Ancient coexistence of norepinephrine, tyramine, and - bioRxiv

Ancient coexistence of norepinephrine, tyramine, and - bioRxiv

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-...

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bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Ancient coexistence of norepinephrine, tyramine, and octopamine signaling in bilaterians

Philipp Bauknecht1 and Gáspár Jékely1*

1

Max Planck Institute for Developmental Biology. Spemannstrasse 35. 72076 Tübingen. Germany Email addresses:

[email protected] [email protected]

*Corresponding author

1

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Abstract Norepinephrine/noradrenaline is a neurotransmitter implicated in arousal and other aspects of vertebrate behavior and physiology. In invertebrates, adrenergic signaling is considered absent and analogous functions are performed by the biogenic amines octopamine and its precursor tyramine. These chemically similar transmitters signal by related families of GPCR in vertebrates and invertebrates, suggesting that octopamine/tyramine are the invertebrate equivalents of vertebrate norepinephrine. However, the evolutionary relationships and origin of these transmitter systems remain unclear. Using phylogenetic analysis and receptor pharmacology, here we establish that norepinephrine, octopamine, and tyramine receptors coexist in some marine invertebrates. In the protostomes Platynereis dumerilii (an annelid) and Priapulus caudatus (a priapulid) we identified and pharmacologically characterized adrenergic α1 and α2 receptors that coexist with octopamine α, octopamine β, tyramine type 1, and tyramine 2 receptors. These receptors represent the first examples of adrenergic receptors in protostomes. In the deuterostome Saccoglossus kowalewskii (a hemichordate), we identified and characterized octopamine α, octopamine β, tyramine type 1, and tyramine 2 receptors, representing the first example of these receptors in deuterostomes. S. kowalewskii also has adrenergic α1 and α2 receptors, indicating that all three signaling systems coexist in this animal. In phylogenetic analysis, we also identified adrenergic and tyramine receptor orthologs in xenacoelomorphs. Our results clarify the history of monoamine signaling in bilaterians. Since all six receptor families (two each for octopamine and tyramine and three for norepinephrine) can be found in representatives of the two major clades of Bilateria, the protostomes and the deuterostomes, all six receptors coexisted in the protostomedeuterostome last common ancestor. Adrenergic receptors were lost from most insects and nematodes and tyramine and octopamine receptors were lost from most deuterostomes. This complex scenario of differential losses cautions that octopamine signaling in protostomes is not a good model for adrenergic signaling in deuterostomes, and that the studies of marine animals where all three transmitter systems coexist will be needed for a better understanding of the origin and ancestral functions of these transmitters.

Background Norepinephrine is a major neurotransmitter in vertebrates with a variety of functions including roles in promoting wakefulness and arousal [1], regulating aggression [2], and autonomic functions such a heart beat [3]. Signaling by the monoamine octopamine in protostome invertebrates is often considered equivalent to vertebrate adrenergic signaling [4] with analogous roles in promoting aggression and wakefulness in flies [5, 6], or the regulation of heart rate in annelids and arthropods [7, 8]. Octopamine is synthesized from tyramine (Figure 1A) which itself also acts as a neurotransmitter or neuromodulator in arthropods and nematodes [4, 9–15]. Octopamine and norepinephrine are chemically similar, are synthesized by homologous enzymes [16, 17], and signal by similar but not orthologous G-protein coupled receptors (GPCRs) [4, 18]. Tyramine also signals by non-orthologous receptors in invertebrates and vertebrates. In insects and nematodes, tyramine signals by a GPCR that is related to octopamine receptors [12, 19]. In vertebrates, tyramine is only present at low levels and signals by the trace-amine receptors, a vertebrate-specific GPCR family only distantly related to the invertebrate tyramine receptors [20, 21]. Given these differences, the precise evolutionary relationships of these monoamine signaling systems are unclear. The evolution of neurotransmitter systems has been analyzed by studying the distribution of monoamines or biosynthetic enzymes in different organisms [22]. This approach has 2

