglia are known to be the source of many of the inputs to the PO(Cooke and Sullivan, 1982; Beltz, 1988; Fingerman, 1992), these somata are one potential source of the MOIH staining seen in the PO of this species,and possibly the others as well.

Patterns of co-localization in the PO of C. productus

In all of the species investigated, the distribution of CHH- and CPRP-like immunoreactivity in the POs was essentially identical with the exception of the appearance of labeling in the three intrinsic somata. To determine the extent of colocalization of the two peptides in C. productus,double-immunolabeling, similar to that in the SG, was undertaken. As Fig. 7 shows, a complete overlap of the two immunoreactivities was found in this tissue (6 preparations). CHH/MOIH (6 preparations) and CPRP/MOIH (6 preparations)double-labelings were also conducted. However, the relative weakness of the MOIH label with respect to either the CHH or CPRP labels made assessment of co-localization difficult. In most POs, the immunoreactivities appeared distinct, whereas in others, weak co-localization in a small population of terminals was evident (data not shown). Given the complete overlap of the CHH and CPRP labels and the fact that they appeared to be derived from a set of somata not labeled by the MOIH antiserum, we believe that the profiles labeled by the MOIH antibody are distinct from those containing CHH/CPRP.

Fig. 5.

Double-immunolabeling of the X-organ (XO) with antisera to crustacean hyperglycemic hormone (CHH)-family members and CHH precursor-related peptide(CPRP) in Cancer productus. (A1-3) Moult-inhibiting hormone(MIH)/mandibular organ-inhibiting hormone (MOIH) double labeling in the XO. This series of micrographs consists of pseudo-colored single optical sections collected from a single focal plane in an XO labeled with both anti-MIH (A1,pseudo-colored green) and anti-MOIH (A2, pseudo-colored red). When A1 and A2 were merged (A3), complete overlap in the labeled structures was revealed(i.e. yellow, but not green or red, coloration is seen in all immunopositive structures in the micrograph), suggesting that the MIH- and MOIH-like labels are contained in a common set of XO somata. The same result was seen in preparations pairing anti-CHH/anti-CPRP (data not shown). (B1-3) MIH/CPRP double labeling in the XO. This series of micrographs consists of pseudo-colored single optical sections collected from a single focal plane in an XO labeled with both anti-MIH (B1, pseudo-colored green) and anti-CPRP (B2,pseudo-colored red). When B1 and B2 were merged (B3), no overlap in the labeled structures was revealed (i.e. a lack of yellow colored structures in the micrograph), suggesting that the MIH- and CPRP-like labels are contained in distinct sets of XO somata. Though not shown, the same result was seen in preparations pairing anti-MIH/anti-CHH, anti-MOIH/anti-CHH and anti-MOIH/anti-CPRP. A1,2 and B1,2 are all shown at the same scale. Likewise A3 and B3 are shown at the same magnification. Scale bars in B2 and B3, 100μm.

Fig. 5.

Double-immunolabeling of the X-organ (XO) with antisera to crustacean hyperglycemic hormone (CHH)-family members and CHH precursor-related peptide(CPRP) in Cancer productus. (A1-3) Moult-inhibiting hormone(MIH)/mandibular organ-inhibiting hormone (MOIH) double labeling in the XO. This series of micrographs consists of pseudo-colored single optical sections collected from a single focal plane in an XO labeled with both anti-MIH (A1,pseudo-colored green) and anti-MOIH (A2, pseudo-colored red). When A1 and A2 were merged (A3), complete overlap in the labeled structures was revealed(i.e. yellow, but not green or red, coloration is seen in all immunopositive structures in the micrograph), suggesting that the MIH- and MOIH-like labels are contained in a common set of XO somata. The same result was seen in preparations pairing anti-CHH/anti-CPRP (data not shown). (B1-3) MIH/CPRP double labeling in the XO. This series of micrographs consists of pseudo-colored single optical sections collected from a single focal plane in an XO labeled with both anti-MIH (B1, pseudo-colored green) and anti-CPRP (B2,pseudo-colored red). When B1 and B2 were merged (B3), no overlap in the labeled structures was revealed (i.e. a lack of yellow colored structures in the micrograph), suggesting that the MIH- and CPRP-like labels are contained in distinct sets of XO somata. Though not shown, the same result was seen in preparations pairing anti-MIH/anti-CHH, anti-MOIH/anti-CHH and anti-MOIH/anti-CPRP. A1,2 and B1,2 are all shown at the same scale. Likewise A3 and B3 are shown at the same magnification. Scale bars in B2 and B3, 100μm.

Fig. 6.

Crustacean hyperglycemic hormone (CHH)-like, CHH precursor-related peptide(CPRP)-like and mandibular organ-inhibiting hormone (MOIH)-like labeling in the segmental nerves (sn) and pericardial organ (PO) of Cancer productus. (A) CHH-like labeling in the sn anterior to the PO. Two bipolar somata were labeled with the CHH antibody in the anterior bar of the PO or in the sns just anterior to it (one stained soma shown;arrow). Staining in these somata was uniform in its distribution, with the label filling the axons emanating from the cell bodies. As this micrograph shows, many small diameter axons and superficially located nerve terminals were also labeled by the CHH antibody in the sn. This micrograph is a brightest pixel projection of 34 optical sections taken at 1.95 μm intervals. (B) CPRP-like labeling in the sn anterior to the PO. As with the CHH antibody, two bipolar somata were labeled in the sn/anterior bar area of the PO by the CPRP antibody (two shown;arrows). Here, labeling within the somata was granular in appearance, and little immunoreactivity was present in the axons emanating from the cell bodies. Similar to the CHH label, many small diameter axons and superficially located nerve terminals in the sn were stained by the CPRP antibody. This micrograph is a brightest pixel projection of 21 optical sections taken at 1.95 μm intervals. (C) CHH-like labeling in the anterior bar of the PO. In addition to the bipolar neurons seen in the sn, a single multipolar soma was routinely labeled by the CHH antibody in the anterior bar of the PO (arrow). As with the other PO somata labeled by this antiserum,staining in this cell body was uniform and diffuse in appearance. In addition to the soma, an extensive network of fine fibers, as well as superficial nerve terminals, were labeled by the CHH antibody within the anterior bar of the PO. Although not shown, the CPRP antibody produced an identical pattern of labeling in this portion of the PO, with the exception that labeling in the multipolar soma was granular in appearance rather than uniformly diffuse. This micrograph is a brightest pixel projection of 37 optical sections taken at 1.95 μm intervals. (D) MOIH-like labeling in the anterior bar of the PO. As with the CHH and CPRP labels, the MOIH antibody stained a network of fine fibers and superficially located nerve terminals in the anterior bar of the PO, though the immunoreactivity was less extensive and weaker than with the other sera. However, no somata were labeled by the MOIH antiserum, nor were any labeled in the sns near the anterior bar. This micrograph is a brightest pixel projection of 26 optical sections taken at 1.95 μm intervals. All scale bars, 100 μm.

