Evolution of the AKH/corazonin/ACP/GnRH receptor superfamily and their ligands in the Protostomia

General and Comparative Endocrinology xxx (2014) xxx–xxx

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General and Comparative Endocrinology

journal homepage: www.elsevier .com/locate /ygcen

Evolution of the AKH/corazonin/ACP/GnRH receptor superfamilyand their ligands in the Protostomia

http://dx.doi.org/10.1016/j.ygcen.2014.07.0090016-6480/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Address: Center for Functional and Comparative InsectGenomics, Department of Biology, University of Copenhagen, Universitetsparken15, DK-2100 Copenhagen, Denmark.

E-mail address: [email protected] (C.J.P. Grimmelikhuijzen).

Please cite this article in press as: Hauser, F., Grimmelikhuijzen, C.J.P. Evolution of the AKH/corazonin/ACP/GnRH receptor superfamily and their ligthe Protostomia. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.07.009

Frank Hauser, Cornelis J.P. Grimmelikhuijzen ⇑Center for Functional and Comparative Insect Genomics, Department of Biology, University of Copenhagen, Denmark

a r t i c l e i n f o a b s t r a c t

Article history:Available online xxxx

Keywords:GPCRNeurohormoneNeuropeptideEcdysozoaLophotrochozoaEvolution

In this review we trace the evolutionary connections between GnRH receptors from vertebrates and thereceptors for adipokinetic hormone (AKH), AKH/corazonin-related peptide (ACP), and corazonin fromarthropods. We conclude that these G protein-coupled receptors (GPCRs) are closely related andhave a common evolutionary origin, which dates back to the split of Proto- and Deuterostomia, about700 million years ago. We propose that in the protostomian lineage, the ancestral GnRH-like receptorgene duplicated as did its GnRH-like ligand gene, followed by diversification, leading to (i) a corazoninreceptor gene and a corazonin-like ligand gene, and (ii) an AKH receptor gene and an AKH-like ligandgene in the Mollusca and Annelida. Subsequently, the AKH receptor and ligand genes duplicated oncemore, yielding the situation that we know from arthropods today, where three independent hormonalsystems exist, signalling with AKH, ACP, and corazonin. Our model for the evolution of GnRH signalingin the Protostomia is a striking example of receptor–ligand co-evolution. This model has been developedusing several bioinformatics tools (TBLASTN searches, phylogenetic tree analyses), which also helped usto annotate six novel AKH preprohormones and their corresponding AKH sequences from the followingmolluscs: the sea hare Aplysia californica (AKH sequence: pQIHFSPDWGTamide), the sea slugTritonia diomedea (pQIHFSPGWEPamide), the fresh water snail Bithynia siamensis goniomphalos(pQIHFTPGWGSamide), the owl limpet Lottia gigantea (pQIHFSPTWGSamide), the oyster Crassostrea gigas(pQVSFSTNWGSamide), and the freshwater pearl mussel Hyriopsis cumingii (pQISFSTNWGSamide). Wealso found AKHs in the tardigrade Hysibius dujardini (pQLSFTGWGHamide), the rotifer Brachionuscalycifloros (pQLTFSSDWSGamide), and the penis worm Priapulus caudatus (pQIFFSKGWRGamide). Thisis the first report, showing that AKH signaling is widespread in molluscs.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction showing that neuropeptides and their GPCRs must have an early

Multicellular animals can be subdivided into two evolutionarylineages, the Deuterostomia, such as vertebrates and some smallergroups of invertebrates, and the Protostomia to which the majorgroups of invertebrates belong, such as arthropods, nematodes,molluscs, and annelids (Fig. 1). The Protostomia and Deuterostomiahave a common evolutionary origin and separated about700 million years (MYR) ago (Douzery et al., 2004).

Neuropeptides and their G protein-coupled receptors (GPCRs)play a central role in the physiology and behavior of all proto-and deuterostomian animals. Some animal groups that originatedbefore the Protostomia/Deuterostomia split, such as the Cnidaria,already have a nervous system that is strongly peptidergic,

evolutionary origin (Grimmelikhuijzen et al., 1996, 2002;Grimmelikhuijzen and Hauser, 2012a; Mirabeau and Joly, 2013;Jekely, 2013).

Traditionally, neuropeptides have been mostly investigated inisolated animal groups, for example only in mammals or onlyinsects and there have been very few examples where neuropep-tides that were first discovered in Deuterostomia were also identi-fied in the Protostomia and the other way around. One suchexample, however, is oxytocin and vasopressin that were first iso-lated and sequenced from cow brains in 1953 (Acher and Chauvet,1953; Du Vigneaud et al., 1953) and that forty years later were alsoidentified in molluscs, annelids, arthropods and nematodes (Prouxet al., 1987; Van Kesteren et al., 1995; Satake et al., 1999; Kandaet al., 2003, 2005; Levoye et al., 2005; Stafflinger et al., 2008;Aikens et al., 2008; Garrison et al., 2012; Beets et al., 2012). Thepresence of oxytocin/vasopressin-related peptides and their GPCRsin Proto- and Deuterostomia implies that these peptides must haveoriginated before the split of these two evolutionary lineages.

ands in

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Cnidaria 9,000(sea anemones, corals,hydras, jellyfishes)

Platyhelmintha 13,000(flatworms)

Nematoda 20,000(roundworms)

Annelida 12,000(earthworms, leeches)

Mollusca 85,000(clams, snails, squids)

Arthropoda 1,500,000(insects, crustaceans,spiders, ticks)

Echinodermata 7,000(starfishes, sea urchins)

Chordata 60,000(vertebrates, mammals,humans)

700 million years ago

Deuterostomia Protostomia

Porifera 5,000(sponges)

Tardigrada 1,200(waterbears)

Priapulida 16(penis worms)

Rotifera 2,000(wheel animals)

Ecdyso-zoa

Lopho-trochozoa

Fig. 1. Simplified phylogenetic tree of multicellular animals. The tree shows two evolutionary lineages, the Prostomia (blue line) and Deuterostomia (red line), that splitabout 700 MYR ago (Douzery et al., 2004). The numbers in green give estimated species numbers belonging to a certain phylum. Most invertebrates belong to the Protostomiawith the Arthropoda being the largest group comprising more than 90% of all multicellular animals. Not all protostomian phyla are shown here; only those that are discussedin the text. The phyla, where we have identified novel AKH peptides (Fig. 7, Table 3) are highlighted in light blue. These results show that AKH signaling is wide-spread inEcdysozoa and Lophotrochozoa.