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

limitations, however, because some of the biosynthetic enzymes are not specific to one substrate [16] and because trace amounts of several monoamines are found across many organisms, even if specific receptors are often absent [22]. For example, even if invertebrates can synthesize trace amounts of norepinephrine, these are not considered to be active neuronal signaling molecules, since the respective receptors are lacking. Consequently, the presence of specific monoamine receptors is the best indicator that a particular monoamine is used in neuronal signaling [11, 23]. To clarify the evolutionary history of adrenergic, octopamine, and tyramine signaling in animals, we decided to undertake a comparative phylogenetic and pharmacological study of these receptor families in bilaterians. Bilaterians, animals with bilateral symmetry, are comprised of protostomes, deuterostomes, and xenacoelomorphs [24]. Deuterostomes include chordates and ambulacrarians (hemichordates and echinoderms), and protostomes are formed by the clades Ecdysozoa, Lophotrochozoa (Spiralia), and Chaetognatha. Ecdysozoa includes arthropods, nematodes, priapulids and other phyla. Lophotrochozoa include annelids, mollusks, and other, mostly marine groups. Xenacoelomorps, a group including acoel flatworms, nemertodermatids, and Xenoturbella, have been proposed to belong to the deuterostomes, or represent a sister group to all remaining bilaterians [25–27]. Here we establish the orthology relationships of adrenergic, octopamine, and tyramine receptors across bilaterians. We find that six receptor families originated at the base of the bilaterian tree. We then pharmacologically characterize adrenergic receptors from an annelid and a priapulid, and octopamine and tyramine receptors from an annelid and a hemichordate. The broad phylogenetic sampling and comparative pharmacology paints a richer picture of the evolution of these receptors, characterized by ancestral coexistence and multiple independent losses.

Results Using database searches, sequence-similarity-based clustering, and phylogenetic analysis, we reconstructed the phylogeny of α1, α2, and β adrenergic, octopamine α, octopamine β, and tyramine type-1 and type-2 receptors. Each family formed well-resolved clusters in a sequence-similarity-based clustering analysis and well-supported clades in molecular phylogenetic analysis (Figure 1B, C and Additional file 1). We identified several invertebrate GPCR sequences that were similar to vertebrate adrenergic α1 and α2 receptors (Figure 1B, C). An adrenergic α1 receptor ortholog is present in the sea urchin Strongylocentrotus purpuratus. Adrenergic α1 and α2 receptors were both present in Saccoglossus kowalewskii, a hemichordate deuterostome (Figure 1B, C and Additional files 1-3), as previously reported [28]. We also identified adrenergic α1 and α2 receptor orthologs in annelids and mollusks (members of the Lophotrochozoa), including Aplysia californica, and in the priapulid worm Priapulus caudatus (member of the Ecdysozoa)(Figure 1B, C and Additional files 1-3). Adrenergic α receptors are also present in a few arthropods, including the crustacean Daphnia pulex and the moth Chilo suppressalis (the Chilo α2 receptor was first described as an octopamine receptor [29]), but are absent from most other insects (Additional files 1-3). Adrenergic α2 receptors are also present in xenacoelomorphs, in Xenoturbella bocki and the nemertodermatid Meara stichopi. M. stichopi also has two adrenergic α1 receptor orthologs (Figure 1C and Additional file 1-3). The identification of adrenergic α1, and α2 receptor orthologs in ambulacrarians, lophotrochozoans, ecdysozoans, and xenacoelomorphs indicates that both families were present in the bilaterian last common ancestor. Adrenergic β receptors are found in chordates, including urochordates and cephalochordates. In addition, we identified an adrenergic β receptor ortholog in the xenacoelomorph M.

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bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

