General and Comparative Endocrinology 175 (2012) 217–233

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General and Comparative Endocrinology

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Review The CHH-superfamily of multifunctional peptide hormones controlling crustacean metabolism, osmoregulation, moulting, and reproduction ⇑ Simon George Webster a, , Rainer Keller b, Heinrich Dircksen c a School of Biological Sciences, Bangor University, LL57 2UW Bangor, UK b Institute for Molecular Biomedicine (LIMES-Institute), University of Bonn, D-53115 Bonn, Germany c Department of Zoology, Stockholm University, S-106 91 Stockholm, Sweden article info abstract

Article history: Apart from providing an up-to-date review of the literature, considerable emphasis was placed in this Received 27 October 2011 article on the historical development of the field of ‘‘crustacean eyestalk hormones’’. A role of the neuro- Accepted 21 November 2011 secretory eyestalk structures of crustaceans in endocrine regulation was recognized about 80 years ago, Available online 29 November 2011 but it took another half a century until the first peptide hormones were identified. Following the identi- fication of crustacean hyperglycaemic hormone (CHH) and moult-inhibiting hormone (MIH), a large Keywords: number of homologous peptides have been identified to this date. They comprise a family of multifunc- Crustacean hyperglycaemic hormone tional peptides which can be divided, according to sequences and precursor structure, into two subfam- superfamily ilies, type-I and -II. Recent results on peptide sequences, structure of genes and precursors are described Gene Transcript and peptide structures here. The best studied biological activities include metabolic control, moulting, gonad maturation, ionic Localization and expression and osmotic regulation and methyl farnesoate synthesis in mandibular glands. Accordingly, the names Biological activities CHH, MIH, and GIH/VIH (gonad/vitellogenesis-inhibiting hormone), MOIH (mandibular organ-inhibiting Signal transduction and receptors hormone) were coined. The identification of ITP (ion transport peptide) in insects showed, for the first time, that CHH-family peptides are not restricted to crustaceans, and data mining has recently inferred their occurrence in other ecdysozoan clades as well. The long-held tenet of exclusive association with the eyestalk X-organ-sinus gland tract has been challenged by the finding of several extra nervous system sites of expression of CHH-family peptides. Concerning mode of action and the question of target tissues, second messenger mechanisms are discussed, as well as binding sites and receptors. Future challenges are highlighted. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction stalk (ES). He identified the sinus gland (SG) as a neurohaemal structure, i.e. as an aggregation of terminals of a cluster of neuro- Crustacean endocrinology began with the discovery, by Koller secretory perikarya that are located in the medulla terminalis [111,112], that colour change in shrimp is caused by blood-borne (MT). This cluster was named the X-organ (XO). For references factors which originated from tissues in the eyestalk. Hanström on the early history see Fingerman [70]. [85,86] provided the morphological basis by describing the anat- During an investigation of a possible hormonal control of carbo- omy of neurosecretory structures in the optic ganglia of the eye- hydrate metabolism in crustaceans, Abramowitz et al. [1] found in 1944 that injection of ES extracts into blue crabs, Callinectes sapi- dus, elicited a dramatic hyperglycaemia. The factor proved to be Abbreviations: AG, androgenic gland; CHH, crustacean hyperglycaemic hor- mone; CPRP, crustacean hyperglycaemic hormone precursor-related peptide; DA, very potent (0.001 ES equivalents giving a significant response), dopamine; ES, eyestalk; GC, guanylyl cyclase; GIH, gonad-inhibiting hormone; heat stable, highly concentrated in the SG, and was named a ‘‘dia- GPCR, G-protein coupled receptor; 5-HT, 5-hydroxy-tryptamine; IBMX, isobutylm- betogenic factor’’. In the course of further studies, this was eventu- ethylxanthine; ITP, ion transport peptide; IPRP, ion transport precursor-related ally replaced by the name crustacean hyperglycaemic hormone peptide; MF, methyl farnesoate; MIH, moult-inhibiting hormone; MO, mandibular organ; MOIH, mandibular organ-inhibiting hormone; NO, nitric oxide; PO, pericar- (CHH). A CHH isolated from the SG of the shore crab, Carcinus mae- dial organ; RIA, radioimmunoassay; SG, sinus gland; TR-FIA, time resolved- nas, was the first to be fully characterized [98]. This was directly fluoroimmunoassay; VIH, vitellogenesis-inhibiting hormone; XO-SG, X-organ-sinus followed by the report of the amino acid sequence of a peptide gland; YO, Y-organ. ⇑ from Homarus americanus [27] which proved to be very similar Corresponding author. Fax: +44 (0)1248 370731/371644. to that of Carcinus-CHH in terms of molecular size (72 amino acids) E-mail addresses: [email protected] (S.G. Webster), [email protected] (R. Keller), [email protected] (H. Dircksen). and sequence identity (61% identical residues). Particularly