Fig. 6.

Crustacean hyperglycemic hormone (CHH)-like, CHH precursor-related peptide(CPRP)-like and mandibular organ-inhibiting hormone (MOIH)-like labeling in the segmental nerves (sn) and pericardial organ (PO) of Cancer productus. (A) CHH-like labeling in the sn anterior to the PO. Two bipolar somata were labeled with the CHH antibody in the anterior bar of the PO or in the sns just anterior to it (one stained soma shown;arrow). Staining in these somata was uniform in its distribution, with the label filling the axons emanating from the cell bodies. As this micrograph shows, many small diameter axons and superficially located nerve terminals were also labeled by the CHH antibody in the sn. This micrograph is a brightest pixel projection of 34 optical sections taken at 1.95 μm intervals. (B) CPRP-like labeling in the sn anterior to the PO. As with the CHH antibody, two bipolar somata were labeled in the sn/anterior bar area of the PO by the CPRP antibody (two shown;arrows). Here, labeling within the somata was granular in appearance, and little immunoreactivity was present in the axons emanating from the cell bodies. Similar to the CHH label, many small diameter axons and superficially located nerve terminals in the sn were stained by the CPRP antibody. This micrograph is a brightest pixel projection of 21 optical sections taken at 1.95 μm intervals. (C) CHH-like labeling in the anterior bar of the PO. In addition to the bipolar neurons seen in the sn, a single multipolar soma was routinely labeled by the CHH antibody in the anterior bar of the PO (arrow). As with the other PO somata labeled by this antiserum,staining in this cell body was uniform and diffuse in appearance. In addition to the soma, an extensive network of fine fibers, as well as superficial nerve terminals, were labeled by the CHH antibody within the anterior bar of the PO. Although not shown, the CPRP antibody produced an identical pattern of labeling in this portion of the PO, with the exception that labeling in the multipolar soma was granular in appearance rather than uniformly diffuse. This micrograph is a brightest pixel projection of 37 optical sections taken at 1.95 μm intervals. (D) MOIH-like labeling in the anterior bar of the PO. As with the CHH and CPRP labels, the MOIH antibody stained a network of fine fibers and superficially located nerve terminals in the anterior bar of the PO, though the immunoreactivity was less extensive and weaker than with the other sera. However, no somata were labeled by the MOIH antiserum, nor were any labeled in the sns near the anterior bar. This micrograph is a brightest pixel projection of 26 optical sections taken at 1.95 μm intervals. All scale bars, 100 μm.

The stomatogastric nervous system

The STNS (Fig. 1) of decapod species is situated over the foregut and is responsible for controlling the movement of food items through this portion of the digestive tract(Selverston and Moulins, 1987; Harris-Warrick et al., 1992). Recently, we identified two neuroendocrine organs within the STNS of C. productus (Christie et al.,2004; Christie and Messinger,2005; Messinger et al.,2005a; Messinger et al.,2005b). One of these, the ACP, is located on the anterior cardiac nerves (acns) overlying the cardiac sac region of the foregut(Christie et al., 2004)(Fig. 1). This site consists of a dense collection of nerve terminals located just below the epineurium covering the acns (Christie et al., 2004), with all innervation to the site originating from four somata, one pair in each commissural ganglion (CoG)(Christie and Messinger, 2005; Messinger et al., 2005b). Based on their innervation of the ACP, these neuron pairs were named anterior commissural neurons 1 and 2 (ACN1/2)(Christie and Messinger, 2005; Messinger et al., 2005b). The other site, the ACO, is located within the anterior medial quadrant of the CoG(Fig. 1) and consists of a dense collection of nerve terminals that envelop an extensive network of hemolymph lacunae that invaginate deep into the ganglion(Messinger et al., 2005a). The origin of the fibers that produce the ACO remain unknown, though somata in the thoracic ganglia are likely candidates(Messinger et al., 2005a). Both the ACP and ACO are also present in the STNSs of C. antennarius, C. anthonyi, C. borealis, C. gracilis, C. irroratus and C. magister (A.E.C., D.I.M. and E. Savage, unpublished observations). To determine if the ACP and/or ACO contain any of the CHH-related or CPRP-like peptides, we immunoprocessed the STNSs from each Cancer species with the same complement of antisera used to map the SG and PO.

Fig. 7.

Co-localization of crustacean hyperglycemic hormone (CHH)-like and CHH precursor-related peptide (CPRP)-like immunoreactivity in the pericardial organ (PO) of Cancer productus. This series of micrographs consists of pseudo-colored single optical sections collected from a single focal plane from a PO labeled with both anti-CPRP (A, pseudo-colored green) and anti-CHH(B, pseudo-colored red). When A and B are merged (C), complete overlap in the labeled structures was revealed (i.e. yellow, but not green or red, coloration is seen in all immunopositive processes in the micrograph), suggesting that the CPRP- and CHH-like labels are contained in a common set of structures in the PO. A and B are shown at the same magnification. Scale bars in B and C, 25μm.

Fig. 7.

Co-localization of crustacean hyperglycemic hormone (CHH)-like and CHH precursor-related peptide (CPRP)-like immunoreactivity in the pericardial organ (PO) of Cancer productus. This series of micrographs consists of pseudo-colored single optical sections collected from a single focal plane from a PO labeled with both anti-CPRP (A, pseudo-colored green) and anti-CHH(B, pseudo-colored red). When A and B are merged (C), complete overlap in the labeled structures was revealed (i.e. yellow, but not green or red, coloration is seen in all immunopositive processes in the micrograph), suggesting that the CPRP- and CHH-like labels are contained in a common set of structures in the PO. A and B are shown at the same magnification. Scale bars in B and C, 25μm.