stichopi (Additional file 4). If xenacoelomorphs are sister to all remaining bilaterians, then this receptor family also originated at the base of Bilateria and was lost from all protostomes. To characterize the ligand specificities of these putative invertebrate adrenergic receptors, we cloned them from S. kowalewskii, P. caudatus, and the marine annelid Platynereis dumerilii. We performed in vitro GPCR activation experiments using a Ca2+-mobilization assay [30, 31]. We found that norepinephrine and epinephrine activated both the adrenergic α1 and α2 receptors from all three species with EC50 values in the high nanomolar range or lower. In contrast, tyramine, octopamine, and dopamine were either inactive or only activated the receptors at approximately two orders of magnitude higher concentrations (Figure 2, Table 1). These phylogenetic and pharmacological results collectively establish these invertebrate receptors as bona fide adrenergic α receptors. To investigate if adrenergic signaling coexists with octopamine and tyramine signaling in protostomes, we searched for octopamine and tyramine receptors in P. dumerilii and P. caudatus. In phylogenetic and clustering analyses, we identified orthologs for tyramine type 1 and type 2 and octopamine α and β receptors in both species (Figure 1B, C and Additional files 5-8). We performed activation assays with the P. dumerilii receptors. The tyramine type 1 and type 2 receptors orthologs were preferentially activated by tyramine with EC50 values in the nanomolar range (Figure 3, Table 1). The P. dumerilii octopamine α receptor was activated by octopamine at a lower concentration than by tyramine and dopamine (Figure 4, Table 1). The P. dumerilii octopamine β receptor was not active in our assay. These results show that specific receptor systems for norepinephrine, octopamine, and tyramine coexist in P. dumerilii and very likely also P. caudatus. When did tyramine and octopamine signaling originate? To answer this, we surveyed available genome sequences for tyramine and octopamine receptors. As expected, we identified several receptors across the protostomes, including ecdysozoans and lophotrochozoans (Additional files 5-8). We also identified tyramine, but not octopamine, receptors in xenacoelomorphs. However, chordate genomes lacked orthologs of these receptors. Strikingly, we identified tyramine type 1 and 2 and octopamine α and β receptor orthologs in the genome of the hemichordate S. kowalewskii (Figure 1B, C, Additional files 5-8). In phylogenetic analyses, we recovered at least one S. kowalewskii sequence in each of the four receptor clades (one octopamine α, one octopamine β, two tyramine type 1, and two tyramine type 2 receptors), establishing these sequences as deuterostome orthologs of these predominantly protostome GPCR families (Additional files 5-8). We cloned the candidate S. kowalewskii tyramine and octopamine receptors and performed ligand activation experiments. The S. kowalewskii type 2 receptors were preferentially activated by tyramine in the nanomolar range. The type 1 receptor was only activated at higher ligand concentrations. The octopamine α and β receptors were preferentially activated by octopamine in the nanomolar range (Figures 3 and 4, Table 1). These data show that octopamine and tyramine signaling also coexists with adrenergic signaling in this deuterostome, as in P. dumerilii and P. caudatus. The presence of tyramine signaling in S. kowalewskii is also supported by the phylogenetic distribution of tyrosine decarboxylase, a specific enzyme for tyramine synthesis [32]. Tyrosine decarboxylase is present in protostomes and S. kowalewskii but is absent from other deuterostomes (Additional file 9). In mammals, aromatic amino acid decarboxylases are involved in synthesizing low amounts of tyramine [33]. We also tested the α adrenergic agonist clonidine and the GPCR antagonists mianserin and yohimbine on several receptors from all three species. These chemicals did not show specificity for any of the receptor types, suggesting these chemicals may not be useful for studying individual biogenic amine receptors in vivo (Table 1. and Additional file 10).

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bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Discussion The discovery of adrenergic signaling in some protostomes and xenacoelomorphs and octopamine and tyramine signaling in a deuterostome changes our view on the evolution of monoamine signaling in bilaterians (Figure 5). It is clear from the phylogenetic distribution of orthologous receptor systems that at least six families of octopamine, tyramine, and adrenergic receptors were present in the bilaterian last common ancestor. These include the adrenergic α1 and α2 receptors, the tyramine type 1 and type 2 receptors, and the octopamine α and β receptors. From the six ancestral families, the octopamine and tyramine receptors were lost from most deuterostomes, and the adrenergic receptors were lost from most ecdysozoans. Interestingly, the xenacoelomorph M. stichopi also has an adrenergic β receptor, representing the only ortholog outside chordates. Octopamine α receptors were likely lost from xenacoelomorphs, since the split of the six receptor families (four with wellresolved xenacoelomorph sequences) predated the divergence of the main lineages of bilaterians (Figure 1C). Although we performed the receptor activation assays in a heterologous system that may not mimic the in vivo situation very well, we find clear evidence of ligand preferences for each receptor. In general, there is a two orders of magnitude difference in the EC50 values between the best ligand and other related ligands for the same receptor measured under the same conditions. We consider these in vitro ligand preferences as indicative of the physiological ligands for these receptors. Furthermore, there is a high congruence between the in vitro ligand specificities and the phylogenetic placement of the different classes of receptors, further strengthening our receptor-type assignments. The most potent ligand of all six orthologous receptor families we analyzed is the same across protostomes and deuterostomes, indicating the evolutionary stability of ligand-receptor pairs, similar to the long-term stability of neuropeptide GPCR ligand-receptor pairs [34, 35]. Understanding the ancestral role of these signaling systems and why they may have been lost differentially in different animal groups will require functional studies in organisms where all three neurotransmitter systems coexist.