2 F. Hauser, C.J.P. Grimmelikhuijzen / General and Comparative Endocrinology xxx (2014) xxx–xxx

Furthermore, the presence of deuterostomian neuropeptides suchas oxytocin and vasopressin and their receptors in protostomiansalso offers the possibility to investigate these neuropeptidesystems in well-established invertebrate models such asCaenorhabditis elegans, and Drosophila melanogaster. These studiesin invertebrate models might eventually contribute to a betterunderstanding of the actions of the corresponding neuropeptidesin mammals (Grimmelikhuijzen and Hauser, 2012a).

One of the reasons that so few common neuropeptides havebeen found in Deuterostomia and Protostomia, might be thatneuropeptide sequences are generally relatively short, making itdifficult to track them using TBLASTN searches in a sequencedanimal genome. However, when instead of the neuropeptidesequences, the receptor sequences (which are much longer thanthe neuropeptide sequences) are used in TBLASTN searches, GPCRorthologues can more readily be identified and evolutionaryrelated hormonal systems more easily be established. In thefollowing paragraphs we will illustrate this phenomenon for threestructurally related insect neuropeptides and their GPCRs.

2. Materials and methods

To identify novel peptides and receptors, TBLASTN searcheswere performed at http://blast.ncbi.nlm.nih.gov/Blast.cgi. Thesame link gives access to the sequence databases of the differentspecies given in Fig. 2. Whole-genome Shotgun Contigs (WGS)and Expressed Sequence Tags (EST) databases were searched withknown protein sequences as query.

Phylogenetic analysis of the receptors was performed with thededuced entire protein sequences using the Maximum Likelihoodmethod at the web service Phylogeny.fr (http://www.phylogeny.

Please cite this article in press as: Hauser, F., Grimmelikhuijzen, C.J.P. Evolutionthe Protostomia. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.yg

fr/version2_cgi/alacarte.cgi; Dereeper et al., 2008). Sequences werealigned with CLUSTALW, using the BLOSUM62 substitution matrix.The alignment was curated with GBlocks allowing small finalblocks, gap positions within the final blocks and less stringentflanking positions. Phylogeny was calculated using PhyML with100 bootstrap replicates. The tree was rooted with human rhodop-sin and rendered using TreeDyn. A second phylogenetic tree of thesame protein sequences was calculated with the Neighbor-Joiningmethod using the MAFFT online version (www.mafft.cbrc.jp; Katohand Standley, 2013). The tree was made with the JTT method andresampled with 100 bootstrap X JTT replicates.

Phylogenetic analysis of the neuropeptides was done with thecleaved, immature neuropeptide sequences (highlighted in yellowand blue in Fig. S5). Sequences were aligned using CLUSTALW anda phylogenetic tree was calculated with the Maximum Likelihoodmethod using the web service Phylogeny.fr with 100 bootstrapreplicates.

3. Adipokinetic hormone and its receptor

Insect adipokinetic hormones (AKHs) are neuropeptides pro-duced by intrinsic neuroendocrine cells of the corpora cardiaca,two small neurohemal organs lying behind the insect brain oneither side of the aorta. These neuropeptides have rather variablestructures in the various insects, although their hallmarks are con-served. These hallmarks are: (i) a length of either 8 or 10 aminoacid residues; (ii) a blocked N terminus (pQ) and C terminus(amide); (iii) an aliphatic amino acid residue in position 2; (iv)an FS, FT or YS sequence at positions 4 and 5; (v) a W residue atposition 8; (vi) either a Wamide (of an 8 amino acid-acid longAKH), or WGXamide C terminus (of a 10 amino-acid long AKH)

of the AKH/corazonin/ACP/GnRH receptor superfamily and their ligands incen.2014.07.009

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://www.phylogeny.fr/version2_cgi/alacarte.cgi

http://www.phylogeny.fr/version2_cgi/alacarte.cgi

http://www.mafft.cbrc.jp


Drosophila simulans (fruit fly)Drosophila sechellia (fruit fly)Drosophila melanogaster (fruit fly)Drosophila yakuba (fruit fly)Drosophila erecta (fruit fly)Drosophila ananassae (fruit fly)Drosophila pseudoobscura (fruit fly)Drosophila persimilis (fruit fly)Drosophila willistoni (fruit fly)Drosophila mojavensis (fruit fly)Drosophila virilis (fruit fly)Drosophila grimshawi (fruit fly)Ceratitis capitata (Mediterranean fruit fly)Glossina morsitans (tsetse fly)Mayetiola destructor (Hessian fly)

Phlebotomus papatasi (sand fly)Lutzomyia longipalpis (sand fly)

Culex pipiens (mosquito)Aedes aegypti (mosquito)Anopheles darlingi (mosquito)Anopheles gambiae (mosquito)

Bombyx mori (silkworm)

Heliconius melpomene (postman butterfly)

Helicoverpa armigera (cotton bollworm)Helicoverpa zea (cotton bollworm)

Manduca sexta (tobacco hornworm)

Tribolium castaneum (flour beetle)Atta cephalotes (leaf-cutter ant)Acromyrmex echinatior (leaf-cutter ant)Pogonomyrmex barbatus (red harvester ant)Solenopsis invicta (red imported fire ant)Camponotus floridanus (Florida carpenter ant)Linepithema humile (Argentine ant)Harpegnathos saltator (Jerdon's jumping ant)Apis mellifera (honey bee)Apis florea (dwarf honey bee)Bombus terrestris (large earth bumble bee)Bombus impatiens (common eastern bumble bee)

Nasonia giraulti (jewel wasp)

Diptera

Lepidoptera

Coleoptera

Hymenoptera

Hemiptera

Cladocera

Ixodida

Phthiraptera

Siphonostomatoida

Nasonia longicornis (jewel wasp)Nasonia vitripennis (jewel wasp)

Pediculus humanus (human body louse)

Acyrthosiphon pisum (pea aphid)Rhodnius prolixus (blood sucking bug)

Lepeophtheirus salmonis (sea louse)

Daphnia pulex (water flea)Dapnia magna (water flea)

divergence time (million years)0600 300

Ixodes scapularis (dear tick)

Galendromus occidentalis (Western predatory mite)Varroa destructor (honey bee mite)

Tetranychus urticae (spider mite)

Hex

apod

a / I

nsec

taC

rust

acea

Che

licer

ata

Megachile rotundata (leafcutter bee)

Strigamia maritima (centipede) Geophilomorpha Myr

iapo

da

order subphylum/class

Mesostigmata

Trombidiformes

Fig. 2. Phylogenetic tree of fifty-three arthropod species, which genomes have been sequenced or are in the process of being sequenced. This tree is not complete and thereare many other arthropod genome projects in the pipeline, for example the ones that are included in the i5k (5000 insects) genome project (Robinson et al., 2011). Adaptedfrom Grimmelikhuijzen and Hauser, 2012b.