Immunohistochemical detection of MOIH, but not CHH, MIH or CPRP in the ACP

In each of the investigated species, no immunoreactivity was seen anywhere in the STNSs processed with the CHH, CPRP or MIH antisera (N≥3 preparations for each antibody in each species). By contrast, in all crabs,extensive labeling was produced throughout using the MOIH antibody(N≥3; Figs 8, 9, 10), including staining of the ACP (Fig. 8) but not the ACO(data not shown).

Regardless of species and in all preparations examined, extensive MOIH-like immunoreactivity was seen in the nerve terminals constituting the ACP(Fig. 8A). In each ACP, the immunopositive terminals originated from four axons projecting from the stomatogastric nerve (stn; Fig. 8B), two traveling from the right superior esophageal nerve(son) and two from the left son(Fig. 8C). Although it was not possible to follow the immunoreactivity in the axons back to their somata of origin, they probably originate from two large (approximately 100 μm in major cross-sectional diameter), weakly immunopositive cell bodies located at the junction of the son and CoG, as these somata are of the same size and location as the ACN1/2s, which are known to be the sole source of innervation to the ACP (Christie et al.,2004; Christie and Messinger,2005; Messinger et al.,2005b).

In addition to the staining just described, in all species the MOIH antiserum also labeled an extensive neuropil in the stomatogastric ganglion that appeared to originate from both the arborizations of two intrinsic immunopositive somata and from the arborizations of projection neurons traveling to the ganglion via the stn(Fig. 9). This, and all other MOIH-like labeling in the STNS, is summarized schematically in Fig. 10.

Members of the CHH family and CPRP are differentially distributed between the neuroendocrine organs of Cancer species

In this study we used antisera generated against Capa-CHH II, Capa-MIH,Capa-MOIH I and Capa-CPRP to assay the neuroendocrine organs of the crabs C. antennarius, C. anthonyi, C. borealis, C. gracilis, C. irroratus, C. magister and C. productus for the presence of isoforms of these peptides. Our results show that in all species, moieties immunologically related to the peptides used for antibody production are present in the SG, PO and ACP, but not in the ACO. Moreover, the patterns of immunoreactivity revealed by these antisera suggest that the native CHH family members and CPRPs are differentially distributed between the neuroendocrine organs of these species, with all members of the CHH family and CPRP present in the SG;CHH, MOIH and CPRP, but not MIH, present in the PO; and only MOIH present in the ACP.

As are all antibodies, the sera used in our study are biological and not chemical reagents and thus are subject to potential cross-reactions with antigens other than that to which they were raised(Saper and Sawchenko, 2003). As Table 1 shows, significant sequence homology is present between the C. pagurus CHH-family members used for antibody production. Despite the sequence similarity of the antigens, the distinct patterns of labeling seen with each antibody strongly suggest that they are detecting different complements of peptides. Furthermore, in all tissues, only adsorption of an antibody by the peptide used for its production completely eliminated labeling. Thus, we feel confident that the staining presented here is an accurate reflection of the distributions of CHH-family members and CPRPs in the neuroendocrine organs of Cancer species.

Fig. 8.

Mandibular organ-inhibiting hormone (MOIH)-like labeling in the anterior cardiac plexus (ACP) of Cancer productus. (A) MOIH-like labeling in the ACP. Immunoprocessing of the stomatogastric nervous system with the MOIH antibody consistently produced labeling in the ACP. This micrograph is a brightest pixel projection of 16 optical sections taken at 1.95 μm intervals. (B) MOIH-like labeling at the junction of the anterior cardiac(acn) and stomatogastric (stn) nerves. Labeling in each ACP arose from four axons that projected into each acn from the stn (axons in the stn denoted by arrows). This micrograph is a brightest pixel projection of 14 optical sections taken at 1.95 μm intervals. (C) MOIH-like labeling at the junction of the stn and the superior esophageal (son) nerves. The four axons that are the source of the MOIH-like labeling in the ACP can be traced through the stn to its junction with the paired sons. Here, two of the four axons enter the left son and two the right son. Within each son, MOIH-like labeling in the axons became weak near the junction of the nerve with the commissural ganglion, and further tracing was not possible from immunoreactivity alone (not shown). This micrograph is a brightest pixel projection of 17 optical sections taken at 1.95 μm intervals. Both B and C are shown at the same magnification. Scale bars in A and C, 75 μm.

Fig. 8.

Mandibular organ-inhibiting hormone (MOIH)-like labeling in the anterior cardiac plexus (ACP) of Cancer productus. (A) MOIH-like labeling in the ACP. Immunoprocessing of the stomatogastric nervous system with the MOIH antibody consistently produced labeling in the ACP. This micrograph is a brightest pixel projection of 16 optical sections taken at 1.95 μm intervals. (B) MOIH-like labeling at the junction of the anterior cardiac(acn) and stomatogastric (stn) nerves. Labeling in each ACP arose from four axons that projected into each acn from the stn (axons in the stn denoted by arrows). This micrograph is a brightest pixel projection of 14 optical sections taken at 1.95 μm intervals. (C) MOIH-like labeling at the junction of the stn and the superior esophageal (son) nerves. The four axons that are the source of the MOIH-like labeling in the ACP can be traced through the stn to its junction with the paired sons. Here, two of the four axons enter the left son and two the right son. Within each son, MOIH-like labeling in the axons became weak near the junction of the nerve with the commissural ganglion, and further tracing was not possible from immunoreactivity alone (not shown). This micrograph is a brightest pixel projection of 17 optical sections taken at 1.95 μm intervals. Both B and C are shown at the same magnification. Scale bars in A and C, 75 μm.

Members of the CHH family and CPRP are differentially distributed within the XO-SG system as well as within the PO

In addition to observing a differential distribution of CHH-family peptides and CPRP among the neuroendocrine organs of various Cancer species,we also found that these peptides are differentially distributed within both the XO-SG and PO of at least C. productus. Using a protocol that allowed double labeling of whole-mount tissue with primary antibodies generated in the same host species, we found complete overlap of labeling for CHH and CPRP in both the XO-SG and the PO. Similarly, a complete overlap of labeling was seen for MIH and MOIH in the XO-SG, but in a different set of neurons than those containing CHH and CPRP. MOIH was also present in the PO,again in a set of structures distinct from those possessing CHH and CPRP.