Conclusions We established the coexistence of adrenergic, octopaminergic, and tyraminergic signaling in the deuterostome S. kowalewskii and the protostomes P. dumerilii and P. caudatus. Signaling by norepinephrine in vertebrates has often been considered as equivalent to signaling by octopamine in invertebrates. Our results change this view and show that these signaling systems coexisted ancestrally and still coexist in some bilaterians. The extent of functional redundancy in species where all six receptor systems coexist will require experimental studies. It may be that some of these monoamines ancestrally had partially overlapping roles. In that case, following the loss of a receptor, functions associated with that ligand-receptor pair may have been taken over by another pair. However, regardless of such potential shifts in function, it is clear that octopamine signaling in invertebrates and adrenergic signaling in vertebrates is not equivalent or homologous from an evolutionary point of view. This has important implications for our interpretation of comparative studies of the function of these neurotransmitter systems and their neural circuits. Our study also contributes to the understanding of nervous system evolution in bilaterians by revealing extensive losses during the history of one of the major classes of neurotransmitter systems.

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bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Methods Gene identification and receptor cloning Platynereis protein sequences were collected from a Platynereis mixed stages transcriptome assembly [36]. GPCR sequences from other species were downloaded from NCBI. GPCRs were cloned into pcDNA3.1(+) (Thermo Fisher Scientific. Waltham. USA) as described before [31]. Forward primers consisted of a spacer (ACAATA) followed by a BamHI or EcoRI restriction site, the Kozak consensus sequence (CGCCACC), the start codon (ATG) and a sequence corresponding to the target sequence. Reverse primers consisted of a spacer (ACAATA), a NotI restriction site, a STOP codon, and reverse complementary sequence to the target sequence. Primers were designed to end with a C or G with 72°C melting temperature. PCR was performed using Phusion polymerase (New England Biolabs GmbH, Frankfurt, Germany). The sequences of all Platynereis GPCRs tested here were deposited in GenBank (accession numbers: α1-adrenergic receptor. KX372342; α2-adrenergic receptor, KX372343 Tyramine-1 receptor, KP293998; Tyramine-2 receptor, KU715093; Octopamine α receptor, KU530199; Octopamine β receptor, KU886229). Tyramine receptor 1 has been previously published [31] as Pdu orphan GPCR 48. The GenBank accession numbers of the S. kowalevskii and P. caudatus sequences tested are: S. kowalevskii α1-adrenergic, ALR88680; S. kovalewskii α2-adrenergic, XP_002734932; P. caudatus α1-adrenergic XP_014662992; P. caudatus α2-adrenergic, XP_014681069; S. kovalewskii Tyramine-1, XP_002742354; S. kovalewskii Tyramine-2A, XP_002734062; S. kovalewskii Tyramine-2B, XP_006812999; S. kowalevskii Octopamine α, XP_006823182; S. kowalevskii Octopamine β, XP_002733926. Cell culture and receptor deorphanization Cell culture assays were done as described before [31]. Briefly, CHO-K1 cells were kept in Ham’s F12 Nut Mix medium (Thermo Fisher Scientific, Waltham, USA) with 10 % fetal bovine serum and penicillin-streptomycin (PenStrep, Invitrogen). Cells were seeded in 96well plates (Thermo Fisher Scientific, Waltham, USA) at approximately 10,000 cells/well. After 1 day, cells were transfected with plasmids encoding a GPCR, the promiscuous Gα-16 protein [37], and a reporter construct GFP-apoaequorin [38] (60 ng each) using 0.375 µl of the transfection reagent TurboFect (Thermo Fisher Scientific. Waltham. USA). After two days of expression, the medium was removed and replaced with Hank’s Balanced Salt Solution (HBSS) supplemented with 1.8 mM Ca2+, 10 mM glucose, and 1 mM coelenterazine h (Promega, Madison, USA). After incubation at 37°C for 2 hours, cells were tested by adding synthetic monoamines (Sigma, St. Louis, USA) in HBSS supplemented with 1.8 mM Ca2+ and 10 mM glucose. Solutions containing norepinephrine, epinephrine or dopamine were supplemented with 100 µM ascorbic acid to prevent oxidation. Luminescence was recorded for 45 seconds in a plate reader (BioTek Synergy Mx or Synergy H4, BioTek, Winooski, USA). For inhibitor testing, the cells were incubated with yohimbine or mianserin (Sigma, St. Louis, USA) for 1 hour. Then, synthetic monoamines were added to yield in each case the smallest final concentration expected to elicit the maximal response in the absence of inhibitor and luminescence was recorded for 45 seconds. Data were integrated over the 45second measurement period. Data for dose-response curves were recorded in triplicate for each concentration. Dose-response curves were fitted with a four-parameter curve using Prism 6 (GraphPad, La Jolla. USA). The curves were normalized to the calculated upper plateau values (100% activation). The different EC50 values for each receptor were compared with the extra sum-of-squares F test in a pairwise manner using Prism 6. Bioinformatics