0016-6480/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2011.11.035 218 S.G. Webster et al. / General and Comparative Endocrinology 175 (2012) 217–233 interesting was the observation that the Homarus-peptide exhib- been demonstrated in Homarus and crayfish species ited both CHH and moult-inhibiting hormone (MIH) activity. This [17,18,205,240]. With regard to biological activities, several novel key finding presaged two aspects of the structure and function of ones have more recently been added to the early recognized roles CHH and homologous peptides: (1) they are multifunctional hor- in the regulation of carbohydrate metabolism (CHH), secretagogue mones and, (2) they often display overlapping biological activities. action on the hepatopancreas, inhibition of moulting (MIH) and go- From C. maenas, a second peptide was soon fully identified [231], nad maturation (GIH/VIH). These comprise: the inhibition of which had distinct moult inhibiting, but no hyperglycaemic activ- methyl farnesoate secretion (MOIH activity) [137,227], water up- ity. MIH had certain structural features which set it apart from Car- take during ecdysis [36], and ionic and osmotic regulation [206]. cinus-CHH, notwithstanding basic similarities in the amino acid These results once again underscore the pleiotropic activities of sequence. After a few more sequences became available, a new CHH-superfamily peptides. Much further work is, however, needed peptide family emerged. Moreover, the CHHs from Carcinus and to elucidate the details and mechanisms of action, at the organ and Homarus on the one hand, and Carcinus MIH and another MIH-re- cellular level, which underlie the variety of biological effects. It is lated peptide from Homarus, VIH [231,235] on the other hand, safe to predict that more biological activities will eventually be could be considered prototypes of two subgroups of this family discovered. [99]. The entire grouping is now usually referred to as the CHH- Finally, an increasing number of studies have established that superfamily. For the two subfamilies, the terms type-I (CHH sensu CHH-superfamily peptides are not confined to neural tissues, par- stricto and ion transport peptides, ITPs) and type-II peptides (MIH ticularly the XO-SG system and the pericardial organs, but are also, and vitellogenesis-inhibiting hormones, VIHs) have been intro- unexpectedly and surprisingly, expressed in non-neural tissues duced [118]. Differences in the gene and precursor structures and organs. A particularly intriguing example is the transient firmly support this division (see below). appearance of secretory CHH-cells (paraneurons) in the fore- and During the last two decades, a remarkable upsurge of interest hindgut epithelium of C. maenas during premoult [36] (see below). has resulted in the identification of approximately 80 family Apart from the attempt to summarize the current state of members from about 40 crustacean species [232], increasingly less knowledge, it has been our aim to show how the field of CHH- by peptide sequencing, but by conceptual translation from cDNAs superfamily peptide research has developed and expanded during or in silico mining of transcriptomes and genomes. All of these the last twenty plus years. The different topics briefly outlined in (with perhaps one exception) can unequivocally be placed in one this introduction will be covered in some detail in this review of the two subfamilies. A remarkable feature from a historical point (For other recent reviews see: [15,23,43,118,200,232]). of view is the fact that putative hormones that had long been postulated from classical ablation and replacement experiments, have now all been identified as members of a common peptide 2. Structures of genes, derived precursors and peptides family. The moult-inhibiting hormone (MIH) was postulated more than 100 years ago by the observation [242], many times repeated, Members of the CHH-superfamily are grouped into type-I (CHH/ of precocious moulting after ES ablation. About 70 years ago, it was ITP) and type-II (MIH, MOIH, VIH/GIH) peptides based on their pre- found that ES ablation resulted in accelerated maturation of the cursor and primary structures [118]. With regard to their gene gonads [173], which led to the postulate of a gonad-inhibiting structures, there are at present no clear-cut rules that distinctly hormone (GIH). More recently, the synonymous term follow this type definition. In general, whereas the now more than vitellogenesis-inhibiting hormone (VIH) was introduced two dozens of identified chh-genes often occur in multiple iso- [29,202,204]. One more term, mandibular organ-inhibiting hor- forms of 3-exon or 4-exon genes in decapods (except a 2-exon gene mone (MOIH) was coined from the discovery that a MIH-type for penaeid Pem-CHH1) [23,31,32,59,79–81,157,223,238], only (Type-II) peptide inhibited the secretion of methyl farnesoate from single itp-genes are found in insect genomes as 3-, 4-, or 5-exon the mandibular organs [135,137,227]. Commonly, the individual genes (Fig. 1A and B) [58,157]. Whilst mih- and moih-genes are peptides, as they appear in the literature, have often been named only found as 3-exon genes in shrimps, giant tiger prawns according to results from single bioassays, illustrating the diversity and crabs (Fig. 1C) [21–23,32,114,157,241], no specific gene struc- of functions. This fact, and also the often observed multifunctional- tures have hitherto been described for vih/gih-genes. In penaeid ity of individual hormones, demonstrates the hazards of classifying shrimps, multiple chh-genes are known to occur in clusters of up these peptides based on single bioassays. to nine simple 3-exon chhA-orchhB-genes in tandem (Fig. 1A) Apart from the identification of many novel CHH-superfamily [23,79–81], and, in several other species, at least multiple, slightly peptides from crustaceans, considerable progress has been made differing copies of chh-genes have been found [42,59]. However, concerning gene structure, biological activities, expression in maximally two single mih-genes only, supposedly on separate non-neural tissues and organs, and occurrence in other arthropod chromosome scaffolds, have been described for several species groups. A remarkable event was the discovery, in the locust [21,22,30,142,166]; apparently only in cancrid crabs, have distinct Schistocerca gregaria, that the so-called ion transport peptide slightly diverged mih- and moih-genes been found in clusters (ITP), turned out to be a CHH-like (Type-I) peptide [7,8,155]. Recent (Fig. 1C) [142]. A single structurally confirmed 4-exon itp-gene genomic studies have revealed the existence and evolution of CHH and two additional atypical but clearly itp-like genes, a 4-exon genes across other arthropods and ecdysozoans in general and a 2-exon gene, have recently been discovered in the genome [34,62,157]. Studies at the DNA level demonstrated multiple copies of the water flea Daphnia pulex [62] but as yet no mih-, moih-or of peptide encoding genes, which explains the existence of iso- vih-genes. forms. Such isoforms have been isolated as mature peptides from Interestingly, only derivatives of the 4-exon and 5-exon genes SGs of single animals, e.g. up to seven slightly different CHHs in are known to undergo alternative splicing processes in crustaceans penaeid shrimp species [54,108,239]. Since they do not differ sig- and insects, leading to long and short splice forms (Fig. 1B) nificantly in hyperglycaemic activity [108,239], any distinctive [13,31,51,59,63,116,215]. Characteristically, the 3-exon chh- and physiological relevance, and their differential expression remains itp-genes, such as e.g. in penaeid shrimp and some insect species, obscure. Another important contribution was the finding that dif- and all the 3-exon mih- and moih-genes have a first phase-0 intron ferent CHH-peptides can be generated by alternative splicing from (no codon-interruption; Fig. 1A–C, i10) usually interrupting the sig- a common transcript [59]. Finally, posttranslational isoform gener- nal peptide-coding region and a second phase-2 intron (codon ation by stereoisomerisation of particular residues in CHHs has interrupted after the second base; Fig. 1A–C, i22) in common. The S.G. Webster et al. / General and Comparative Endocrinology 175 (2012) 217–233 219

Fig. 1. Gene, precursor and primary structures of type-I (CHHs/ITPs) and type-II (MIH/MOIH/VIH/GIH) peptides. (A) Clusters of shrimp Metapenaeus ensis chhA-genes (blue) and chhB-genes (green) in tandem arrangements occur on two different scaffolds. All genes are 3-exon genes as enlarged for the chhA1’’-gene containing a first phase-0 (i10) intron splitting the signal peptide (SP) message in exon-1 and -2 (e1, e2) and a second phase-2 intron (i22) splitting the first part of the CHHA (until aa 40 in e2; orange) and the second part in exon-3 (aa 41 + to ⁄STOP; yellow). Note regulatory elements upstream of exon-1 [79,80]. (B) Alternative splicing of chh- and itp-gene messages into long and short splice forms in shore crab Carcinus maenas (CarmaPOCHH = CHHL, SGCHH = CHH [59]), water flea Daphnia pulex (DappuITP/ITPL [62,157]) and fruit fly Drosophila melanogaster (DromeITP/ITPL1-L2 [63]). Note the additional exons in the 4- or 5-exon genes coding for the second parts of the long CHH (POCHH) or ITPL messages and the corresponding phase-2 introns. (C) Crab Cancer pagurus (Canpa) mih-/moih1-gene cluster in tail-to-tail arrangement of closely related typical unspliced 3-exon genes with exon/intron structures similar to the shrimp chh-genes in A. (D) Alignment of type-I long and short CHH and ITP splice form peptides and type-II MIH/VIH peptides and their cystine-bridges. Gap introduced into type-I peptides to show the important position of the additional Gly (yellow) in position 5 after the first Cys in all type-II peptides. Note also the invariant Val (yellow) in position 13 after the first Cys in type-II peptides, the alpha-helices (a; first helix aaaa in blue is missing in CHHs) and 3D-stabilising aas (x)