Ours is the first study to show co-localization of CHH- and CPRP-like immunoreactivities in either the XO-SG or the PO, but the association of these two peptides is not surprising given that both peptides are produced from a common preprohormone in C. productus(de la Iglesia et al., 2005; Hsu et al., 2005a). More interesting is the complete overlap of the MIH and MOIH labels in the XO-SG,which are encoded by different genes (Lu et al., 2000). As stated earlier, the peptides used for the production of the MIH and MOIH antibodies exhibit significant amino acid sequence identity (approximately 61%; Table 1), and thus it is possible that each serum was cross-reacting with a common peptide. However, we were unable to completely block the MIH staining in the XO-SG with any peptide other than Capa-MIH, including Capa-MOIH I. Similarly, the XO-SG label produced by the MOIH antibody was suppressed only after adsorption by Capa-MOIH and not by Capa-MIH. These results suggest that the MIH- and MOIH-like labelings were specific for their respective antigens. Clearly, molecular and/or biochemical characterization of the complement of native C. productus CHH-family peptides will be necessary to resolve this issue unambiguously.

Fig. 9.

Mandibular organ-inhibiting hormone (MOIH)-like labeling in the stomatogastric ganglion (STG) of Cancer productus. (A) MOIH-like labeling in the STG. Among the regions of the stomatogastric nervous system labeled by the MOIH antibody was the STG. Here, staining was present in two intrinsic somata as well as in the neuropil within the ganglion. The immunopositive neuropil appears to originate from both the arborizations of the intrinsic immunopositive somata as well as from the arborizations of projection neurons, which send axons to the STG via the stomatogastric nerve (stn). (B) Higher magnification image of the MOIH-immunopositive somata shown in A. Note that the staining in these cell bodies is cytoplasmic and distinctly punctate. The micrographs shown in both A and B are single optical sections. Scale bars in A and B, 75 μm.

Fig. 9.

Mandibular organ-inhibiting hormone (MOIH)-like labeling in the stomatogastric ganglion (STG) of Cancer productus. (A) MOIH-like labeling in the STG. Among the regions of the stomatogastric nervous system labeled by the MOIH antibody was the STG. Here, staining was present in two intrinsic somata as well as in the neuropil within the ganglion. The immunopositive neuropil appears to originate from both the arborizations of the intrinsic immunopositive somata as well as from the arborizations of projection neurons, which send axons to the STG via the stomatogastric nerve (stn). (B) Higher magnification image of the MOIH-immunopositive somata shown in A. Note that the staining in these cell bodies is cytoplasmic and distinctly punctate. The micrographs shown in both A and B are single optical sections. Scale bars in A and B, 75 μm.

Fig. 10.

Schematic representation of mandibular organ-inhibiting hormone (MOIH)-like immunoreactivity in the stomatogastric nervous system (STNS) of Cancer species. In this diagram, filled circles represent immunopositive somata, thick lines within nerves represent immunopositive axons and tangles of thin lines represent regions of immunopositive neuropil or neuroendocrine release sites. In addition to labeling the anterior cardiac plexus (ACP) and the axons innervating it, MOIH-like labeling was also evident in other regions of the STNS. In brief, approximately a dozen somata were labeled in each commissural ganglion (CoG), as were two in the stomatogastric ganglion (STG). No immunopositive somata were present in the esophageal ganglion (OG) nor were any seen in the nerves of the STNS. Immunopositive neuropil was present in the CoGs and the STG. Extraganglionic neuropil was present in the superior esophageal nerves (sons; most commonly in the vicinity of the dorsal posterior esophageal nerve (dpon)], at the junction of the sons, the esophageal nerve (on) and the stomatogastric nerve (stn), as well as in the stn proper[commonly near the insertion point of the anterior cardiac nerves(acns)]. The immunopositive neuropil in the STG probably originates from both the arborizations of the intrinsic somata as well as from the arborizations of approximately six axons projecting from the stn(approximately three from each son). Immunopositive axons were also present in the circumesophageal connectives (cocs), which link the STNS to the supraesophageal and thoracic ganglia, as well as occasionally in the anterior lateral nerves (alns; one axon in each aln)that emanate from the STG and innervate muscles of the gastric mill region of the foregut.

Fig. 10.

Schematic representation of mandibular organ-inhibiting hormone (MOIH)-like immunoreactivity in the stomatogastric nervous system (STNS) of Cancer species. In this diagram, filled circles represent immunopositive somata, thick lines within nerves represent immunopositive axons and tangles of thin lines represent regions of immunopositive neuropil or neuroendocrine release sites. In addition to labeling the anterior cardiac plexus (ACP) and the axons innervating it, MOIH-like labeling was also evident in other regions of the STNS. In brief, approximately a dozen somata were labeled in each commissural ganglion (CoG), as were two in the stomatogastric ganglion (STG). No immunopositive somata were present in the esophageal ganglion (OG) nor were any seen in the nerves of the STNS. Immunopositive neuropil was present in the CoGs and the STG. Extraganglionic neuropil was present in the superior esophageal nerves (sons; most commonly in the vicinity of the dorsal posterior esophageal nerve (dpon)], at the junction of the sons, the esophageal nerve (on) and the stomatogastric nerve (stn), as well as in the stn proper[commonly near the insertion point of the anterior cardiac nerves(acns)]. The immunopositive neuropil in the STG probably originates from both the arborizations of the intrinsic somata as well as from the arborizations of approximately six axons projecting from the stn(approximately three from each son). Immunopositive axons were also present in the circumesophageal connectives (cocs), which link the STNS to the supraesophageal and thoracic ganglia, as well as occasionally in the anterior lateral nerves (alns; one axon in each aln)that emanate from the STG and innervate muscles of the gastric mill region of the foregut.

Biologically, it is interesting to postulate why MIH and MOIH are present in the same set of XO somata. If MIH and MOIH are contained in the same secretory vesicles, then all signals triggering their release would probably result in the coordinated inhibition of both somatic growth (via the inhibition of steroid production in the Y-organ by MIH) and gonadal growth(via the inhibition of methyl farnesoate production in the mandibular organ by MOIH). If, however, the two peptides are contained in distinct sets of vesicles, then the two peptides might well be released in response to distinct physiological cues. Clearly, the former versus the latter situation would have profound physiological consequences for an animal, and with future studies it will be interesting to determine the physiological significance of the co-localized peptides.

Do multiple tissue localizations imply pleiotropic functions for the CHH family members and CPRP in Cancer species?