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bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Protein sequences were downloaded from the NCBI. Redundant sequences were removed from the collection using CD-HIT [39] with an identity cutoff of 70%. Sequence cluster maps were created with CLANS2 [40] using the BLOSUM62 matrix and a P-value cutoff of 1e-70. For phylogenetic trees, protein sequences were aligned with MUSCLE [41]. Alignments were trimmed with TrimAI [42] in “Automated 1” mode. The best amino acid substitution model was selected using ProtTest 3 [43]. Maximum likelihood trees were calculated with RAxML [44] using the CIPRES Science Gateway [45] or with IQ-TREE and automatic model selection (http://www.iqtree.org/). Bootstrap analysis in RAxML was done and automatically stopped [46] when the Majority Rule Criterion (autoMRE) was met. The resulting trees were visualized with FigTree (http://tree.bio.ed.ac.uk/software/figtree/). The identifiers of deorphanized adrenergic, octopamine, and tyramine receptors [12, 29, 47–59] were tagged with _AA1, AA2, _Oa, _Ob, _T1, or _T2. The trees were rooted on 5HT receptors. Funding The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ European Research Council Grant Agreement 260821. PB is supported by the International Max Planck Research School (IMPRS) ‘‘From Molecules to Organisms.’’ The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data, and in writing the manuscript. Acknowledgments We thank John Gerhart for Saccoglossus DNA and the image of Saccoglossus. We thank Mattias Hogvall for Priapulus DNA and the image of Priapulus, and Anne-C. Zakrzewski for the Xenoturbella image. We also thank Elizabeth Williams for comments on the manuscript.

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33. Lovenberg W, Weissbach H, Udenfriend S: Aromatic L-amino acid decarboxylase. J Biol Chem 1962, 237:89–93. 34. Jékely G: Global view of the evolution and diversity of metazoan neuropeptide signaling. Proc Natl Acad Sci U S A 2013, 110:8702–8707. 35. Mirabeau O, Joly JS: Molecular evolution of peptidergic signaling systems in bilaterians. Proc Natl Acad Sci U S A 2013, 110:E2028–E2037. 36. Conzelmann M, Williams EA, Krug K, Franz-Wachtel M, Macek B, Jékely G: The neuropeptide complement of the marine annelid Platynereis dumerilii. BMC Genomics 2013, 14:906. 37. Offermanns S, Simon MI: G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C. J Biol Chem 1995, 270:15175–15180. 38. Baubet V, Le Mouellic H, Campbell AK, Lucas-Meunier E, Fossier P, Brúlet P: Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level. Proc Natl Acad Sci U S A 2000, 97:7260–7265. 39. Li W, Godzik A: Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 2006, 22:1658–1659. 40. Frickey T, Lupas A: CLANS: a Java application for visualizing protein families based on pairwise similarity. Bioinformatics 2004, 20:3702–3704. 41. Edgar RC: MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 2004, 5:113. 42. Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T: trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 2009, 25:1972– 1973. 43. Darriba D, Taboada GL, Doallo R, Posada D: ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 2011, 27:1164–1165. 44. Stamatakis A: RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014, 30:1312–1313. 45. Miller MA, Pfeiffer W, Schwartz T: Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In 2010 Gateway Computing Environments Workshop (GCE). IEEE; 2010:1–8. 46. Pattengale ND, Alipour M, Bininda-Emonds OR, Moret BM, Stamatakis A: How many bootstrap replicates are necessary? J Comput Biol 2010, 17:337–354. 47. Balfanz S, Jordan N, Langenstück T, Breuer J, Bergmeier V, Baumann A: Molecular, pharmacological, and signaling properties of octopamine receptors from honeybee (Apis mellifera) brain. J Neurochem 2014, 129:284–296.