F. Hauser, C.J.P. Grimmelikhuijzen / General and Comparative Endocrinology xxx (2014) xxx–xxx 3

(Gäde et al., 1997) (Table 1). The AKHs play a role in carbohydrate(trehalose) homeostasis in insect larvae (Kim and Rulifson, 2004)and mobilize lipids and carbohydrates from the fat body duringflight or intense locomotion in adult insects (Gäde et al., 1997).Thus, although structurally not related, the AKH neuropeptideshave the role in insects that glucagon and adrenaline have in ver-tebrates (Kim and Rulifson, 2004).

The AKHs occur in every insect with a sequenced genome(Fig. 2), with some insects having two copies of the AKH gene(Gäde et al., 1997; Noyes and Schaffer, 1990; Hansen et al., 2010).


The AKHs also occur in crustaceans where they are named red-pig-ment-concentrating-hormone (RPCH), which has a function inbackground color adaption (camouflage) (Fernlund and Josefsson,1972; Carlsen et al., 1976; Dircksen et al., 2011; Christie et al.,2011). The evolutionary origin of the AKH peptides in arthropodsbelow the level of crustaceans (Fig. 2) is unclear, because in annota-tion analyses of the genome from the spider mite Tetranychus urtica(Chelicerata) or of an expressed sequence tag (EST) library from thetick Ixodes scapularis (Chelicerata) no obvious AKH genes could beidentified (Christie, 2008; Veenstra et al., 2012).



Table 1Comparison of the amino acid sequences of the locust (L. migratoria) AKH-I as an example of an AKH having 10 amino acid residues, the mosquito (A. gambiae) AKH (having 8residues), ACP, corazonin, CCAP, and the mouse (Mus musculus) GnRH. Gaps are introduced to give maximal alignments. Amino acid residues that are identical in at least three ofthe six peptides are highlighted in red. Residues that are identical in two of the six peptides are highlighted in green and conserved residues are highlighted in blue. Thesealignments show that AKH, ACP, and corazonin are structurally related, with ACP being intermediate between AKH and corazonin.

Neuropeptide name Amino acid sequence Neuropeptide length (number of residues) Species

AKH-I (Adipokinetic Hormone-I) pQLNF--TPNWGTamide 10 L. migratoria

AKH (Adipokinetic Hormone) pQLTF--TPAW--amide 8 A. gambiae

ACP (AKH/corazonin-related peptide) pQVTF--SRDWNAamide 10 A. gambiae

Corazonin pQ-TFQYSRGWTNamide 11 A. gambiae

GnRH (Gonadotropin-Releasing Hormone) pQ--HWSYGLRPGamide 10 M. musculus

CCAP (Crustacean Cardioactive Peptide)

PFCNAFTGC----amide9 A. gambiae


From this very short overview given above, the main message isthat the AKHs probably occur in all insects and crustaceans andthat they play a role in carbohydrate homeostasis, lipid and carbo-hydrate mobilization, and probably also other physiological adap-tations related to locomotion and stress.

In 1998, we cloned a G protein-coupled receptor from D. mela-nogaster that was structurally related to the GnRH receptors frommammals (Hauser et al., 1998) and in 2002 we purified and iden-tified its endogenous ligand from D. melanogaster third instar lar-vae. To our surprise, this ligand turned out to be AKH (Staubliet al., 2002), a conclusion that half a year later was confirmed byother workers in the field (Park et al., 2002). These results left uswith the question what carbohydrate homeostasis and carbohy-drate and lipid mobilization (AKH) in insects have to do with sexand reproduction (GnRH), a question that still has not been solved(Staubli et al., 2002).

Fig. S1 (Supplementary Data) shows an alignment of variousarthropod AKH receptors. This alignment shows several blocks ofidentical amino acid residues especially in the TM I to TM VIIregion. Furthermore, the AKH receptor genes have five commonintrons. Two of them are unique for the AKH receptors (one inthe TM II and another one shortly after TM III), while one intron(positioned in TM IV) is common to the AKH, ACP, corazonin, andmammalian GnRH receptors (Fig. S1). This last finding is remark-able and confirms that the AKH, ACP, corazonin, and GnRH recep-tors are evolutionarily related (see also below).

4. Corazonin and its receptor

Corazonin (from corazón, Spanish for heart) is an eleven aminoacid-long neuropeptide that was originally isolated from the cor-pora cardiaca of the cockroach Periplaneta americana, because ofits cardio-excitatory actions on the isolated cockroach heart(Veenstra, 1989). Corazonin has some structural similarities withthe AKHs, but it is one to three amino acid residues longer (Table 1).In contrast to the AKHs, the structure of corazonin in insects is rel-atively invariable, with the structure shown in Table 1 ([Arg7]-corazonin) occurring in most insects, while only a few isoformsare known: [His7]-corazonin (for example in locusts), [Gln10]-corazonin (American crane fly), [His4, Gln7]-corazonin (for examplein Manthis religiosa), [Thr4, His7]-corazonin (the honey bee Apismellifera), and [Tyr3, Gln7, Gln10]-corazonin (the bumble bee Bom-bus soroeensis) (Verleyen et al., 2006; Predel et al., 2007). The beetleTribolium castaneum and the pea aphid Acyrthosiphon pisum do nothave corazonin (Li et al., 2008; Richards et al., 2008, 2010;Huybrechts et al., 2010).

[Arg7]-corazonin is also present in crustaceans (Ma et al., 2008;Dircksen et al., 2011; Christie et al., 2011), but in Chelicerata(Fig. 2) it could not be identified in the spidermite (Veenstraet al., 2012), while it was identified in an EST library from the tickI. scapularis (Christie, 2008).


Although corazonin was originally isolated because of its car-dio-excitatory actions on the isolated heart, these activities have,so far, only been documented in P. americana and the blood-suck-ing bug Rhodnius prolixus (Veenstra, 1989; Patel et al., 2014). Inother insects, a wide spectrum of biological roles has been assignedto corazonin, ranging from a regulator of insulin producing cells inthe brain of D. melanogaster (Nässel et al., 2013), a coordinator ofsperm transfer and copulation duration in male D. melanogaster(Tayler et al., 2012), an inducer of a black body coloration pattern(melanization) characteristic for crowding and pre-swarming inlocusts (Tawfik et al., 1999), and an initiator of ecdysis in moths(Kim et al., 2004). It is difficult to find a common denominatorfor all these activities, but the role of corazonin might be relatedto feeding, stress, and perhaps to starvation-induced stress(Veenstra, 2009; Boerjan et al., 2010).