As just discussed, we have found that the CHH, MOIH and CPRP are each present in at least two neuroendocrine sites. With such multiple tissue localizations, the potential clearly exists for distinct cues triggering the release of a given hormone from each site. In addition, distinct isoforms of the CHH, MOIH and CPRP may be present in each tissue, as has been shown for CHH in several crab species (Dircksen et al., 2001; Toullec et al.,2006). In Carcinus maenas, the CHH of the PO, unlike its SG counterpart, does not possess hyperglycemic activity nor does it appear to inhibit ecdysteroid synthesis (Dircksen et al., 2001). Instead, this CHH isoform is proposed to serve either a sensory or local modulatory role in this species(Dircksen et al., 2001). Although unproven, the differential distributions of CHH, CPRP and MOIH revealed in our study suggest the possibility of pleiotropic functions for these peptides in Cancer species (e.g. their classically ascribed functions versus possible roles as locally released neuromodulators). As additional studies are undertaken, it will be interesting to see if this hypothesis is borne out.

Conclusions

In conclusion, the data we present show that members of the CHH family and CPRPs are differentially distributed both between and within the neuroendocrine organs of crabs of the genus Cancer. These results represent the most complete immunohistochemical survey of CHH-family peptides and CPRP in the neuroendocrine organs of crustaceans and provide a foundation for future studies directed at the identification of the native isoforms present in each species and tissue, as well as the elucidation of their physiological roles in members of this genus. The finding of CHH, CPRP and MOIH-like labeling in multiple neuroendocrine tissues suggest that these peptides may be released into the circulatory system in response to varied cues and that some of their isoforms may possess distinct tissue-specific functions.

    List of abbreviations
     
  • acn

    anterior cardiac nerve

  •  
  • ACO

    anterior commissural organ

  •  
  • ACP

    anterior cardiac plexus

  •  
  • CHH

    crustacean hyperglycemic hormone

  •  
  • CoG

    commissural ganglion

  •  
  • CPRP

    crustacean hyperglycemic hormone precursor-related peptide

  •  
  • GIH

    gonad-inhibiting hormone

  •  
  • MIH

    moult-inhibiting hormone

  •  
  • MOIH

    mandibular organ-inhibiting hormone

  •  
  • PO

    pericardial organ

  •  
  • SG

    sinus gland

  •  
  • sn

    segmental nerve

  •  
  • son

    superior esophageal nerve

  •  
  • STG

    stomatogastric ganglion

  •  
  • stn

    stomatogastric nerve

  •  
  • STNS

    stomatogastric nervous system

  •  
  • VIH

    vitellogenesis-inhibiting hormone

  •  
  • XO-SG

    X-organ-sinus gland system

John Edwards, Lilliam Ambroggio, Rachel Latham, Minhui Lin, Christina Ngo and Trendafil Halatchev are thanked for their help with some of the immunohistochemistry and confocal microscopy. John Weller, Curtis Easton and Christopher Goiney are thanked for their assistance in animal collection. Dr William Stegeman (Jackson ImmunoResearch Laboratories) is thanked for his help in designing and trouble-shooting the double-immunohistochemical studies as well as providing the reagents necessary for conducting them. Y.A.H. gratefully acknowledges financial support from the University of Washington Graduate Opportunities & Minority Advancement Program for Graduate Opportunity Program Fellowship. Some of the animals used here were collected under the auspices of Washington Department of Fish and Wildlife Scientific Collection Permits #03-018a and #04-021 (to A.E.C.).