[95,163] as well as posttranslational modifications of N-terminal pyroglutamate (pQ) and C-terminal amidations (–NH2). Blue line indicates position of typical joined exon or splice sites between aas 40/41 and 41/42. C0,I0 CHH-/ITP-precursor related peptides; ⁄STOP codon. second exon always terminates in the codon for the 40th or 41st respectively. This is also the case for all 4- and 5-exon itp-genes of amino acid (aa) of every mature CHH and MIH/MOIH/VIH peptide, insects which contain one (or two in Drosophila) additional phase-2 220 S.G. Webster et al. / General and Comparative Endocrinology 175 (2012) 217–233 intron as a basis for the usually observed alternative splicing of pyro-Glu, a common posttranslational modification protecting hnRNAs (Fig. 1B), combining in all cases the second coding parts peptides against aminopeptidase’s degradation, is another of peptides (aas 41 + or 42+) to either long or short splice forms: structural determinant that is a distinctive difference between Thus, long peptide splice forms, such as PO-CHHs of crabs or long many CHHs and all hitherto known ITPs [58], shrimp and freshwa- CHH-Ls of shrimp and insect ITP-L isoforms arise from the unsp- ter prawn CHHs [31,32], and all type-II peptides. As has been liced exons 1–4 (or 5inDrosophila [63]), whereas the short splice shown in shore crabs, C. maenas CHHs with blocked and unblocked forms, i.e. bioactive short CHHs sensu stricto and ITPs, arise from N-termini display identical bioactivity [38]. (5) The aromatic aas hnRNAs with exon-3 (or both, exon-3 plus exon-4 in Drosophila) (Phe or Tyr) in position 3 (or 2) of CHHs and ITPs (4 or 3 in dipteran being spliced out (crustaceans: [31,59,116,217,218], insects: ITPs) of the N-terminal alpha-helix appear to be a conserved [13,51,63,155]. Of note, the exon-4-derived second coding parts feature important for the biological activity of both CHHs and ITPs of the short splice forms occur as silent elements in the 30 untrans- [81,94,95,159,244]. However, in some shrimp CHHs, the N- lated region of the mRNAs of the long splice forms (Fig. 1B). terminus, even if artificially elongated, appears to be of minor An important aspect clearly distinguishing chh-/itp-genes from importance for bioactivity [81,96]. About 15 years ago, sinus gland mih-genes is the occurrence in the exons-2 of so-called CHH- or CHHs of American lobsters were found to occur in two stereoiso- ITP-precursor related peptides (CPRPs, IPRPs) of unknown biologi- mer variants differing by the configuration of L- and D-Phe3 cal significance. Single copies of these peptides with largely vary- [205]. This conformational polymorphism occurs also in several ing lengths (7–54 aas) always precede the mature CHH/ITP crayfish species (Orconectes limosus, Astacus leptodactylus, Cherax peptides in their precursors and are clearly separated by dibasic destructor and two Procambarus species), in which CHHs share cleavage sites, but are completely absent in all mih-/moih-gene the same N-terminal sequence pQVFDQ-. The biological response derived precursors (Fig. 1A–C) and VIH/GIH-precursors [67,162]. to the D-Phe3-forms is larger and longer lasting when compared Surprisingly, IPRPs are also absent from the two atypical, probably with that of the L-Phe3-forms. Therefore, this posttranslational because of mutation and deletion, N-terminally elongated ITP-like modification was interpreted as an efficient protection against peptide precursors of D. pulex, although they ‘‘retain’’ all other aminopeptidases [200]. A similar situation is likely met with structural characteristics of DappuITP/ITPLs with which they D-Trp4-VIHs occurring in Homarus species [171]. (6) The highest perfectly align, much better than with any known type-II peptide number of identities or close similarities of aas are restricted to a precursor [62]. Montagné et al. [157] have proposed various evolu- core structure of the first 40 or 41 amino acids (i.e. the exon-2 tionary scenarios leading from the origin of ancestral either 2-exon derivatives), containing two out of five important characteristic or 3-exon genes to 3-exon and 4-exon genes in crustaceans or structural motifs in CHHs and ITPs flanked by the conserved cys- insects and to the recently confirmed Drosophila 5-exon gene tine-bound loops I and II [32,65,118]. Structurally similar motifs structure (Strauß and Dircksen, unpublished). These scenarios pos- have been described for the second and third regions of type-II tulate intragenic exon duplications. Interestingly, all genes contain peptides, especially in VIHs [118]. (7) The importance of distinctive upstream-regulatory sequences that allow for binding of several signatures between type-I and –II peptides has been derived from transcription factors (e.g. Pit-1a, CAAT-binding factor, TATA-box), detailed 3D-structural analyses of MIH from the Kuruma prawn, including second messenger cAMP-responsive element binding Marsupenaeus japonicus [95]. The characteristic Gly12 insertion proteins (CREB), as well as juvenoid (methyl farnesoate; retinoic and the invariant Val20 occur in all type-II peptides, i.e. in positions acid receptors (RXRs), and ecdysteroid receptors (EcRs). This shows 5 and 13, respectively, of the 16 amino acid spacer between the that the genes are possibly under control by other factors or feed- first and the second cysteine (yellow marked in Fig. 1D). These fea- back mechanisms (Fig. 1A; e.g. [80,141]). tures, that are lacking or changed in all type-I peptides, lie in the All precursors and mature peptides of CHH-superfamily mem- first of five alpha-helical structures, which are only found in bers have several characteristic features in common (as partly type-II peptides [94,95,163]. Artificial introduction of an additional demonstrated in Fig. 1D): (1) The conformation with 6 cysteines glycine into shrimp CHH in this position led to at least tenfold low- in the same positions putatively leads to the same three common er hyperglycaemic bioactivity [94,95]. Furthermore, the 3D-struc- disulphide bonds. For type-I/-II, respectively, because type-II pep- ture of MarjaMIH and supposedly also CHHs shows that this first tides have an additional Gly (see below), these are: C7–C43/C44, alpha-helical part of type-II (and most likely also this non-helical C23/C24–C39/C40,C26/C27–C52/C53. Whilst usually being inferred only part in type-I) peptides comes into close apposition to the C-termi- on the basis of homology, a few studies have unequivocally as- nus, a structural constraint which is thought to be critical for the signed these cystine bridges with the aid of tryptic fragmentation distinctive biological activity of the peptide(s) [95]. of native (or synthetic) non-reduced peptides followed by Ed- man-degradation and/or mass spectrometric analysis (e.g. 3. Biological activities [59,97,98,110,240]. (2) The normal length is 72 aas for type-I bio- active CHHs and almost all insect ITPs, excepting dipteran species 3.1. Type-I peptides: crustacean hyperglycaemic hormones which have one more N-terminal amino acid (Ser or Asn in position 2, i.e. are 73 aas in length). Instead, the type-II peptides, MIHs, The defining role of CHH is concerned with control of carbohy- MOIHs and VIHs have 77–83 aas (usually 78; [67,162,200]. (3) drate metabolism: It has long been known that injection of crude All CHHs and ITPs sensu stricto but none of the long splice forms eyestalk or sinus gland homogenates or purified CHH leads to are C-terminally amidated, which may protect them from carboxy- hyperglycaemia, which is group- or species-specific [101,132]. This peptidase’s degradation. In shrimp and crayfish CHHs, amidation response is initiated rapidly (within 15–20 min) and is sustained has been shown to confer increased bioactivity over non-amidated for long periods (1–3 h). Hyperglycaemia is a result of net mobili- peptide analogues [96,159]. For recombinant locust ITP (rITP), the zation of glycogen in target tissues such as the midgut gland and bioactivity of amidated rITP was shown to be two orders of magni- abdominal muscle as a consequence of activation of phosphorylase tude higher than rITP having a free C-terminus but only 1 order of and inhibition of glycogen synthase [190,193,195]. Although the magnitude higher than rITP having a C-terminal glycine residue hyperglycaemic response is seemingly adaptive in that it makes [228]. Although never protected N-terminally and usually not C- readily metabolisable energy stores available throughout the terminally, some MIHs and VIHs have identified C-terminal amida- organism, it has been suggested that this may be an artefact tions [19,145,164,171,180]. However, the functional significance of reflecting leakage of glucose from the cells following generalised this posttranslational modification is still unknown. (4) N-terminal mobilization of glycogen [184,185]. S.G. Webster et al. / General and Comparative Endocrinology 175 (2012) 217–233 221