A corazonin receptor has been cloned and deorphanized in D.melanogaster (Cazzamali et al., 2002; Park et al., 2002) and in themalaria mosquito Anopheles gambiae (Belmont et al., 2006).Fig. S2 shows an alignment of the corazonin receptors from thesame species as given in Fig. S1 (except for the beetle T. castaneum,which does not have a corazonin signaling system). This alignmentshows that the arthropod corazonin receptors are closely related.This conclusion is confirmed at the genomic level by the presenceof three common introns (Fig. S2). As mentioned earlier, the intronin TM IV is common to the superfamily of corazonin, AKH, ACP, andGnRH receptors, while the introns shortly after TM V and TM VI arecorazonin gene-specific.

5. Adipokinetic hormone/corazonin-related peptide (ACP) andits receptor

When we identified the AKH and corazonin receptors from A.gambiae, we also cloned two other structurally related receptorsfrom this mosquito and identified one of them as crustacean cardi-oactive peptide (CCAP) receptor (Cazzamali et al., 2003), while thesecond was an orphan receptor, where no ligand could be found inour insect neuropeptide library (Belmont et al., 2006). CCAP is asmall cyclic neuropeptide that is not related to AKH and corazonin(Table 1). Thus, although the CCAP receptor is structurally relatedto the AKH and corazonin receptors, the CCAP receptor ligand isquite different from AKH and corazonin. This suggests a ligandexchange during the evolution of these three receptors.

To find a possible ligand for the above-mentioned mosquitoorphan receptor, we searched the genome database from A. gam-biae, using TBLASTN and various insect AKH and corazoninsequences. These searches resulted in the identification of a neuro-peptide gene that, after processing, yielded a ten amino acid-longneuropeptide with a structure that was intermediate between A.gambiae AKH and A. gambiae corazonin (Table 1). Based on thesestructural relationships, we dubbed the peptide AKH/corazonin-related peptide, or ACP (Hansen et al., 2010).




In subsequent experiments, A. gambiae ACP turned out to be theendogenous ligand for the A. gambiae orphan receptor (Hansenet al., 2010). Moreover, A. gambiae ACP did not activate the A. gam-biae AKH and corazonin receptors, while A. gambiae AKH andcorazonin did not activate the A. gambiae ACP receptor (Hansenet al., 2010). Thus AKH, corazonin, and ACP and their specific recep-tors are three independent hormonal systems. Yet, these receptorsare structurally closely related as are their ligands (Table 1). Thesefindings, therefore, suggest a co-evolution of receptors and ligandsby means of gene duplication and subsequent diversification

ACPR

CRZR

CRZR

ACPR

A

CRZR AKHR

a-GnRHR

Octopus, Crassostrea, Lottia, Aplysia, Capitella, Helobdella

Mollusca/Annelida

ArthropodaDrosophila, Apis, Daphnia

mosquitoes, Bombyx, Nasonia, Rhodnius

Tribolium , pea aphid

Fig. 3. A proposed scenario of the evolution and coevolution of the AKH/corazonin/ACreceptors or corazonin receptor-like receptors; ACPR = ACP receptors; AKHR = AKH recpropose that ancestral GnRH-like receptors and ligands were present during early protostbefore the emergence of Mollusca and Annelida and diverged, leading to corazonin-recligands. In molluscs and Ecdysozoa, the AKH-like ligand evolved to canonical AKHs (Thormonal system duplicated once again before the emergence of the Arthropoda, leadinNote that in some arthropod species either the corazonin or ACP signalling system was

Table 2A comparison of the amino acid sequences of the chicken and sea urchin GnRHs with that ofresidues in common with either chicken GnRH-1 or -2. This figure shows that all sequencegrouped in a different way, showing that some peptides that were originally published as bFigs. 7 and 8.

Neuropeptide name Amino acid sequence Species name

Chicken GnRH-1 pQ--HWSYGLQPG---amide Gallus gallusChicken GnRH-2 pQ--HWSHGWYPG---amide Gallus gallusSea urchin GnRH pQVHHRFSGWRPG---amide StrongylocentrOctopus GnRH pQNYHFSNGWHPG---amide Octopus vulgaCrassostrea GnRH pQNYHFSNGWQP----amide Crassostrea giAplysia GnRH pQNYHFSNGWYA----amide Aplysia califorLottia GnRH pQHYHFSNGWKS----amide Lottia giganteaCapitella GnRH-1 pQAYHFSHGWFP----amide Capitella teletaCapitella GnRH-2 pQ-FSFSLPGKWGN--amide Capitella teletaCapitella GnRH-3 pQGFSFSLPGKWGGA-amide Capitella teletaHelobdella GnRH pQ-FSFTPPGKWPFGTamide Helodelda robC. elegans AKH-GnRH pQ-MTFTDQWT CaenorabditisDrosophila AKH pQ-LTFSPDW------amide Drosophila meCrab RPCH pQ-LNFSPGW------amide Cancer borealiAplysia AKH pQ-IHFSPDWGT----amide Aplysia califorLottia AKH pQ-IHFSPTWGS----amide Lottia giganteaCrassostrea AKH pQ-VSFSTNWGS----amide Crassostrea giDrosophila corazonin pQTFQYSRGWTN----amide Drosophila me


(Fig. 3). The model of Fig. 3 will be discussed in more detail atthe end of this review.