Basu, A. C. and Kravitz, E. A. (
2003
). Morphology and monoaminergic modulation of crustacean hyperglycemic hormone-like immunoreactive neurons in the lobster nervous system.
J. Neurocytol.
32
,
253
-263.
Beltz, B. S. (
1988
). Crustacean neurohormones. In
Invertebrate Endocrinology, Vol. 2, Endocrinology of Selected Invertebrate Types
(ed. H. Laufer and R. G. H. Downer), pp.
235
-258. New York: Alan R. Liss.
Bliss, D. E. (
1951
). Metabolic effects of sinus gland or eyestalk removal in the land crab, Gecarcinus lateralis.
Anat. Rec.
111
,
502
-503.
Chan, S. M., Chen, X. G. and Gu, P. L. (
1998
). PCR cloning and expression of the molt-inhibiting hormone gene for the crab(Charybdis feriatus).
Gene
224
,
23
-33.
Chan, S. M., Gu, P. L., Chu, K. H. and Tobe, S. S.(
2003
). Crustacean neuropeptide genes of the CHH/MIH/GIH family:implications from molecular studies.
Gen. Comp. Endocrinol.
134
,
214
-219.
Chang, E. S., Chang, S. A., Beltz, B. S. and Kravitz, E. A.(
1999
). Crustacean hyperglycemic hormone in the lobster nervous system: localization and release from cells in the subesophageal ganglion and thoracic second roots.
J. Comp. Neurol.
414
,
50
-56.
Charmantier-Daures, M., Charmantier, G., Janssen, K. P., Aiken,D. E. and van Herp, F. (
1994
). Involvement of eyestalk factors in the neuroendocrine control of osmoregulation in adult American lobster Homarus americanus.
Gen. Comp. Endocrinol.
94
,
281
-293.
Chen, S. H., Lin, C. Y. and Kuo, C. M. (
2004
). Cloning of two crustacean hyperglycemic hormone isoforms in freshwater giant prawn (Macrobrachium rosenbergii): evidence of alternative splicing.
Mar. Biotechnol.
6
,
83
-94.
Chen, S. H., Lin, C. Y. and Kuo, C. M. (
2005
). In silico analysis of crustacean hyperglycemic hormone family.
Mar. Biotechnol
.
7
,
193
-206.
Christie, A. E. and Messinger, D. I. (
2005
). Hormonal signaling from the crustacean stomatogastric nervous system.
Comp. Biochem. Physiol.
141A
,
S157
.
Christie, A. E., Baldwin, D. H., Marder, E. and Graubard, K.(
1997
). Organization of the stomatogastric neuropil of the crab, Cancer borealis, as revealed by modulator immunocytochemistry.
Cell Tissue Res.
288
,
135
-148.
Christie, A. E., Edwards, J. M., Cherny, E., Clason, T. A. and Graubard, K. (
2003
). Immunocytochemical evidence for nitric oxide- and carbon monoxide-producing neurons in the stomatogastric nervous system of the crayfish Cherax quadricarinatus.
J. Comp. Neurol
.
467
,
293
-306.
Christie, A. E., Cain, S. D., Edwards, J. M., Clason, T. A.,Cherny, E., Lin, M., Manhas, A. S., Sellereit, K. L., Cowan, N. G., Nold, K. A. et al. (
2004
). The anterior cardiac plexus: an intrinsic neurosecretory site within the stomatogastric nervous system of the crab Cancer productus.
J. Exp. Biol.
207
,
1163
-1182.
Chung, J. S., Wilkinson, M. C. and Webster, S. G.(
1996
). Determination of the amino acid sequence of the moult-inhibiting hormone from the edible crab, Cancer pagurus.
Neuropeptides
30
,
95
-101.
Chung, J. S., Wilkinson, M. C. and Webster, S. G.(
1998
). Amino acid sequences of both isoforms of crustacean hyperglycemic hormone (CHH) and corresponding precursor-related peptide in Cancer pagurus.
Regul. Pept.
77
,
17
-24.
Chung, J. S., Dircksen, H. and Webster, S. G.(
1999
). A remarkable, precisely timed release of hyperglycemic hormone from endocrine cells in the gut is associated with ecdysis in the crab Carcinus maenas.
Proc. Natl. Acad. Sci. USA
96
,
13103
-13107.
Cooke, I. M. and Sullivan, R. E. (
1982
). Hormones and neurosecretion. In
The Biology of Crustacea, Vol. 3. Neurobiology: Structure and Function
(ed. H. L. Atwood and D. C. Sandeman), pp.
206
-278. New York: Academic Press.
Cruz-Bermúdez, N. D., Fu, Q., Kutz-Naber, K. K.,Christie, A. E., Li, L. and Marder, E. (
2006
). Mass spectrometric characterization and physiological actions of GAHKNYLRFamide, a novel FMRFamide-like peptide from crabs of the genus Cancer.
J. Neurochem.
97
,
784
-799.
Davey, M. L., Hall, M. R., Willis, R. H., Oliver, R. W., Thurn,M. J. and Wilson, K. J. (
2000
). Five crustacean hyperglycemic family hormones of Penaeus monodon: complementary DNA sequence and identification in single sinus glands by electrospray ionization-Fourier transform mass spectrometry.
Mar. Biotechnol.
2
,
80
-91.
de Kleijn, D. P. and van Herp, F. (
1995
). Molecular biology of neurohormone precursors in the eyestalk of Crustacea.
Comp. Biochem. Physiol.
112B
,
573
-579.
de Kleijn, D. P., Janssen, K. P., Martens, G. J. and van Herp,F. (
1994a
). Cloning and expression of two crustacean hyperglycemic-hormone mRNAs in the eyestalk of the crayfish Orconectes limosus.
Eur. J. Biochem
.
224
,
623
-629.
de Kleijn, D. P., Sleutels, F. J., Martens, G. J. and Van Herp,F. (
1994b
). Cloning and expression of mRNA encoding prepro-gonad-inhibiting hormone (GIH) in the lobster Homarus americanus.
FEBS Lett
.
353
,
255
-258.
de Kleijn, D. P., de Leeuw, E. P., van den Berg, M. C., Martens,G. J. and van Herp, F. (
1995
). Cloning and expression of two mRNAs encoding structurally different crustacean hyperglycemic hormone precursors in the lobster Homarus americanus.
Biochim. Biophys. Acta.
1260
,
62
-66.
de la Iglesia, H. O., Hsu, Y. W., Weller, J. R. and Christie, A. E. (
2005
).
Molecular cloning of cDNAs encoding crustacean hyperglycemic hormone (CHH) and its precursor-related peptide(CPRP) from the sinus gland of the crab Cancer productus
. Program No. 30.6. 2005 Abstract Viewer/Itinerary Planner. Washington, DC:Society for Neuroscience, 2005. Online.
Dircksen, H., Bocking, D., Heyn, U., Mandel, C., Chung, J. S.,Baggerman, G., Verhaert, P., Daufeldt, S., Plosch, T., Jaros, P. P. et al.(
2001
). Crustacean hyperglycaemic hormone (CHH)-like peptides and CHH-precursor-related peptides from pericardial organ neurosecretory cells in the shore crab, Carcinus maenas, are putatively spliced and modified products of multiple genes.
Biochem. J.
356
,
159
-170.
Edomi, P., Azzoni, E., Mettulio, R., Pandolfelli, N., Ferrero,E. A. and Giulianini, P. G. (
2002
). Gonad-inhibiting hormone of the Norway lobster (Nephrops norvegicus): cDNA cloning,expression, recombinant protein production, and immunolocalization.