The adaptive significance of hyperglycaemia is evident when CHH titres have been measured (RIA, EIA or TR-FIA) in crustaceans exposed to perceived stressful episodes, such as during emersion, hypoxia or thermal stress [25,39,42,105,113,230], when rapid in- creases in CHH are seen. Similarly, exposure to bacterial endotox- ins [140], parasitism by dinoflagellates [211–213], heavy metal pollutants [139] lead to CHH release and hyperglycaemia. For noc- turnal crustaceans such as the crayfish, A. leptodactylus, hyper- glycaemia is a result of release of CHH at the beginning of the scotophase [75,91,92]. In particular, exercise seems to be a potent elicitor of CHH release. In the Christmas Island red crab, Gecarcoi- dea natalis, forced exercise (sufficient to induce hypoxia and subse- quent hyperlactaemia) results in CHH release within a few minutes, which leads to sustained hyperglycaemia (Fig. 2). Raised glucose levels appear to negatively regulate CHH release: Injection studies have shown that glucose inhibits CHH release in C. maenas [185]. The ecophysiological significance of this negative feedback loop is vividly illustrated in G. natalis, where it is absent in the dry season, when crabs are fossorial and quite inactive, yet during the annual reproductive migration, when crabs must complete an energetically demanding migration of several km in a few days, glucose levels are tightly controlled by this mechanism, thus ensuring economy in glycogen reserve utilization [158]. The nega- tive feedback loop has been elegantly demonstrated in isolated CHH neurones in Cancer borealis, where bath application of D- glucose at concentrations that reflect in vivo levels, results in dose-dependent hyperpolarisation, and presumably, inhibition of CHH release [74]. Measurement of CHH in unstressed crabs shows that hormone release is episodic with short half-life in circulation of a few minutes [39]. Although the N-terminally blocked and unblocked isoforms of CHH most commonly seen in crabs are equally biologically active [38], in crustaceans such as crayfish and lobsters where additional stereo inversion of CHH via the L-Phe3 to D-Phe3-configuration occurs [17,71,201,203,205,240], the D-Phe3 isoform is more potent in eliciting prolonged hyperglycaemia compared with the L-Phe3 stereoisomer [205] (e.g. see above), in crayfish Procambarus clarkii, the D-Phe3 isoform is about ten times more potent in repressing ecdysteroid synthesis than the L-Phe3 isoform [240]. CHH has been shown to act as a secretagogue in that it stimu- lates amylase release from the midgut gland in Orconectes limosus and C. maenas [189], and it may also be involved in elevation of free fatty acids and phospholipids [187]. Thus the role of CHH as an adaptive hormone central to metabolism is clear. However, recent studies which are highlighted below have shown that there are many other physiologically relevant functions for CHH that not only show that these hormones are truly pleiotropic, but also vividly illustrate the functional evolution of the CHH group pep- tides in arthropods. Whilst type-II peptides (MIH) that profoundly inhibit ecdyster- oid synthesis by the Y-organs seem to be universal in crabs, in many crayfish, penaeid shrimp, and in lobsters (H. americanus), CHH acts as a MIH [27], but in the crab C. maenas, CHH is between 10–20 times less potent in inhibiting ecdysteroid synthesis com- pared to MIH [237]. Additionally, a further type-II peptide which Fig. 2. The effect of exercise on CHH, glucose and lactate levels in Gecarcoidea inhibits methyl farnesoate synthesis (mandibular organ-inhibiting natalis. Crabs were exercised for 10 min (horizontal black bar), followed by a hormone, MOIH) has been identified in the edible crab Cancer 110 min. recovery period (horizontal white bar). Black columns: exercised crabs (n = 5), grey columns: controls (n = 5). Error bars: +1 SEM. Inset shows individual pagurus [227], and in other cancrids (Webster, unpublished). profiles from exercised (solid lines) and control (dotted lines) crabs. ⁄P < 0.05, Nevertheless, in the other crustaceans thus far studied, Libinia ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001. Reprinted from [158] with permission. emarginata, C. maenas, CHH fulfils this role [104,137]. A little studied biological activity is the inhibitory effect of the to the CHH content of the extract [20]. With view to the inhibition eyestalk on the androgenic gland (AG). Eyestalk removal causes a of the mandibular organs by CHH alluded to earlier, this appears to dramatic hypertrophy of the AG of Cherax quadricarinatus, and an be another example of the negative regulation of a peripheral increased incorporation of 14C-Leu into total AG protein, as endocrine gland by a type-I peptide. measured by organ culture in vitro [107], which is inhibited by Type-II peptides that inhibit vitellogenin synthesis or addition of SG extract. This inhibitory activity appears to be due uptake into oocytes, (vitellogenesis-or gonad-inhibiting hormones, 222 S.G. Webster et al. / General and Comparative Endocrinology 175 (2012) 217–233

VIH/GIH, hereafter referred to as VIH) have been described in the and these observations led to the hypothesis that the X-organ sinus lobster, H. americanus [202] and woodlouse, Armadillidium vulgare gland system was the source of a moult-inhibiting hormone (MIH), [78]. However, in penaeid shrimps, similar peptides have not yet which negatively regulated moulting hormone (ecdysteroid) pro- been identified, and in these crustaceans, CHHs appear to fulfil duction by the Y-organ, thus controlling preparations for moult comparable roles [10,108,222]. It is also noteworthy that none of and ecdysis [174]. More recent studies have repeatedly shown that the type-II peptides have hyperglycaemic activity, thus, their eyestalk removal leads to increases in circulating ecdysteroids con- rather specific circumscribed biological activity contrasts vividly sequent upon increased ecdysteroid synthesis by the Y-organs with those of the CHHs. [28,90,106]. Nevertheless, the nature of MIH remained elusive until A novel function of CHH concerns its role in osmo/ionoregula- development of the in vitro Y-organ bioassay [153,199,234], which tion. Perfusion of posterior (ion regulatory) gills of the euryhaline measured inhibition of ecdysteroid synthesis by Y-organs in the crab, Pachygrapsus marmoratus with SG extracts or CHH dramati- presence of SG extracts. Subsequent identification of MIH from C. cally increases transepithelial potential and Na+ influx maenas quickly followed [237], fully substantiated by Edman micro [66,179,206], and injection of CHH increases haemolymph osmo- sequencing of the hormone [231,235], which revealed that this larity and Na+ levels in eyestalk-less crayfish, A. leptodactylus hormone was structurally related to CHH. As alluded to earlier, [196]. In the penaeid shrimp, Litopenaeus vannamei, elevated levels MIH has no hyperglycaemic activity, yet in crabs, CHH has limited of a putative splice variant of CHH equivalent to that found in the activity in repressing ecdysteroid synthesis, although such biolog- pericardial organ [59] are found in shrimp exposed to reduced ical activity is not due to cross-reactivity at the receptor level: salinity, and dsRNAi showed a lethal phenotype, exhibiting gill Y-organ plasma membranes possess discrete high affinity, haemorrhage at high dsRNA concentrations [215]. Another osmo- saturable binding sites for both hormones [233]. Circulating levels regulatory role of CHH, concerns its release from cells in the fore- of MIH have been measured by RIA in C. maenas. Typically these and hindgut of C. maenas from where a massive and highly tempo- are very low (<5 fmol/ml), but occasionally reach levels of rally coordinated release of hormone from these paraneurons at 20–40 fmol/ml, during episodic release with higher levels during the start of ecdysis results in dipsogenesis and subsequent water the night than during daytime [39]. Surprisingly, there is no evi- absorption that is critical to successful ecdysis and subsequent dence to suggest that MIH levels are higher in intermoult, com- swelling to postmoult dimensions [36]. pared to premoult in this species, an observation which differs However, in an evolutionary and functional context, the discov- from the situation in crayfish, P. clarkii, where MIH levels measured ery of a peptide in locusts, S. gregaria, named ion-transport peptide using an ultra sensitive TR-FIA were about 6 fmol/ml during