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48. Verlinden H, Vleugels R, Marchal E, Badisco L, Pflüger HJ, Blenau W, Broeck JV: The role of octopamine in locusts and other arthropods. J Insect Physiol 2010, 56:854–867. 49. Gross AD, Temeyer KB, Day TA, Pérez de León AA, Kimber MJ, Coats JR: Pharmacological characterization of a tyramine receptor from the southern cattle tick, Rhipicephalus (Boophilus) microplus. Insect Biochem Mol Biol 2015, 63:47–53. 50. Kastner KW, Shoue DA, Estiu GL, Wolford J, Fuerst MF, Markley LD, Izaguirre JA, McDowell MA: Characterization of the Anopheles gambiae octopamine receptor and discovery of potential agonists and antagonists using a combined computationalexperimental approach. Malar J 2014, 13:434. 51. Wu SF, Yao Y, Huang J, Ye GY: Characterization of a β-adrenergic-like octopamine receptor from the rice stem borer (Chilo suppressalis). J Exp Biol 2012, 215(Pt 15):2646– 2652. 52. Huang J, Wu SF, Li XH, Adamo SA, Ye GY: The characterization of a concentrationsensitive α-adrenergic-like octopamine receptor found on insect immune cells and its possible role in mediating stress hormone effects on immune function. Brain Behav Immun 2012, 26:942–950. 53. Lind U, Alm Rosenblad M, Hasselberg Frank L, Falkbring S, Brive L, Laurila JM, Pohjanoksa K, Vuorenpää A, Kukkonen JP, Gunnarsson L, Scheinin M, Mårtensson Lindblad LG, Blomberg A: Octopamine receptors from the barnacle balanus improvisus are activated by the alpha2-adrenoceptor agonist medetomidine. Mol Pharmacol 2010, 78:237–248. 54. Chen X, Ohta H, Ozoe F, Miyazawa K, Huang J, Ozoe Y: Functional and pharmacological characterization of a beta-adrenergic-like octopamine receptor from the silkworm Bombyx mori. Insect Biochem Mol Biol 2010, 40:476–486. 55. Blais V, Bounif N, Dubé F: Characterization of a novel octopamine receptor expressed in the surf clam Spisula solidissima. Gen Comp Endocrinol 2010, 167:215–227. 56. Chang DJ, Li XC, Lee YS, Kim HK, Kim US, Cho NJ, Lo X, Weiss KR, Kandel ER, Kaang BK: Activation of a heterologously expressed octopamine receptor coupled only to adenylyl cyclase produces all the features of presynaptic facilitation in aplysia sensory neurons. Proc Natl Acad Sci U S A 2000, 97:1829–1834. 57. Gerhardt CC, Bakker RA, Piek GJ, Planta RJ, Vreugdenhil E, Leysen JE, Van Heerikhuizen H: Molecular cloning and pharmacological characterization of a molluscan octopamine receptor. Mol Pharmacol 1997, 51:293–300. 58. Wu SF, Xu G, Ye GY: Characterization of a tyramine receptor type 2 from hemocytes of rice stem borer, Chilo suppressalis. J Insect Physiol 2015, 75:39–46. 59. Jezzini SH, Reyes-Colón D, Sosa MA: Characterization of a prawn OA/TA receptor in Xenopus oocytes suggests functional selectivity between octopamine and tyramine. PLoS ONE 2014, 9:e111314.

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Figures, tables additional files EC50 (M)/IC50 (M) P. dumerilii α1adrenergic 95% CI

Tyramine

Octopamine

Clonidine

inactive

inactive

P. dumerilii α2adrenergic 95% CI

8.4E-05

S. kowalevskii α1adrenergic 95% CI S. kovalewskii α2adrenergic 95% CI P. caudatus α1adrenergic 95% CI

Dopamine

Epinephrine

Yohimbine

Mianserin

inactive

Norepinephri ne 2.1E-07 ***

1.2E-04 ***

3.7E-07 n.s.

4.4E-06

3.7E-06

2.7E-06 ***

2.6E-06

1.0E-007 to 4.2E-007 8.2E-09 ***

2.7E-005 to 0.00056 1.6E-06

1.3E-007 to 1.1E-006 1.1E-08 n.s.

2.3E-006 to 8.2E-006 5.7E-06

1.9E-006 to 7.2E-006 2.5E-05

2.8E-005 to 0.00024 inactive

6.683E-007 to 1.0E-005 inactive

2.4E-007 to 2.7E-005 inactive

5.7E-009 to 1.1E-008 1.7E-08 ***

8.3E-007 to 3.2E-006 3.8E-06 ***

5.0E-009 to 2.2E-008 1.9E-08 n.s.