The A. gambiae ACP amino acid sequence is identical to thatfrom other mosquitoes (Aedes aegypti, Culex pipiens), the blood-sucking bug R. prolixus, and the crustacean Speleonectes lucayensis,while it is very similar to ACPs found in other arthropods (Hansenet al., 2010; Christie, 2014). In fact, most arthropods have theN-terminal sequence pQVTFSRDW in common with A. gambiaeACP, while only the C-terminal XXamide sequence varies (Table 1)(Hansen et al., 2010). Thus, in contrast to the AKHs, the ACPs are

AKHR

AKHR

AKHR

ACP

CRZ

CRZ AKH

AKH

AKH

ACP

B

CRZ AKH

a-GnRH

P/GnRH receptors (A) and their ligands (B) in the Protostomia. CRZR = corazonineptors; a-GnRHR = ancestral GnRH receptor; a-GnRH = ancestral GnRH. Here, weome evolution (bottom part of the figure). The genes for these molecules duplicatedeptor-like receptors and AKH-receptor-like receptors and corazonin- and AKH-likeable 3), implicating that also their receptors are genuine AKH receptors. The AKHg to the AKH and ACP systems, in addition to the corazonin system that persisted.lost (Hansen et al., 2010).

protostome GnRH-like sequences. The amino acid residues highlighted in red indicates are related in various degrees to chicken GnRH. The peptide sequences can also beeing GnRHs are, in fact, more AKH- or corazonin related. Such groupings are shown in

References Phylum

King and Millar (1982) ChordataMiyamoto et al. (1984) Chordata

otus purpuratus Rowe and Elphick (2012) Echinodermataris Iwakoshi et al. (2002) Molluscagas Bigot et al. (2012) Molluscanica Zhang et al. (2008) Mollusca

Veenstra (2010) MolluscaVeenstra (2011) AnnelidaVeenstra (2011) AnnelidaVeenstra (2011) Annelida

usta Veenstra (2011) Annelidaelegans Lindemans et al. (2009) Nematodalanogaster Schaffer et al. (1990) Arthropodas Li et al. (2003) Arthropodanica This paper Mollusca

This paper; Roch et al. (2014) Molluscagas This paper Molluscalanogaster Veenstra (1994) Arthropoda




highly conserved, a property that it has in common withcorazonin.

Fig. S3 shows that the structures of the ACP receptors from A.gambiae, and A. aegypti are nearly identical (when the TM I to VIIregions are compared), despite the fact that these mosquito speciesseparated 150–200 million years ago (Krzywinski et al., 2006). Alsothe structures from the other arthropod ACP receptors are verysimilar to the mosquito receptors (Fig. S3). The ACP receptor geneshave one unique intron in common (just after TM II), which is notfound in the AKH or corazonin receptor genes. It is interesting thatmost ACP receptor genes share one intron with the AKH receptorgenes (located in TM VI). This intron is not present in any of thecorazonin receptor genes (Fig. S2), which would suggest that theACP receptor genes are evolutionarily more related to the AKHreceptor genes, which is also reflected in our cartoon of Fig. 3.

So far, nothing is known about the biological actions of ACP,except that it activates its own specific receptor (Hansen et al.,2010). Patel et al. (2014) found that ACP does not elevate hemo-lymph lipid concentrations in R. prolixus, while R. prolixus AKHdoes. Also, corazonin increases heart-beat frequency in vitro, whileR. proxilus ACP and AKH fails to do so (Patel et al., 2014). Thus,although structurally very similar, the actions of ACP do apparentlynot coincide with those of AKH and corazonin.

6. Gonadotropin releasing hormone (GnRH)

A companion paper of this Special Issue is focusing on the evo-lution of deuterostomian GnRHs and their receptors (Sherwoodet al., in preparation; see also Roch et al., 2011, 2014). We willtherefore confine ourselves to only giving a short overview ofGnRH-like peptides and their receptors in the Protostomia.

Protostomian GnRH-like peptides have first been identified inseveral molluscan species and later in nematodes and annelids(Fig. 1). These peptides were named GnRHs or GnRH-like peptides,although the similarities between them and vertebrate GnRHswere not always very convincing (Iwakoshi et al., 2002; Zhanget al., 2008; Onitsuka et al., 2009; Lindemans et al., 2009;Veenstra, 2010, 2011; Bigot et al., 2012) (Table 2). The GnRH-likepeptide from Octopus vulgaris is the one that mostly resembles ver-tebrate GnRH, although this molluscan peptide is two amino acidresidues longer than its vertebrate counterparts (Table 2). Table 2also shows that it is hard to assign the molluscan and annelid pep-tides to being GnRH-, AKH-, ACP-, or corazonin-like and to drawconclusions about the evolution of these neurohormones. The evo-lutionary relationships between these four hormonal systems,however, become much clearer, when also their receptors are com-pared. Such comparisons are carried out in the next chapter.

7. Phylogeny of the protostomian AKH, ACP, corazonin, andGnRH receptors

To establish the evolutionary relationships between arthropodAKHs, ACPs, corazonins and the protostomian and vertebrateGnRHs, we compared their GPCRs. Fig. 4 shows an alignment ofthe transmembrane regions (TM I-VII) of the AKH, ACP, and corazo-nin receptors from four insect species (the wasp Nasonia vitripen-nis, the mosquito A. gambiae, the silkworm Bombyx mori, and thefruitfly D. melanogaster), one mollusc (the oyster Crassostrea gigas),and the human GnRH receptor sequence. This figure shows thatthere are remarkable amino acid residue identities and conserva-tions between the four receptors, as can be seen in the sequencelogo’s given in Fig. 4. Several of these identical residues belong tothe hallmarks of the neuropeptide family A GPCRs (marked byfilled triangles in Fig. 4; Venkatakrishnan et al., 2013), while othersappear to be characteristic for the AKH/ACP/corazonin/GnRHreceptor superfamily and might be part of its signature (marked


by asterisks in Fig. 4). These apparent superfamily-specific residuesare: Trp (at arbitrary position 72 of Fig. 4); Pro (position 117); Gln(position 145); Phe (position 149); Gln (position 166); Tyr (posi-tion 182); Leu (position 195); Ala (position 252); Leu (position257); Tyr (position 274); and Gly (position 315). Many of thesesuperfamily-specific residues (asterisks) are located in one of theextracellular loops of the GPCRs (red lines in Fig. 4) and are goodcandidates for being ligand binding residues. Gln-145 and Phe-149, for example, have already been recognized as ligand-bindingresidues in the human GnRH receptor (Forfar and Lu, 2011).

The presence of the superfamily-specific residues in Fig. 4already suggests that the AKH, ACP, corazonin, and GnRH receptorsare evolutionarily related. To obtain a better insight into their evo-lutionary relationships, we carried out phylogenetic tree analyses,using two different calculation methods (Figs. 5 and 6). The resultsof these analyses are presented in Fig. 5 and show several GPCRclusters, which are summarized, from top to bottom, as follows:

1. An arthropod AKH receptor cluster (highlighted in light blue).2. A molluscan (Crassostrea, Lottia), annelid (Helobdella, Capitella),

and nematode (Caenorabditis) AKH receptor cluster (also high-lighted in light blue).

3. An arthropod ACP receptor cluster (highlighted in dark blue)4. An arthropod corazonin receptor cluster (highlighted in dark

brown).5. An annelid (Capitella) and molluscan (Crassostrea, Lottia,

Octopus) receptor cluster (highlighted in light brown), whichis located between the corazonin receptor and the GnRH recep-tor cluster. This group is, therefore, named corazonin receptor/GnRH receptor cluster.