(ITP), which stimulated Isc of ileal preparations in Ussing chamber intermoult, decline to about 1.3 fmol/ml during early premoult, assays (as a result of increased Cl transport) in a dose-dependent but subsequently increase to levels comparable to intermoult manner, is notable [9]. Initial (partial) Edman micro sequencing levels in late premoult and postmoult [167]. These observations, indicated that ITP was similar to CHH and full identification via and furthermore, the finding that during premoult, Y-organs cDNA library screening subsequently firmly established this pep- become entirely refractive to the inhibitory influence of MIH tide as a member of the type I CHH peptide family [155]. Since fluid [41,165,167], clearly do not support but rather deeply challenge transport across the ileum is dependent on Cl [125,126] it seems the classically accepted model of moult control. likely that an important function of ITP is concerned with water The vitellogenesis or gonad-inhibiting hormone (VIH/GIH), is conservation in xeric insects, such as locusts, and it is intriguing another Type-II hormone, long postulated from eyestalk removal to note that CHH release prior to and during ecdysis in crabs essen- experiments in decapods, where, depending on season, and physi- tially has an analogous function – water uptake by the gut [36].To ological status, this operation results in accelerated secondary date the biochemical identity of ITP (mature processed peptide) vitellogenesis due to increased synthesis or uptake of vitellogenin has only been unequivocally demonstrated in locusts and fruit flies (Reviews: [2,3,156]). A candidate VIH was first characterised in H. [7,8,63]. However, immunohistochemical studies have shown that americanus [204] and subsequently fully identified by micro ITP/CHH-like peptides are common in insects and other arthropods sequencing [202] and cDNA cloning [56]. This work has also re- (myriapods, arachnids) thus far examined [13,51,63,65,123,214] as cently been extended to include VIH in the European lobster H. also indicated from in silico analysis of expressed sequence tags gammarus [171]. To date, the only authentic VIH to be fully identi- and genomic information [33,35]. Thus, a widespread if not univer- fied in other malacostracans is from the woodlouse A. vulgare [78]. sal distribution of ITP/CHH-like peptides in arthropods seems However, it is notable that in the penaeids, identifiable Type-II hor- likely. mones corresponding to VIH type molecules appear to be absent, An interesting feature of both CHH and ITP concerns the pres- yet several CHHs can inhibit vitellogenin mRNA synthesis [108]. ence of splice variants, as discussed earlier. The CHH produced A CHH peptide (Pej-SGP-III) from SG of M. japonicus is particularly by intrinsic cells in the pericardial organs (PO-CHH) of C. maenas active in this respect [221]. Additionally, in keeping with the view seems to have no hyperglycaemic activity and does not inhibit that Type-II peptides have rather circumscribed roles, a well char- ecdysteroid synthesis [42,59]. Similarly, the corresponding locust acterised MIH (Pej-SGP-IV) from this species has no VIH activity ITP isoform (ITP-L) is inactive in the ileal bioassay, and it has been [221]. Conversely, lobster VIH has no hyperglycaemic activity postulated that this peptide may antagonise the action of ITP by [204], nor does crab MIH [237]. Nevertheless, a recent study has competing at the receptor [177]. In this context, it is interesting shown that MIH increases vitellogenin mRNA expression in midgut to note that in the blue crab, C. sapidus, saturable and displaceable gland explants of C. sapidus during mid-late vitellogenesis [247]. binding sites for PO-CHH, that are poorly competed for by SG-CHH The mandibular organ (MO) of decapods, first described by Le are present in membrane preparations from a variety of tissues, Roux in 1968 [124], is the site of synthesis of methyl farnesoate, and it was notable that the greatest number of binding sites for the un-epoxidated precursor of insect juvenile hormone III PO-CHH occur in scaphognathite preparations [93]. [16,120,122,219]. MF titres can be correlated with ovarian matura- tion in the spider crab, L. emarginata [120], and injection of JH 3.2. Type-II hormones: moult-inhibiting hormone, vitellogenesis/gonad mimics induce changes in ovarian follicle cell morphology, remi- inhibiting hormone, mandibular organ-inhibiting hormone niscent of juvenile hormone-induced patency of equivalent ovarian cells in insects [87]. Increased levels of MF have been observed in It has long been known that eyestalk removal in many decapod eyestalk-less decapods [121,122,220]. Thus, it has been suggested crustaceans often (but not always) results in accelerated moulting, that MF plays a pivotal role in crustacean reproduction [119].In S.G. Webster et al. / General and Comparative Endocrinology 175 (2012) 217–233 223 particular, these observations strongly supported the existence of a in the CNS of malacostracans include the tapetal cells in the retina mandibular organ-inhibiting hormone (MOIH). Candidate mole- of juvenile crayfish P. clarkii, which intriguingly undergo daily cules (MOIH-I, II) which inhibit MF synthesis by MO in vitro, were rhythms of immunoreactive content [69], and it has been sug- identified in the edible crab, C. pagurus by micro sequencing [227], gested that the secretion dynamics of these cells may be modu- cDNA and genomic cloning [142]. Whilst MIH and MOIH have aris- lated by serotonin release from adjacent retinular cells [68]. en by gene duplication in C. pagurus [142] and exhibit considerable With regard to the expression of CHH-like peptides in the ner- sequence identity (62%; Fig. 1D), MOIH shows only moderate activ- vous systems of non-malacostracan crustaceans, little is as yet ity in repressing ecdysteroid synthesis. These molecules are widely known, due in part to their small size, which makes dissection ex- distributed within this genus (Webster, unpublished), but structur- tremely challenging. Patterns of CHH-immunopositive neurones ally equivalent hormones have not been found in other decapods. have been determined in the branchiopods Daphnia magna and Art- Indeed, candidate MOIHs in L. emarginata and C. maenas appear emia salina [243]. Whilst immunopositive structures comparable to to be CHHs [104,136,137]. Clearly, further studies are now needed those seen in the XO-SG system are absent, peripheral neurones to clarify this situation, since it would be unusual if MOIH has only that project centrally to the nerve cord and to appendage muscles evolved in cancrid crabs. and maxillary somatic muscles may be comparable to the multipo- lar cells in the PO of malacostracans. 4. Sites of hormone synthesis and release Expression of CHH in non-neural tissues of crustaceans was first demonstrated in C. maenas. During premoult, many thousands of The most important site of synthesis and release of CHH-super- ‘‘open-type’’ paraneurons, intrinsic to the fore- and hindgut ex- family neurohormones in malacostracan crustaceans is the X-or- press a XO-CHH. In the foregut, these cells surround muscle inser- gan sinus gland system. In particular, the CHH neurosecretory tions in the pyloric stomach, suggesting a mechanoreceptive system has been described in detail by immunohistochemical tech- function [236]. In the hindgut, the same cell type is found between niques in many species of malacostracan crustaceans (For a list see the luminal epithelial cells overlying the entire longitudinal mus- [232]). In general, these studies have shown distinct localization of culature [236]. During the start of ecdysis, these cells transiently CHH in X-organ perikarya and neurohaemal endings in the sinus release their entire content of CHH resulting in a spectacular surge gland. Type-I peptide CHH immunoreactivities rarely overlap with in CHH levels [36] as shown in Fig. 4. In embryos of C. maenas, seri- those of Type-II (MIH, VIH/GIH, MOIH) peptides (Fig. 3), but in spe- ally iterated peripheral CHH-cells are found at the insertions of the cies in which more than one Type-II peptide has been identified, abdominal flexor muscles, again suggesting a mechanoreceptive co-localisation always occurs, for example, MOIH- and MIH-immu- function [40]. Expression of mRNA encoding PO-CHH has been ob- noreactivities completely overlap in SG of C. pagurus (Fig. 3d–f). In served in heart, gills and antennal gland of Macrobrachium rosen- a few instances, co-expression of Type-I and Type-II peptides oc- bergii [31] and epidermis gill and gut of L. vannamei [215]. curs, for example in M. japonicus (CHH and MIH, [197]) and H. Recently, unusual CHH encoding transcripts that uniquely do not americanus (CHH and VIH, [55,170,182])(Fig. 3g–i). In the latter include a CPRP, have been identified by in situ hybridization in case, patterns of expression seem to be particularly complicated: the epithelial cells of the spermatophore sac in Fenneropenaeus Different populations of X-organ secrete L- or D-aminoacyl stereo- chinensis [133] but as yet, it is not known whether any these tran- isomer of both neurohormones [170], although the functional sig- scripts are translated, or what may be their functional significance. nificance of such differential synthesis (and presumably release) Distribution of ITP-immunopositive neurones in the CNS of in- remains unexplored. sects has demonstrated that perikarya in the pars lateralis of the During embryonic development of C. maenas mRNA encoding brain project axons to the corpora cardiaca and allata, where re- CHH and MIH mRNA are first seen at about 50% development, lease occurs, complex arrangements of ITP interneurons in the and two pairs of perikarya in each eye, expressing these hormones brain, suboesophageal and abdominal ganglia, and neurosecretory are seen at about 75–80% development (Fig 3c) [40]. During the neurones associated with the neurohaemal organs of the periphe- larval (zoeal) stages MIH cell numbers remain invariant (4 neuro- ral nervous system [51,58,63,65]. In a situation analogous to that nes per eyestalk) [235], and in adult crabs these number 62–65 for seen in crabs, in the flour beetle T. castaneum, large numbers of CHH and 28–36 for MIH [64]. In the Norway lobster, Nephrops nor- ITP expressing paraneurons have been detected in the midgut epi- vegicus, CHH-immunopositive axon endings have been observed in thelium of late stage larvae [13]. the sinus gland of embryos at 90% development, however at this stage no VIH-immunopositive structures were seen [73]. In the 5. Control of CHH secretion lobster, both CHH and VIH-immunoreactive structures have been observed in larval stages I–IV [182]. In pursuit of the question how release of CHH-superfamily pep- With regard to the distribution of CHH-superfamily peptides in tides is regulated in the XO-SG system, investigations have focused other tissues of the central and peripheral nervous system, almost on biogenic amines and enkephalins as possible transmitters of all studies indicate that Type-II peptides are restricted to the eye- stimulatory or inhibitory activities in the nervous system. stalk neurosecretory system, although MOIH-immunoreactivity, including perikarya, axons and secretory terminals have been ob- served in the stomatogastric ganglion and anterior cardiac plexus 5.1. Serotonin (5-Hydroxy-tryptamine; 5-HT) of Cancer productus [88], and MIH-mRNA expression has been ob- served in the cerebral ganglia of C. pagurus [141]. In contrast, The role of 5-HT as a CHH releasing agent is well established. CHH is expressed in a wide variety of neural and non-neural tissues Following the initial observation that 5-HT injection in O. limosus in crustaceans. Over 25 years ago, CHH-like-immunoreactive produced marked hyperglycaemia in intact, but not in eyestalk- material was detected in extracts from PO of C. maenas by RIA less animals [102], several studies on different species have [103], and later in the ventral nerve cord of H. americanus [24]. confirmed and refined this result [127,128,138,146,186]. The effect For crabs, the source of the immunoreactive material was shown was also demonstrated more directly by measuring haemolymph to be from intrinsic multipolar neurones of the PO which express CHH levels [138,186] or CHH released in vitro from eyestalk tissue the splice variant of CHH encoded by exons I–IV [42,59,61], and incubated with 5-HT [127]. in lobsters, from neurones in the so-called second thoracic roots Pharmacological experiments have elucidated the 5-HT role of the ventral nerve cord [12,24]. Other sites of synthesis of CHH in more detail. Fluoxetine, a 5-HT reuptake inhibitor, caused signif- 224 S.G. Webster et al. / General and Comparative Endocrinology 175 (2012) 217–233