Gene
284
,
93
-102.
Fanjul-Moles, M. L. (
2006
). Biochemical and functional aspects of crustacean hyperglycemic hormone in decapod crustaceans:review and update.
Comp. Biochem. Physiol.
142C
,
390
-400.
Fingerman, M. (
1992
). Decapod crustacean glands. In
Microscopic Anatomy of Invertebrates, Vol. 10. Decapod Crustacea
(ed. F. W. Harrison and A. G. Humes), pp.
345
-394. New York: Wiley-Liss.
Fu, Q., Christie, A. E. and Li, L. (
2005a
). Mass spectrometric characterization of crustacean hyperglycemic hormone precursor-related peptides (CPRPs) from the sinus gland of the crab, Cancer productus.
Peptides
26
,
2137
-2150.
Fu, Q., Goy, M. F. and Li, L. (
2005b
). Identification of neuropeptides from the decapod crustacean sinus glands using nanoscale liquid chromatography tandem mass spectrometry.
Biochem. Biophys. Res. Commun
.
337
,
765
-778.
Fu, Q., Kutz, K. K., Schmidt, J. J., Hsu, Y. W., Messinger, D. I., Cain, S. D., de la Iglesia, H. O., Christie, A. E. and Li, L.(
2005c
). Hormone complement of the Cancer productussinus gland and pericardial organ: an anatomical and mass spectrometric investigation.
J. Comp. Neurol
.
93
,
607
-626.
Gu, P. L. and Chan, S. M. (
1998
). Cloning of a cDNA encoding a putative molt-inhibiting hormone from the eyestalk of the sand shrimp Metapenaeus ensis.
Mol. Mar. Biol. Biotechnol.
7
,
214
-220.
Gu, P. L., Tobe, S. S., Chow, B. K., Chu, K. H., He, J. G. and Chan, S. M. (
2002
). Characterization of an additional molt inhibiting hormone-like neuropeptide from the shrimp Metapenaeus ensis.
Peptides
23
,
1875
-1883.
Hanström, B. (
1931
). Neue untersuchengenüber sinnesorgane und nervernsystem der crustaceen I.
Z. Morph. Ökol. Tiere
.
23
,
80
-236.
Hanström, B. (
1933
). Neue untersuchengenüber sinnesorgane und nervernsystem der crustaceen II.
Zool. Jahrb. Abt. Anat. Ontog. Tiere
.
56
,
378
-520.
Hanström, B. (
1935
). Preliminary report on the probable connection between the blood gland and the chromatophore activator in decapod crustaceans.
Proc. Natl. Acad. Sci. USA
21
,
584
-585.
Hanström, B. (
1937
). Die sinusdrüse und der hormonal bedingte fabwechesl der crustaceen.
Kungl. Svenska Vetenskap. Handl
.
3
,
1
-99.
Harris-Warrick, R. M., Marder, E., Selverston, A. I. and Moulins, M. (ed.) (
1992
).
Dynamic Biological Networks: The Stomatogastric Nervous System
. Cambridge: MIT Press.
Hsu, Y. A., Weller, J. R., de la Iglesia, H. O. and Christie, A. E. (
2004
).
Immunocytochemical evidence for crustacean hyperglycemic hormone-related peptides in the neuroendocrine organs of Cancer crabs
. Program No. 274.4. 2004 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2004. Online.
Hsu, Y. A., de la Iglesia, H. O. and Christie, A. E.(
2005a
). Crustacean hyperglycemic hormone-related peptides in Cancer productus.
Comp. Biochem. Physiol.
141A
,
S157
.
Hsu, Y. A., Messinger, D. I., Christie, A. E. and de la Iglesia,H. O. (
2005b
).
Molecular characterization and putative circadian cycling of β-pigment dispersing hormone (β-PDH) in the sinus gland of the crab Cancer productus
. Program No. 30.3. 2005 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2005. Online.
Keller, R. (
1992
). Crustacean neuropeptides:structures, functions and comparative aspects.
Experientia
48
,
439
-448.
Khayat, M., Yang, W., Aida, K., Nagasawa, H., Tietz, A.,Funkenstein, B. and Lubzens, E. (
1998
). Hyperglycaemic hormones inhibit protein and mRNA synthesis in in vitro-incubated ovarian fragments of the marine shrimp Penaeus semisulcatus.
Gen. Comp. Endocrinol.
110
,
307
-318.
Klein, J. M., Mangerich, S., de Kleijn, D. P., Keller, R. and Weidemann, W. M. (
1993
). Molecular cloning of crustacean putative molt-inhibiting hormone (MIH) precursor.
FEBS Lett.
334
,
139
-142.
Koller, G. (
1925
). Fabwechsel bei Crangon vulgaris.
Verh. Dtsch. Zool. Ges.
30
,
128
-132.
Koller, G. (
1927
). Über chromatophorensystem, farbensinn und farbwechsel bei Crangon vulgaris.
Z. Vergl. Physiol
.
5
,
191
-246.
Krungkasem, C., Ohira, T., Yang, W. J., Abdullah, R., Nagasawa,H. and Aida, K. (
2002
). Identification of two distinct molt-inhibiting hormone-related peptides from the giant tiger prawn Penaeus monodon.
Mar. Biotechnol.
4
,
132
-140.
Lacombe, C., Greve, P. and Martin, G. (
1999
). Overview on the sub-grouping of the crustacean hyperglycemic hormone family.
Neuropeptides
33
,
71
-80.
Lee, K. J., Elton, T. S., Bej, A. K., Watts, S. A. and Watson,R. D. (
1995
). Molecular cloning of a cDNA encoding putative molt-inhibiting hormone from the blue crab, Callinectes sapidus.
Biochem. Biophys. Res. Commun
.
209
,
1126
-1131.
Lu, W., Wainwright, G., Webster, S. G., Rees, H. H. and Turner,P. C. (
2000
). Clustering of mandibular organ-inhibiting hormone and moult-inhibiting hormone genes in the crab, Cancer pagurus, and implications for regulation of expression.
Gene
253
,
197
-207.
Lu, W., Wainwright, G., Olohan, L. A., Webster, S. G., Rees, H. H. and Turner, P. C. (
2001
). Characterization of cDNA encoding molt-inhibiting hormone of the crab, Cancer pagurus;expression of MIH in non-X-organ tissues.
Gene
278
,
149
-159.
Marco, H. G., Hansen, I. A., Scheller, K. and Gade, G.(
2003
). Molecular cloning and localization of a cDNA encoding a crustacean hyperglycemic hormone from the South African spiny lobster, Jasus lalandii.
Peptides
24
,
845
-851.
Messinger, D. I., Kutz, K. K., Le, T., Verley, D. R., Hsu, Y. W., Ngo, C. T., Cain, S. D., Birmingham, J. T., Li, L. and Christie, A. E.(
2005a
). Identification and characterization of a tachykinin-containing neuroendocrine organ in the commissural ganglion of the crab Cancer productus.
J. Exp. Biol.
208
,
3303
-3319.
Messinger, D. I., Verley, D. R., Fu, Q., Kutz, K. K., Hsu, Y. A., Li, L., Birmingham, J. T. and Christie, A. E. (
2005b
).
Structural and functional characterization of the anterior cardiac neuron 1/2-anterior cardiac plexus (ACN1/2-ACP) neuroendocrine system of the crab Cancer productus.
Program No. 30.8. 2005 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2005. Online.