6. A chordate/urochordate/echinoderm GnRH receptor cluster(highlighted in yellow).

An especially interesting finding from the analysis shown inFig. 5 is the presence of two clearly different receptor clusters inboth molluses and annelids: one that is AKH receptor-like (lightblue in Fig. 5) and the other that is corazonin receptor-like (lightbrown in Fig. 5). This finding suggests that two types of GnRH-likereceptors (one AKH-like, one corazonin-like) already evolved at thelevel of Mollusca and Annelida (Fig. 3A). A similar conclusion canbe drawn when phylogenetic tree analyses are carried out, usinga different calculation method (Fig. 6).

Would this split into the two receptor types in molluses andannelids also be associated with the emergence of two types ofligands, one perhaps being more corazonin- and the other moreAKH-like (Fig. 3B)? We tried to clarify this question by carryingout phylogenetic tree analyses of the peptide sequences given inTable 2 plus several other newly annotated neuropeptidesequences from arthropods and other ecdysozoans, molluscs andannelids belonging to the AKH, ACP, corazonin, or GnRH peptidefamily. Fig. 7 shows such analyses. Before discussing Fig. 7, wewould like to give two caveats. First, one has to be very careful car-rying out phylogenetic tree analyses using short (8–12 amino acidresidues) peptide sequences. Such analyses will not yield robustresults comparable to the phylogenetic tree analyses of receptors(Fig. 5), which are on average 400 amino acid residues long. Second,the phylogenetic tree software programs will only give an overalljudgement of the short sequence, without taking into considerationthat certain amino acid residues are crucial for receptor binding.This might lead to wrong assignments (clustering) of very closelyrelated peptides (for example ACP and AKH). Thus, we should beprepared to make manual corrections for these wrong assignments.

With all these warnings in mind, the outcome of Fig. 7 looksvery exciting and fits reasonably well with the receptor data ofFigs. 5 and 6. The results of Fig. 7 can be summarized as follows(from top to bottom):



Fig. 4. Alignment of some established or annotated insect and molluscan AKH, ACP, and corazonin receptors and the human GnRH receptor. Cgig = Crassostrea gigas;Nvt = Nasonia vitripennis; Agam = Anopheles gambiae; Bmor = Bombyx mori; Dmel = Drosophila melanogaster; Hsap = Homo sapiens. The other abbreviations are as in Fig. 3. Theseven transmembrane helices are indicated by TM 1–7. The extracellular parts of the receptor are indicated by a red line. Most of the extracellular N termini and theintracellular C termini of the receptors are not shown in this figure, because they do not show significant sequence similarities in the alignment. Filled triangles above theamino acid residues indicate residues that are always identical in members of the neuropeptide family A GPCRs (Venkatakrishnan et al., 2013). Asterisks indicate residues thatare always identical in the insect AKH, ACP, corazonin and mammalian GnRH receptors and that might represent residues that form the signature of this receptor superfamily.


Please cite this article in press as: Hauser, F., Grimmelikhuijzen, C.J.P. Evolution of the AKH/corazonin/ACP/GnRH receptor superfamily and their ligands inthe Protostomia. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.07.009


ACPR

AKHR

GnRHR

CRZR

CRZR/GnRHR

Mollusca

Annelida

Nematoda

Chordata

Urochordata

Echinodermata

Annelida Molusca

Arthropoda

Arthropoda

Arthropoda

Fig. 5. Phylogenetic tree analysis of protostomian ACP receptors, AKH receptors, corazonin receptors and deuterostomian GnRH receptors. These receptor sequences wereretrieved from NCBI and most of them have been published (see, for example, Hauser et al., 2008; Sherwood et al., this issue; Rodet et al., 2005, 2008; Vadakkadath Meethalet al., 2006: Tsutsumi et al., 1992; Kanda et al., 2006; Sun et al., 2001; Lindemans et al., 2009). The complete GPCR amino acid sequences were used for each receptor (given inFig. S4) together with a software program based on maximal likelihood phylogeny (Dereeper et al., 2008). The tree is rooted with human rhodopsin. Bootstrap values (1–100)are given at each branch. AKHR = AKH receptor; ACPR = ACP receptor; CRZR = corazonin receptor; GnRHR = GnRH receptor. The major new finding of this analysis is that trulyAKH receptors appear to occur in molluscs, annelids, and nematodes. Receptors related to corazonin receptors occur in annelids and molluscs.


1. Two clusters of AKH-like peptides from arthropods, other ecdy-sozoans, molluscs, and annelids (highlighted in light blue).These clusters are likely ligands for the AKH receptor cluster(highlighted in light blue) in Fig. 5 (which, in the literature,has already been confirmed for many of them). A manual


curation has to be carried out for the two annelid (Capitellaand Helobdella) peptides (marked with asterisks in Fig. 7): theseare not genuine (sensu stricto) AKHs, although they clustertogether with the established AKHs. Yet, they are promisingcandidate ligands for annelid AKH receptors shown in Fig. 5.



Chordata

Arthropoda

Mollusca

Arthropoda

Annelida

Nematoda

Echino-dermata

Urochordata

Mollusca

Annelida

Arthropoda

ACPR

AKHR

GnRHR

CRZR

CRZR/

GnRHR

Fig. 6. Phylogenetic tree analysis of the same sequences used in Fig. 5, but now applying the neighbor-joining calculation method (Katoh and Standley, 2013). The sameabbreviations and color codes are used as in Fig. 5. The results from Fig. 6 are similar to that of Fig. 5. They show that nematodes, molluscs, and annelids have receptors thatappear to be truly AKH receptors (AKHR), similar to arthropods, and that molluscs and annelids have another group of receptors that are located between the GnRH receptorsand corazonin receptors (CRZR/GnRH).


Also the Nasonia peptide marked with an asterisk in Fig. 7 is anACP, not AKH.

2. A cluster of ACP-like peptides from arthropods (highlighted indark blue), which are excellent candidates for the cluster ofACP receptors (highlighted in dark blue) of Fig. 5. For several


of them it has already been published that they are the ligands.The manual corrections to be made are: Drosophila AKH andBrachionus AKH are genuine AKHs and not ACPs.