Fig. 3. Immunohistochemical localisation of CHH, MIH, VIH and MOIH in crustaceans. (a–c) Carcinus maenas. (a) Section of sinus gland double immunostained for CHH (brown, peroxidase anti peroxidase/diaminobenzidine), and MIH (black, silver enhanced immunogold. (b) Double immunostained (as in a) section of XO. Insert shows transverse section of XO-SG tract. (c) Double labelled whole mount preparation of embryo at mid-eye stage (70–85% development). Two pairs of CHH (red, Cy3) and MIH (green, FITC)-immunoreactive neurones are seen. Both peptides are not co-localised, but the sinus gland appears yellow due to stacking of the confocal image. (d–f) Cancer pagurus. Adjacent semi thin (1 lm) sections immunolabelled to show complete co-localisation of MIH-ir (d, green, FITC), MOIH-ir axon profiles (e, red, Cy 3) demonstrated by merged confocal images (f). (g–i) Homarus americanus. (g) Stacked confocal image of X-organ showing small L-VIH somata (red, Alexa 568), and larger D-VIH somata (green and yellow, Alexa 488). (h) Image of X-organ showing L-CHH (green,) L-, and D-CHH somata (orange). (i) Image of X-organ showing D-CHH (red), D-VIH (green) somata and a population of cells producing both D-isomers (orange). Scale bars a,b, d–i = 50 lm, inset b,c = 25 lm. (a,b) adapted from [64], (c) adapted from [40], (d–f) S.G. Webster (unpublished), g–i adapted from [170]. a–c, g–i with permission. icant hyperglycaemia in the crab, Chasmagnathus granulata and the an increase of CHH in the haemolymph and hormone release from crayfish, O. limosus. In the latter species, the effect was also shown XO/SG complexes in vitro [248]. However, in another study on the by elevation of haemolymph CHH [186]. Further, release of CHH same species, the opposite effect was observed: DA caused hypo- from ES-tissue in vitro was inhibited by the 5-HT-receptor antago- glycaemia in vivo, an effect that was blocked by spiperone, a DA nist methysergide [127], and employment of a number of 5-HT- receptor blocker, and DA also reduced CHH release from eyestalk receptor agonists and antagonists in P. clarkii in vivo led to the con- tissue in vitro [188]. DA was found to be without hyperglycaemic clusion that 5-HT-induced hyperglycaemia may be mediated by 5- or CHH-releasing effect in other studies on O. limosus, Palaemon

HT1- and 5-HT2-like receptor types [128]. Although several crusta- elegans [102,138,147]. Studies on the distribution of relatively cean 5-HT receptors (both 5-HT1 and 5-HT2 types) have now been large amounts of DA (tyrosine-hydroxylase immunoreactivity) in fully identified [47,172,210,225] and partly localised to eyestalk the eyestalk and especially the medulla terminalis of crayfish tissue including medulla terminalis [209], the identity of the 5- Procambarus clarkii, together with electrophysiological reactivities HT receptor type involved in control of CHH release is unknown. of small (excited by DA) and large (inhibited by DA) XO-cells [4] indicate that DA is likely to be involved in differential regulation 5.2. Dopamine of CHH- and MIH/VIH-neurons. Clearly, studies based on recently identified crustacean-specific DA receptors (both types DA1 and A role of dopamine (DA) in the release of CHH is not clear. A DA2 [45,46]) are now needed to dissect the differential dopaminer- positive response was reported in P. clarkii, in which DA caused gic neuromodulation of XO-cells under physiological conditions. S.G. Webster et al. / General and Comparative Endocrinology 175 (2012) 217–233 225

embedding electron microscopic labelling techniques showed that Leu-enkephalin- together with proctolin-immunoreactivities were both co-localised with MIH-ir granules in SG-terminals of C. mae- nas [57]. Combined EM-double immunogold labelling and autora- diography of tritiated naloxone binding sites showed localization of naloxone only at the membranes of CHH-containing axon termi- nals in the SG, whereas no binding was observed at MIH-contain- ing axon terminals [83].