3.5E-006 to 9.1E-006 1.3E-05

1.2E-005 to 5.1E-005 4.5E-06

3.7E-06

1.9E-06

3.6E-08

1.0E-008 to 2.7E-008 1.2E-13 ***

1.9E-007 to 7.4E-005 5.6E-09

9.0E-009 to 4.1E-008 2.3E-09 ***

7.6E-006 to 2.2E-005 3.3E-07

1.6E-006 to 1.1E-005 inactive

2.0E-006 to 6.8E-006 inactive

2.5E-007 to 1.4E-005 inactive

6.7E-009 to 1.9E-007 inactive

6.7E-014 to 1.9E-013 7.5E-09

3.3E-009 to 9.4E-009 inactive

1.1E-009 to 4.6E-009 inactive

2.6E-007 to 4.0E-007   inactive

inactive

4.0E-009 to 1.3E-008 4.7E-07 *

inactive

4.5E-07 n.s

inactive

9.8E-07

1.7E-007 to 1.2E-006 1.7E-05

P. caudatus α2adrenergic 95% CI

inactive

P. dumerilii Tyramine-1 95% CI

1.1E-08 ***

2.7E-06 ***

1.1E-06 * p=0.021 4.5E-007 to 2.4E-006 2.1E-06

7.8E-06

1.8E-007 to 1.0E-006   3.1E-05

2.1E-06

4.3E-007 to 2.2E-006 4.7E-05

7.6E-009 to 1.6E-008 7.0E-09 ***

1.1E-006 to 6.1E-006 7.8E-07 ***

1.0E-006 to 4.1E-006 5.3E-06

1.0E-005 to 2.8E-005 1.1E-04

1.5E-006 to 3.9E-005 3.9E-06

9.8E-006 to 9.9E-005 4.8E-05

7.0E-007 to 6.0E-006 5.4E-05

1.7E-005 to 0.00012 6.4E-06

3.0E-009 to 1.6E-008 8.6E-05 n.s.

3.8E-007 to 1.5E-006 inactive

2.1E-006 to 1.3E-005 2.9E-04 n.s.

2.9E-005 to 0.00038 inactive

2.1E-006 to 7.0E-006 0.57

8.6E-006 to 0.00026 inactive

3.6E-005 to 7.9E-005 1.7E-06

3.9E-006 to 1.0E-005 1.7E-05

2.8E-005 to 0.00025 1.0E-09 ***

very wide

8.6E-08 ***

0.00013 to 0.00065 1.4E-06

2.1E-006 to 0.00017 inactive

7.1E-007 to 3.9E-006 inactive

7.7E-006 to 3.7E-005 1.6E-04

6.6E-010 to 1.5E-009 5.9E-09 ***

4.0E-008 to 1.8E-007 1.6E-06 ***

7.4E-007 to 2.6E-006   1.6E-05

2.4E-009 to 1.4E-008 1.3E-05

6.5E-007 to 3.7E-006 2.6E-07 *

6.2E-006 to 4.0E-005 1.4E-07 n.s.

4.2E-006 to 4.1E-005 1.7E-05

8.4E-008 to 7.7E-007 6.9E-07 *

3.0E-006 to 9.5E-005 inactive

1.8E-007 to 2.4E-006 6.4E-08 ***

6.7E-008 to 3.0E-007 1.6E-07 * p=0.048 7.6E-008 to 3.5E-007 inactive

P. dumerilii Tyramine-2 95% CI S. kovalewskii Tyramine-1 95% CI S. kovalewskii Tyramine-2A 95% CI S. kovalewskii Tyramine-2B 95% CI P. dumerilii Octopamine α 95% CI S. kowalevskii Octopamine α 95% CI S. kowalevskii Octopamine β 95% CI

inactive

4.0E-008 to 1.0E-007

inactive

7.2E-08

1.2E-04

1.4E-008 to 3.5E-007 1.4E-06

2.8E-05

2.1E-05

5.4E-008 to 0.47 1.9E-05

3.6E-005 to 0.00036 3.5E-06 * p=0.003 1.8E-006 to 6.7E-006 5.3E-05

9.0E-007 to 2.2E-006 inactive

5.1E-006 to 0.00015 8.8E-06

1.1E-005 to 3.6E-005 9.0E-09

1.1E-005 to 3.0E-005 1.6E-06

2.6E-04

2.5E-006 to 3.0E-005 1.8E-05

4.1E-009 to 1.9E-008 7.8E-06

9.7E-007 to 2.6E-006 2.2E-05

1.5E-005 to 0.00018 3.5E-06 ***

3.4E-006 to 0.02 inactive

7.1E-006 to 4.7E-005 inactive

3.1E-006 to 1.8E-005 1.6E-04

1.2E-005 to 3.6E-005 6.4E-06

1.0E-005 to 0.0023

3.1E-006 to 1.3E-005

1.4E-006 to 8.1E-006

Table 1. EC50 (M)/IC50 (M) values of all tested GPCRs with the indicated ligands or inhibitors. The most effective natural ligand for each receptor is shown in bold. 95% confidence intervals for the EC50 (M)/IC50 (M) values are given in every second line. The lowest EC50 value for each receptor was compared to the next lowest one using the extra sum-of-squares F test. ***, p<0.0001; *, p<0.05; n.s., not significant. Significance values are shown for the compared pairs.