3. A cluster of peptides with structural properties of both corazo-nin and GnRH (highlighted in light brown). These peptides are



ACP

AKH

CRZ

GnRH

AKH

CRZ/GnRH

*

* *

*

*

*

Fig. 7. Phylogenetic tree analysis of ACP, AKH, corazonin, and GnRH sequences from chordates, echinoderms, molluscs, annelids, nematodes and arthropods. The peptidesequences used are generally short (8–14 amino acid residues) and are given in Fig. 8. Their preprohormone structures and gene identification numbers are given in Fig. S5.The peptide sequences indicated by an asterisk belong to a neuropeptide group, which is different from the ones, in which they cluster in the analysis. For example, DrosophilaAKH has the consensus sequence for AKH and not for ACP. The same is true for Branchionus AKH. Similarly, the Nasonia ACP has the consensus sequence for ACP and not forAKH. When a tree like Fig. 7 is made, each amino acid residue weights independently of its biological function, when being grouped into one of the clusters, for example theAKH or ACP clusters. For receptor binding, however, some residues are critical (and therefore invariable in for example insects), while other residues are unimportant (andtherefore variable in insects). These critical residues of the ligands (also called consensus sequences of AKH or ACP) should, therefore, ‘‘overrule’’ the assignments made basedon a simple phylogenetic tree analysis. The software program used is based on maximal likelihood phylogeny (Dereeper et al., 2008).


good candidate ligands for the receptor cluster (highlighted inlight brown) that is located between the corazonin receptorsand GnRH receptors in Fig. 5.


4. A cluster of genuine corazonins (highlighted in dark brown).Here we have to make one manual curation: the Branchiostomapeptide (Table 3) is a GnRH and not a corazonin (Roch et al.,



Table 3Novel AKHs identified in this paper from various molluscs, the tardigrade Hysibius dujardini, the rotifer Brachionus calycifloros, and the penis worm Priapulus caudatus. Locustamigratoria AKH-1, the first insect AKH isolated (Stone et al., 1976), is included for comparison. The AKHs from Aplysia californica and Lottia gigantea were independently discoveredby us in our present study, and by Roch and coworkers (Fig. 4 of Roch et al., 2011; Fig. 2 of Roch et al., 2014). We initially overlooked these AKH structures in the papers of Rochet al. (2011, 2014). The peptides from Tritonia, Brachionus, and Priapulus are not sensu stricto AKHs, because, although being 10 amino acid residues long and having most of theother AKH hallmarks, they do not have the canonical GXamide C-terminal sequence, but instead, EPamide, SGamide, and RGamide, respectively.


2014). This group of corazonins are the likely ligands for thecluster of corazonin receptors highlighted in dark brown inFig. 5. For several of them, this match has already beenpublished.

5. A cluster of established GnRHs (highlighted in yellow), whichcorresponds to the GnRH receptors of Fig. 5 (highlighted inyellow).

In an earlier version of Fig. 7 we only used published peptidesequences (given in Table 2). However, when we found potentialAKH receptors in molluscs and annelids (highlighted in light bluein Fig. 5) we thought that it would be highly exciting also to findtheir ligands. The molluscan and annelid ligands published, sofar, were all named GnRH-like (Table 2). Therefore, we screenedthe sequenced genomes of three molluscan species (Aplysia califor-nica, Lottia gigantea, and C. gigas) and two annelid species (Capitellateleta, and Helobdella robusta), using TBLASTN and various arthro-pod AKH, ACP, and corazonin sequences. These analyses yieldedone novel AKH preprohormone each for A. californica, L. gigantea,and C. gigas (Table 3, Fig. S3). Surprisingly, the three AKHs con-tained in these three preprohomornes are structurally truly AKHs,fulfilling all the structural requirements for arthropod AKHs andRPCHs, including their sizes (10 amino acid residues long) (Table 3).We have to add, however, that the AKHs from A. californica and L.gigantea were independently discovered by us in our present study,and by Roch and coworkers (a peptide sequence shown in Fig. 4 ofRoch et al., 2011; and in Fig. 2 of Roch et al., 2014). We initiallyoverlooked these sequences in the publications of Roch et al.(2011, 2014).

To find out whether other molluscs, which do not have a com-pleted genome sequence, also have AKHs we screened ExpressedSequence Tag (EST) databases from a large number of molluscanspecies. We were able to identify AKH peptides from the sea slugTritonia diomeda (pQIHFSPGWEPamide), the freshwater snail Bithy-nia siamensis goniomphalos (pQIHFTPGWGSamide), and the fresh-water pearl mussel Hyriopsis cumingii (pQISFSTNWGSamide)(Table 3). These results show that not only are AKH genes presentin the genomes of molluscs, but they are also expressed in consid-erable amounts. The presence of structurally truly AKHs in


molluscs, therefore, appears to be established. Five out of six mol-luscan AKHs given in Table 3 have a WGXamide C terminus, fulfill-ing the structural requirement for arthropod AKHs (see chapter 3),while the Tritonia C-terminal sequence is WEXamide, which makesthis peptide slightly different.

Inspired by our success in Mollusca, we also screened the gen-omes and EST databases of other non-arthropod protostomians forthe presence of AKHs and found an AKH peptide in the tardigradeHysibius dujardini (pQLSFSTGWGHamide). These data suggest thatgenuine AKHs are widespread in Mollusca and Ecdysozoa.

During our screen we also found a peptide in the rotiferBrachionus calyciflorus (Rotifera; pQLTFSSDWSGamide), and thepenis worm Priapulus caudatus (Priapulida; pQIFFSKGWRGamide)(Table 3). These peptides resemble AKHs, but have an XGamideinstead of a GXamide C terminus. We should, therefore, perhapscall these peptides AKH-like.

We were unable to find genuine AKHs when blasting the gen-ome sequences of the annelids H. robusta, and C. teleta, and theEST databases of several other annelid species. However, thereis one group of published annelid GnRH-like peptides (HelobdelaGnRH; Capitella GnRH-1, -2, -3; see Table 2) that are not obviousAKHs, because several of the AKH hallmarks are lacking, but thatnevertheless are located within the clusters of AKH peptides inFig. 7. These peptides might represent a transition betweenancestral GnRHs and the genuine AKHs that evolved later(Fig. 3B). Also, these peptides might be the ligands for the Helobd-ella and Capitella AKH receptors given in Fig. 5 (highlighted inlight blue).

Fig. 8 gives an overview of all the peptide sequences used inFig. 7. They are highlighted with the same color code as in Fig. 7with the exception of the annelid peptides, which are highlightedin green, because, although they cluster within the AKH peptidegroup (Fig. 7), they are not genuine AKHs.

8. Discussion

In this review we have tried to trace the evolution of AKH, ACP,corazonin, GnRH and their receptors below the level of arthropods.