6. Second messengers and signal transduction pathways

Early studies to determine the involvement of cyclic nucleotides in CHH signalling showed that injection of CHH in O. limosus, increased levels of cGMP in several tissues [195]. Subsequently, it has been shown that CHH increases cGMP in a time- and dose-dependent manner [37,76,77]. Since the effects of CHH are potentiated by phosphodiesterase inhibitors, and are associated Fig. 4. Release patterns of CHH during ecdysis in Carcinus maenas. Moult stages with increases in guanylyl cyclase (GC) activity in membrane, but have been subdivided according to % emergence from carapace in stage E (ecdysis), not cytosolic preparations of H. americanus, it seems clear that and time after emergence (min.) in post-ecdysis, stage A. n = 7–17 at each for CHH, signal transduction pathways involve a membrane bound stage.⁄P < 0.05c,d, ⁄⁄P < 0.01 a,b, ⁄⁄⁄P < 0.001 d,e, e,f. S.G. Webster, unpublished. GC, which is presumably the CHH-receptor. With regard to down- Is there evidence for secretion control of other members of the stream events involved in CHH signalling, rather less is known. CHH-superfamily by 5-HT? The inhibitory effect of ES-ganglia Eyestalk ablation increases glycogen synthase activity (conversion conditioned medium on ecdysteroid production in Y-organs of C. of inactive to active enzyme without increase in total activity) in O. antennarius cultured in vitro, which was enhanced by 5-HT in the limosus muscle tissue and this phenomenon is rapidly reversed by medium, was interpreted as evidence for a release of MIH [147]. injection of CHH [195]. Incubation of midgut gland tissues with However, at the time of this study neither the structure of CHH CHH reduces the incorporation of glucose into glycogen, again sug- nor MIH was known, and it is possible that CHH was in fact in- gesting that it reduces glycogen synthase activity, leading to glyco- volved, whose inhibitory action on Y-organ ecdysteroidogenesis genolysis [192]. Additionally, it has been suggested that CHH is now known [237]. At present, little is known about the anatomy activates phosphorylase ([195], review: [190]). Recent molecular of the serotoninergic input into the XO/SG system, apart from studies are beginning to dissect mechanisms involved in CHH sig- electron microscopic evidence for direct synaptic contacts of nalling, and indicate that CHH is involved in transcriptional regula- immunoreactive 5HT-terminals (though of unknown origin) onto tion of enzymes involved in glucose regulation. It has recently been X-organ CHH-cell dendrites in the crayfish medulla terminalis shown that in M. japonicus, eyestalk ablation increases glycogen [224]. In another neurosecretory pathway, involving cells in the synthase transcript levels, and decreases glycogen phosphorylase 2nd roots of the thoracic ganglion and the suboesophageal transcript number in midgut gland tissues (but not muscle), thus ganglion of H. americanus, which produce PO-CHH [12,24,60],a neatly complementing and extending the earlier models of CHH close apposition of 5-HT neurons to these neurons was demon- action [190,191,195]. Additionally, since eyestalk ablation did not strated by double immunolabelling [12], thus providing evidence change levels of phosphoenolpyruvate carboxykinase or fructose for a direct regulation of PO-CHH release. 1, 6-bisphosphatase transcript levels suggests that CHH is not in- volved in gluconeogenesis [161]. With regard to second messenger involvement in ITP signalling, 5.3. Enkephalins it seems likely that cAMP is a relevant signalling molecule, since it mimics the action of the peptide in that it stimulates ileal Cl There is sufficient evidence that identified enkephalins, whose transport, Na+,K+ transport and isosmotic fluid resorption [175]. presence in the crustacean eyestalk and other parts of the nervous Nevertheless, there are some important differences: Exogenous system has been well documented [57,89,143,144,181], inhibit re- cAMP does not inhibit acid secretion in the ileum, conversely ITP, + lease of CHH. In intact Uca pugilator, injection of Leu-enkephalin re- unlike cAMP does not stimulate ileal NH4 secretion [6,7]. A model sulted in a decrease of the resting glucose level, an effect which for the control of ion transport across the locust ileum has been was completely abolished by naloxone [181]. Release of CHH from proposed [48,176,178]. C. maenas ES-tissue in vitro was also inhibited by Leu-enkephalin, The emerging scenario that second messenger pathways, and as has been shown by a drastically reduced hyperglycaemic effect thus the cognate receptors of CHH and ITP are entirely different, when injected into intact Uca pugilator [181]. Corresponding re- namely that crustaceans use a membrane bound GC whilst insects sults were obtained with in vitro incubated ES-tissue from O. limo- use a GPCR given the similarity if the corresponding ligands seems sus and direct determination of CHH by immunoassay [169]. axiomatic, but would contradict contemporary views on co-evolu- Anatomical evidence of enkephalinergic input to the XO/SG sys- tion of peptides, receptors and cell signalling systems in arthro- tem supports the physiological experiments. By double immunola- pods [44]. belling, many close appositions between dendrites of CHH-XO/SG Regarding the second messengers involved in MIH signalling, a neurons and enkephalinergic fibres were demonstrated, suggesting large and somewhat disparate literature exists, and both cAMP inhibitory input via synaptic or synaptoid contacts. No enkephalin- and cGMP have been implicated in regulation of ecdysteroidogene- immunoreactivity was detected in the SG of O. limosus [169]. Other sis in a variety of crustacean species (Reviews: [50,166,207,208]). authors did, however, detect enkephalin-ir granules in distinct Pharmacological approaches, in which ecdysteroid synthesis has axon terminals in the SG of three crab species, i.e. C. maenas, U. been measured following application of cell permeant/slowly pugilator and Eriocheir sinensis by use of immunogold electron hydrolysable cyclic nucleotide analogues, demonstrate that in microscopy [57,84,89]. Interestingly, low-temperature post crayfish (P. clarkii, O. limosus) both cyclic nucleotides inhibit 226 S.G. Webster et al. / General and Comparative Endocrinology 175 (2012) 217–233