Mettulio, R., Edomi, P., Ferrero, E. A., Lorenzon, S. and Giulianini, P. G. (
2004
). The crustacean hyperglycemic hormone precursors a and b of the Norway lobster differ in the preprohormone but not in the mature peptide.
Peptides
25
,
1899
-1907.
Ohira, T., Watanabe, T., Nagasawa, H. and Aida, K.(
1997a
). Cloning and sequence analysis of a cDNA encoding a crustacean hyperglycemic hormone from the Kuruma prawn Penaeus japonicus.
Mol. Mar. Biol. Biotechnol
.
6
,
59
-63.
Ohira, T., Watanabe, T., Nagasawa, H. and Aida, K.(
1997b
). Molecular cloning of a molt-inhibiting hormone cDNA from the kuruma prawn Penaeus japonicus.
Zool. Sci.
14
,
785
-789.
Ohira, T., Katayama, H., Tominaga, S., Takasuka, T., Nakatsuji,T., Sonobe, H., Aida, K. and Nagasawa, H. (
2005
). Cloning and characterization of a molt-inhibiting hormone-like peptide from the prawn Marsupenaeus japonicus.
Peptides
26
,
259
-268.
Passano, L. M. (
1951
). The X-organ-sinus gland system in crabs.
Anat. Rec.
111
,
502
.
Santos, E. A., Nery, L. E., Keller, R. and Goncalves, A. A.(
1997
). Evidence for the involvement of the crustacean hyperglycemic hormone in the regulation of lipid metabolism.
Physiol. Zool.
70
,
415
-420.
Saper, C. B. and Sawchenko, P. E. (
2003
). Magic peptides, magic antibodies: guidelines for appropriate controls for immunohistochemistry.
J. Comp. Neurol.
465
,
161
-163.
Sedlmeier, D. (
1982
). The mode of action of the crustacean neurosecretory hyperglycemic hormone (CHH). II. Involvement of glycogen synthase.
Gen. Comp. Endocrinol
.
47
,
426
-432.
Sedlmeier, D. (
1988
). The crustacean hyperglycemic hormone (CHH) releases amylase from the crayfish midgut gland.
Regul. Pept.
20
,
91
-98.
Sedlmeier, D. and Keller, R. (
1981
). The mode of action of the crustacean neurosecretory hyperglycemic hormone. I. Involvement of cyclic nucleotides.
Gen. Comp. Endocrinol
.
45
,
82
-90.
Selverston, A. I. and Moulins, M. (ed.) (
1987
).
The Crustacean Stomatogastric System
. Berlin:Springer.
Serrano, L., Blanvillain, G., Soyez, D., Charmantier, G.,Grousset, E., Aujoulat, F. and Spanings-Pierrot, C. (
2003
). Putative involvement of crustacean hyperglycemic hormone isoforms in the neuroendocrine mediation of osmoregulation in the crayfish Astacus leptodactylus.
J. Exp. Biol.
206
,
979
-988.
Sonobe, H., Nishimura, T., Sonobe, M., Nakatsuji, T.,Yanagihara, R., Kawakami, T. and Aimoto, S. (
2001
). The molt-inhibiting hormone in the American crayfish Procambarus clarkii:its chemical synthesis and biological activity.
Gen. Comp. Endocrinol
.
121
,
196
-204.
Soyez, D. (
1997
). Occurrence and diversity of neuropeptides from the crustacean hyperglycemic hormone family in arthropods. A short review.
Ann. N. Y. Acad. Sci
.
814
,
319
-323.
Spanings-Pierrot, C., Soyez, D., Van Herp, F., Gompel, M.,Skaret, G., Grousset, E. and Charmantier, G. (
2000
). Involvement of crustacean hyperglycemic hormone in the control of gill ion transport in the crab Pachygrapsus marmoratus.
Gen. Comp. Endocrinol
.
119
,
340
-350.
Sun, P. S. (
1994
). Molecular cloning and sequence analysis of a cDNA encoding a molt-inhibiting hormone-like neuropeptide from the white shrimp Penaeus vannamei.
Mol. Mar. Biol. Biotechnol.
3
,
1
-6.
Sun, P. S. (
1995
). Expression of the molt-inhibiting hormone-like gene in the eyestalk and brain of the white shrimp Penaeus vannamei.
Mol. Mar. Biol. Biotechnol
.
4
,
262
-268.
Tang, C., Lu, W., Wainwright, G., Webster, S. G., Rees, H. H. and Turner, P. C. (
1999
). Molecular characterization and expression of mandibular organ-inhibiting hormone, a recently discovered neuropeptide involved in the regulation of growth and reproduction in the crab Cancer pagurus.
Biochem. J.
343
,
355
-360.
Tensen, C. P., de Kleijn, D. P. and van Herp, F.(
1991
). Cloning and sequence analysis of cDNA encoding two crustacean hyperglycemic hormones from the lobster Homarus americanus.
Eur. J. Biochem.
200
,
103
-106.
Toullec, J. Y., Vinh, J., Le Caer, J. P., Shillito, B. and Soyez, D. (
2002
). Structure and phylogeny of the crustacean hyperglycemic hormone and its precursor from a hydrothermal vent crustacean:the crab Bythograea thermydron.
Peptides
23
,
31
-42.
Toullec, J. Y., Serrano, L., Lopez, P., Soyez, D. and Spanings-Pierrot, C. (
2006
). The crustacean hyperglycemic hormones from an euryhaline crab Pachygrapsus marmoratus and a fresh water crab Potamon ibericum: eyestalk and pericardial isoforms.
Peptides
27
,
1269
-1280.
Umphrey, H. R., Lee, K. J., Watson, R. D. and Spaziani, E.(
1998
). Molecular cloning of a cDNA encoding molt-inhibiting hormone of the crab, Cancer magister.
Mol. Cell. Endocrinol
.
136
,
145
-149.
van Herp, F. (
1998
). Molecular, cytological and physiological aspects of the crustacean hyperglycemic hormone family. In
Recent Advances in Arthropod Endocrinology
(ed. G. M. Coast and S. G. Webster), pp.
53
-70. Cambridge:Cambridge University Press.
Wainwright, G., Webster, S. G., Wilkinson, M. C., Chung, J. S. and Rees, H. H. (
1996
). Structure and significance of mandibular organ-inhibiting hormone in the crab, Cancer pagurus. Involvement in multihormonal regulation of growth and reproduction.
J. Biol. Chem
.
271
,
12749
-12754.
Webster, S. G. (
1996
). Measurement of crustacean hyperglycaemic hormone levels in the edible crab Cancer pagurus during emersion stress.
J. Exp. Biol.
199
,
1579
-1585.
Webster, S. G. (
1998
). Neuropeptides inhibiting growth and reproduction in crustaceans. In
Recent Advances in Arthropod Endocrinology
(ed. G. M. Coast and S. G. Webster), pp.
33
-53. Cambridge: Cambridge University Press.
Weidemann, W., Gromoll, J. and Keller, R.(
1989
). Cloning and sequence analysis of cDNA for precursor of a crustacean hyperglycemic hormone.
FEBS Lett
.
257
,
31
-34.
Wilcockson, D. C., Chung, S. J. and Webster, S. G.(
2002
). Is crustacean hyperglycaemic hormone precursor-related peptide a circulating neurohormone in crabs?
Cell Tissue Res.
307
,
129
-138.
Yang, W. J. and Rao, K. R. (
2001
). Cloning of precursors for two MIH/VIH-related peptides in the prawn, Macrobrachium rosenbergii.
Biochem. Biophys. Res. Commun
.
289
,
407
-413.