ACP

AKH

CRZ

GnRH

Arthropoda

Tardigrada

Nematoda

Mollusca

Annelida

Priapulida

Arthropoda

Arthropoda

Mollusca

Annelida

Priapulida

Rotifera

Chordata

Urochordata

Echinodermata

CRZ/GnRH

AKH/GnRH

Fig. 8. An overview of the peptide sequences used in Fig. 7. Most peptide sequences have been published, but other peptides have been discovered in the present study(Table 3). The same color code has been used as in Fig. 7 except for two peptides, highlighted in green, that that group within the cluster of AKH peptides in Fig. 7, but that arenot sensu stricto AKHs.


Phylogenetic tree analyses of the receptors for these neuropeptidesgive a rather clear picture, where ACP receptors first originated inthe Arthropoda (Fig. 5). Also the AKH and corazonin receptors arepresent in the Arthropoda, but they must have emerged earlier,because a molluscan, annelid and nematode AKH receptor clustercan be identified in our phylogenetic tree analysis of Fig. 5


(highlighted in light blue). For the corazonin receptors we founda molluscan and annelid cluster (highlighted light brown inFig. 5) located between the canonical corazonin receptors andGnRH receptors. These molluscan/annelid receptors might beregarded as an evolutionary transition between GnRH receptorsand canonical corazonin receptors. Many of these findings can be




summarized in a scheme for AKH/ACP/corazonin/GnRH receptorevolution as shown in Fig. 3A.

The next question is, can we create such an evolutionaryscheme as in Fig. 3A also for the ligands? This is relatively easywithin the Arthropoda, where AKH and corazonin form discretepeptide families and ACP is structurally intermediate betweenAKH and corazonin, although it cannot be decided ‘‘by eye’’whether ACP is more AKH- than corazonin-related (Table 1,Table 2). Phylogenetic tree analyses of the peptide sequences(Fig. 7), however, locate the ACP cluster between two AKH clusters,suggesting that ACP is more AKH- than corazonin-like. This findingsupports our evolutionary model (Fig. 3B), where the ACP geneoriginated by gene duplication from the AKH gene.

Another remarkable feature from these analyses is that the pep-tide from C. elegans (Table 2) that was called AKH-GnRH-like(Lindemans et al., 2009) is, in fact, a genuine AKH, because struc-turally it falls within the cluster of AKH peptides and it is clearlynot related to GnRH (Fig. 7). This conclusion fits reasonably wellwith the position of the C. elegans receptor, which is situatedwithin the AKH receptor cluster (Fig. 5). These findings, therefore,suggest, that AKH signalling already exists below the level ofArthropoda, at least at the level of Nematoda (Fig. 1).

A highly remarkable and clear-cut result of our present study isthe discovery that AKH peptides already exist in lower Ecdysozoa,such as Tardigrada and Priapulida and even in Mollusca, such asvarious bivalves, gastropods, and cephalopods (Table 3). Thesefindings, together with the presence of AKH receptors in these ani-mals (highlighted in light blue in Fig. 5) show that AKH signalinghas already emerged at the level of Mollusca.

The other GnRH-related peptides (Table 2) that other research-ers identified in the molluscs A. californica, C. gigas, O. vulgaris, andL. gigantea (Zhang et al., 2008; Bigot et al., 2012; Veenstra, 2010;Iwakoshi et al., 2002) and in the annelid C. teleta. (CapitellaGnRH-3, Veenstra, 2011) cluster together in a rather homogenouspeptide family (highlighted light brown in Fig. 7), neighboringcorazonin and GnRH. This finding is consistent with the positionof the corazonin-/GnRH-receptor cluster in Fig. 5. Thus, alsocorazonin signaling might have evolved in Mollusca and Annelida(Fig. 3). We have to stress, however, that these molluscan andannelid corazonins are not corazonins sensu stricto, becausethey are lacking many of the structural properties discussed inchapter 4.

We investigated other phyla that emerged before the Molluscaand Annelida, such as platyhelminthes (Fig. 1). The genome of theplatyhelminth Schmidtea mediterranea is in the process of beingsequenced (www.ncbi.nlm.nih.gov/genome/232). We tried to blast(TBLASTN) the genomic data of this species with our receptor andpeptide sequences, however, without success, meaning that nodata are available yet for the Platyhelmintha.

There exist several completed genome projects from cnidarians(Fig. 1), for example that from the freshwater polyp Hydra magni-papilla (Chapman et al., 2010), the sea anemone Nematostella vect-ensis (Putman et al., 2007), and the elkhorn coral Acropora digitifera(Shinzato et al., 2011). Furthermore, there have been reports on theoccurrence of GnRH-like peptides in the sea pansy Renilla köllikeri(Anctil, 2000) and N. vectensis (Anctil, 2009). We have blasted thegenome sequences of the three cnidarians for the presence ofAKH-, ACP-, corazonin-, and GnRH-like receptors and were unableto find significant hits. Also a screening using TBLASTN for AKH-,ACP-, corazonin- and GnRH-like peptides did not givepositive results. Anctil (2009) published the sequence of aGnRH-like peptide in N. vectensis, but this sequence has only thePG-amide C-terminal two amino acid residues in common withGnRHs (Table 2) and, in our view, cannot be regarded as a genuineGnRH-like molecule. Thus, there are no indications that GnRHsignaling occurs in Cnidaria (Fig. 1).


9. Conclusions

GnRH-like peptides and receptors have originated before thesplit of proto- and deuterostomians about 700 MYR ago. In the Pro-tostomia, the GnRH-like peptide and receptor genes have dupli-cated and significantly diversed, giving rise to two receptor typesin the Annelida/Mollusca (corazonin-receptor-like receptors andAKH receptors) and three receptor types in the Arthropoda (AKH,ACP, and corazonin receptors), each with their specific ligands.Thus, the corazonin and AKH hormonal systems already evolvedbefore the split of Arthropoda and Annelida/Mollusca (Fig. 1). Theevolution of the AKH/corazonin/ACP/GnRH receptor superfamilyand their ligands is summarized in Fig. 3.

Acknowledgments

We thank Drs. Dan Larhammer and João Cardoso for inviting usto write a review paper on the evolution of AKH receptors, whichled us to the discovery of novel AKH peptides in molluscs, NoahKassem for typing the manuscript, and the Danish Research Agency(FNU), Lundbeck Foundation, and Carlsberg Foundation for finan-cial support.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ygcen.2014.07.009.

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Evolution of the AKH/corazonin/ACP/GnRH receptor superfamily and their ligands in the Protostomia

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