maenas and G. lateralis [49,160] coupled with observations that Y-organs express both the catalytic (b) subunit of GC-I and a Ca2+/Calmodulin dependent NO synthase clearly support the involvement of NO in the MIH signal transduction cascade [109,129,131,154]. An attractive model for the MIH signal trans- duction cascade, which neatly accounts for many of the above observations, has recently been proposed by Chang and Mykles [26] (Fig. 5), which is analogous to the signalling mechanism of cardio acceleratory peptide 2b in Drosophila Malpighian tubules (Review: [14]). However, critical analyses of all the available data do not always support this model, as has been reiterated by Nak- atsuji et al. [166], and in particular, full elucidation of cognate MIH (and CHH) receptors are now urgently needed. Whilst the MIH-mediated signalling mechanisms alluded to here relate to (facultative) allosteric regulation by phosphorylation of enzymes involved in ecdysteroidogenesis, which typically act on a time scale of minutes, it is also worthwhile to consider the evi- dence suggesting long term regulation of ecdysteroid synthesis by transcriptional or translational control, i.e. constitutive control of ecdysteroid synthesis, which acts over a much longer time scale. It has been known for many years that eyestalk ablation in- creases rates of protein and RNA synthesis by the Y-organs [72,198], and that protein synthesis by Y-organs increases during premoult [216], which is coincident with dramatic, large-scale changes in patterns of gene expression [131]. Incubation of Y-or- gans with crude sinus gland extracts specifically inhibits incorpo- ration of amino acids into protein [52,72,152]. Additionally, since cAMP analogues, isobutylmethylxanthine (IBMX) or forskolin inhi- bit protein synthesis by Y-organs [82,152] it has been proposed that MIH might be involved in such constitutive regulation, acting via this second messenger. However, these studies used crude eye- stalk or SG extracts; so, it is entirely possible that these effects are unrelated to MIH (or indeed CHH). In one study [117], a proteomic Fig. 5. Model of MIH signalling in crab Y-organs, proposed by Chang and Mykles approach was used to show that C. maenas MIH specifically down- [26]. MIH binds to a G-protein coupled receptor (MIH-R), activating adenylyl cyclase (AC), resulting in cAMP production and protein kinase A (PKA) activation. regulates expression of a transaldolase. Since this enzyme is part of Phosphorylation of enzymes via PKA may be important in constitutive control of the pentose phosphate pathway involved in NADPH production, ecdysteroid synthesis, but also by facultative regulation by phosphorylation of Ca2+ which is mandatory for cytochrome P450 production, and in view channels, leading to calmodulin (CaM) activation of nitric oxide synthase (NOS) of the observation that expression of one of these (CYP4C15) is either directly, or indirectly via calcineurin (CaN). Calmodulin can additionally upregulated in premoult Y-organs in O. limosus [53], it is feasible abrogate increases in cAMP via activation of phosphodiesterase 1 (PDE1). A nitric oxide sensitive soluble guanylyl cyclase (GC-1) then increases cGMP synthesis, that this sort of constitutive regulation may be an important part activating protein kinase G (PKG) which inhibits ecdysteroid synthesis. Phospho- of MIH signalling pathways. In view of the revolution in contempo- diesterase 5 (PDE5) can abrogate increases in cGMP, potentially modulating this rary proteomic approaches, for example those involving nano-scale inhibitory pathway during for example, premoult, when the Y-organ becomes identification of proteins from in- gel digestion of Coomassie refractory to MIH. Reprinted from [26] with permission. stained 2D polyacrylamide gels by limited MS-based sequencing and homology-based database interrogation (viz. [229]), there are ecdysteroidogenesis [165,168,194]. Likewise in crabs (C. sapidus, C. now attractive possibilities for comprehensively identifying maenas) similar effects are seen [49,183], and forskolin (a potent hormone and moult-cycle related changes in Y-organ protein adenylyl cyclase activator) profoundly inhibits ecdysteroid expression to identify MIH-mediated control points in ecdysteroi- synthesis [148,151,152,165,168,183,194]. However, in the rock crab dogenesis pathways. C. antennarius, only cAMP analogues are effective in inhibiting With regard to the second messengers and signal transduction ecdysteroidogenesis [149–152], yet in C. sapidus, the opposite is true pathways used in MOIH and VIH signalling, very little is known. where only cGMP analogues are effective [165]. Furthermore, it has In C. pagurus, there is evidence implicating cAMP as a relevant sec- been observed that both the qualitative and quantitative effect of ond messenger, since cAMP analogues mimic the action of MOIH, cyclic nucleotide analogues upon inhibition of ecdysteroidogenesis and small but dose- dependent increases in levels of this second is dependent on moult stage, and results are often contradictory messenger occur within 5 min of hormone administration [226]. ([183], reviewed in [50]). cGMP does not appear to be relevant in this signalling pathway, Measurements of profiles of intracellular cyclic nucleotides in a dichotomy which is surprising, given that MIH and MOIH appear several species of crab and crayfish following administration of to have arisen by gene duplication. purified MIH (rather than crude SG extracts) have shown that fol- lowing a rather small and transient (2-fold, 1–4 min) increase in cAMP, a few minutes after hormone addition, there is a large and 7. Binding sites and receptors sustained (20–60-fold, 30–60 min) increase in cGMP levels [11,41,165,168]. Overall, these results clearly suggest that MIH 7.1. Binding sites activates a soluble GC (viz. an NO dependent guanylyl cyclase, GC-I). Indeed, since a variety of NO donors (SNAP, SE175) and the Classical binding studies, employing 125I-labelled CHH or MIH, GC-I agonist YC-1 can inhibit ecdysteroid synthesis in vitro in C. have revealed specific, saturable and displaceable binding sites S.G. Webster et al. / General and Comparative Endocrinology 175 (2012) 217–233 227 from a considerable number of tissues and several species, e.g. O. was further suggested that structural elucidation of this putative limosus [115] C. maenas [37,115,233], C. sapidus [93] and M. japoni- receptor should be possible by cloning based on the sequence of cus [5] (for review see [43]). The tissues include Y-organs, hepato- the catalytic domain of the enzyme, which is conserved in known pancreas, fore-, mid- and hindgut, skeletal muscle (abdominal and membrane cyclases from several species. The first cloning of a scaphognathite), heart and gills. These studies clearly suggest mul- membrane guanylyl cyclase from the abdominal muscle of the tiple target tissues, underscoring the pleiotropic effects of CHH- crayfish P. clarkii by this strategy was reported by Liu et al. superfamily peptides. Kd values range, in most cases, between 4 [134]. This enzyme has the typical domain structure of known and 7 1010 M [43]. These values are functionally consistent with membrane cyclases, a predicted molecular mass of 151,185 Da, resting levels of CHH in the haemolymph, which are typically with- and was found to be expressed in muscle, hepatopancreas, heart, in 1 and 25 1011 M [25,36,37,105,115,158]. and gills – tissues which have previously been shown to respond Specificity of CHH-binding sites was first demonstrated with to CHH with an increase of cGMP in vitro and in vivo [37,195]. hepatopancreas membranes from C. maenas and O. limosus [115]. More guanylyl cyclases, both membrane bound and soluble, In the crab, the binding sites have a 10-times lower affinity for have been cloned from the land crab, Gecarcinus lateralis [130]. Orconectes-CHH compared to Carcinus-CHH, and there was almost Another putative membrane receptor guanylyl cyclase (Cs GC-YO no binding of Carcinus-CHH to the Orconectes membranes. This is 1) was identified by cloning from Y-organs of C. sapidus [245].In consistent with the observation that the CHH of each of the species this study, an antibody was raised against the extracellular domain has very little hyperglycaemic activity in others [100], and it also of the enzyme, and employed to localize the antigen in Y-organ tis- indicates that this species-specificity is not only due to the evolu- sue sections by immunohistochemistry, which showed the antigen tion of CHHs, but also to co-evolution of the putative receptors. to be restricted to the peripheral margin of cells. In addition, it was The occurrence of different CHH-superfamily peptides in single shown that pre-incubation of Y-organs with the antibody in vitro species should be paralleled by the existence of binding sites with blunted the inhibitory response to MIH in terms of ecdysteroid different specificity. This has been first demonstrated in C. maenas production. This result, together with the finding that the receptor Y-organs which have clearly distinct binding sites for CHH and MIH cyclase was more strongly expressed during intermoult and less in [233]. Katayama and Chung [93] (see also review: [43]) studied premoult are consistent with the hypothesis of the authors that Cs binding of eyestalk (ES)-CHH in comparison to PO-CHH in different GC-YO 1 is an MIH-receptor. tissues: hepatopancreas, gills, heart, abdominal muscle, scaphog- Although cGMP appears to be the predominant second messen- nathites, mid- and hindgut of C. sapidus. Binding sites for ES-CHH ger in the action of CHH-superfamily peptides, cAMP responses with similar affinities were confirmed in all tissues, while affinities have also been demonstrated, e.g. in O. limosus hepatopancreas for PO-CHH were found to be tissue-specific. Binding sites in and heart after injection of CHH [195]. Recently, Zmora et al. abdominal and scaphognathite muscle displayed the highest affin- [246,247] have provided evidence that MIH binding sites increase ity, whereas, e.g. the membrane preparations from hepatopancreas markedly in the hepatopancreas from C. sapidus during an ad- and gills do not appear to have affinity for PO-CHH. It remains to be vanced ovarian stage, and that they are signalling via cAMP. As established whether this reflects the existence of real, structurally shown by in vitro incubation of hepatopancreas fragments, these different receptor types [93]. Differences between target tissues binding sites appear to mediate a cAMP response to MIH and are have also been demonstrated in the number of binding sites unresponsive to CHH, whilst CHH elevates cGMP in the same prep- (Review: [43]). aration by binding to a different receptor, i.e. probably the mem- A structural difference between binding sites for CHH and MIH brane guanylyl cyclase. The nature of this MIH receptor is was shown by photo affinity binding of labelled ligand and subse- unknown. In fact, no cAMP signalling receptor for CHH-superfam- quent SDS–PAGE of the resulting ligand/receptor complex, which ily peptides, presumably a G-protein coupled receptor (GPCR), has yielded values of 51 kDa for MIH- and 61 kDa for CHH-binding thus far been identified in any crustacean species. sites in Y-organs and hepatopancreas of C. sapidus [246]. The same technique yielded a mass of 70 kDa for MIH-binding sites in Y-organs of M. japonicus [5]. However, it is unclear how these mass 8. Concluding remarks values relate to the molecular mass (151 kDa) of the cloned puta- tive membrane receptor guanylyl cyclase from crayfish muscle From a large number of crustacean species, many (about 80) [134] (see below). CHH-superfamily peptides have thus far been fully identified by various methods. In comparison to the number of publications on 7.2. Receptor(s) identification, studies on biological activities remain limited. Undoubtedly, many more biological activities are awaiting discov- As discussed above, signalling via cGMP appears to be the pre- ery. We hope it is clear from the foregoing text that this field offers dominant second messenger mechanism in the action of CHH- much opportunity for further rewarding work. Moreover, many of superfamily peptides. This suggests guanylyl cyclase(s) as recep- the already studied actions require more in-depth- analysis. This is tors. An important first contribution is the detailed study by Goy vividly illustrated by recent studies on MIH regulation of Y-organs in 1990 [76] on H. americanus skeletal muscle. He observed a dras- which make it increasingly clear that this is far more complex than tic (50–100 fold) increase of cGMP in muscle fragments incubated the simple, long-held hypothesis suggested, namely that Y-organ with CHH in vitro. That the response was potentiated by the phos- activity is regulated solely by MIH levels in the haemolymph phodiesterase inhibitor IBMX showed that the primary effect of [26]. Indeed, our knowledge of the central role of CHH in blood glu- CHH on cGMP levels is due to stimulation of the cyclase rather than cose regulation is still incompletely understood, but transcriptomic to an inhibition of phosphodiesterase. After fractionation of muscle studies are now beginning to shed light on cellular regulatory homogenate into particulate/membrane (100,000 g sediment) and mechanisms [131,161,229]. soluble fractions and incubation with CHH, it was found that CHH If we compare biological activities in different species, results enhanced cGMP production in the membrane fraction, with little are sometimes inconsistent or even contradictory. Considering or no effect on soluble guanylyl cyclase activity (which is reason- this, we should be aware of the fact that studies have been, and still ably abundant in lobster muscle). In conclusion, it was shown that are, performed on various crustacean species which differ in terms a CHH-responsive membrane-bound guanylyl cyclase exists which of affiliation to often only distantly related systematic groups and appears to explain the marked increase of cGMP in intact muscle. It consequently disparate life histories and physiologies. There has 228 S.G. 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