12

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

A

B Tyrosine decarboxylase (TDC)

Tyrosine Tyrosine-3monooxygenase

1E-20

Tyramine Aromatic amino acid decarboxylase (AADC)

L-DOPA

1E-180

Dopamine-D2

Tyramine betahydroxylase

Octopamine

α2−adrenergic

Phenylethanolamine N-methyltransferase (PNMT)

Dopamine betahydroxylase

Noradrenaline

Dopamine

β-adrenergic

Octopamine α

Deuterostome Protostome P. dumerilii S. kowalevskii P. caudatus Xenacoelomorph

α1−adrenergic

Adrenaline

Tyramine-1

Octopamine β

Tyramine-2

C KF460458.1_Chilo_suppressalis Pca_AA2 Pdu_AA2 Sko_AA2 100 Msti_m.26298 98 Xboc_m.19526 Xboc_m.10057 49 Msti_m.20273 69 Xboc_m.13939 100 Sko_T1-2 NP_001024335.1_Caenorhabitis_elegans_T1 70 49 Pdu_T1 70 EF490687.1_Rhipicephalus_microplus_T1 81 CAQ48240.1_Periplaneta_americana_T1 CAA64865.1_Bombyx_mori_T1 94 76 BAB71788.1_Drosophila_melanogaster_T1 72 CAB76374.1_Apis_mellifera_T1 94 Q25321.1_Locusta_migratoria_T1 61

68

α2-adrenergic

94

100

tyramine-1

Xboc_m.21892 78 100

61

75

NP_650652.1_Drosophila_melanogaster_T2 ADK91078.1_Chilo_suppressalis_T2 BAI52937.1_Bombyx_mori_T2 100

tyramine-2

Pdu_T2 Sko_T2-1 Sko_T2-2

100 100

100 57 83 56

99 100 93

96

91 100

Msti_m.26958 Msti_m.21331

Sko_AA1 Pca_AA1 Pdu_AA1

100

α1-adrenergic

AAA35496.1_Homo_sapiens_AA1A U62771.1_Lymnaea_stagnalis_Oa 96 Pdu_Oa GU074418.1_Balanus_improvisus_Oa EGK96547.1_Anopheles_gambiae_Oa 99 AAC17442.1_Drosophila_melanogaster_Oa 99 100 JN641302.1_Chilo_suppressalis_Oa 100 98 BAF33393.1_Bombyx_mori_Oa 99 AAP93817.1_Periplaneta_americana_Oa CAD67999.1_Apis_mellifera_Oa Sko_OA1 XP_006825733.1_Sko_AA3 Msti_m.23488

octopamine-α

100 35

84

46

76

AY055377.1_Spisula_solidissima_Ob AF117654.1_Aplysia_kurodai_Ob 100 AF222978.1_Aplysia_californica_Ob Sko_OB1 100 GU074422.1_Balanus_improvisus_Ob GU074419.1_Balanus_improvisus_Ob 100 58 GU074420.1_Balanus_improvisus_Ob 80 82 HF548211.1_Apis_mellifera_Ob 100 54 HF548212.1_Apis_mellifera_Ob GU074421.1_Balanus_improvisus_Ob 100 55 CCO13922.1_Apis_mellifera_Ob Q4LBB9.2_Drosophila_melanogaster_Ob 96 JN620367.1_Chilo_suppressalis_Ob 100 AB470228.1_Bombyx_mori_Ob AAQ91625.1_Branchiostoma_floridae 100

octopamine-β

64 100

Msti_m.21580 63

5HTR 0.4

Figure 1. Biosynthesis of monoamines and phylogeny of adrenergic, tyramine, and octopamine GPCR sequences. (A) Biosynthesis of tyramine, octopamine, norepinephrine, and epinephrine from tyrosine. The enzymes catalyzing the reaction steps are indicated. (B) Sequence-similarity-based cluster map of bilaterian octopamine, tyramine, and adrenergic GPCRs. Nodes correspond to individual GPCRs and are colored based on taxonomy. Edges correspond to BLAST connections of P value >1e-70. (C) Simplified phylogenetic tree of bilaterian adrenergic, tyramine, and octopamine GPCR sequences. The tree is rooted on 5HT receptors. Abbreviations: Pdu, P. dumerilii; Pca, P. caudatus; Sko, S. kowalevskii; Msti, M. stichopi; Xboc, X. bocki. 13

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Figure 2. Dose-Response curves of adrenergic GPCRs from P. dumerilii, P. caudatus, and S. kowalevskii treated with varying concentrations of ligand. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3). EC50 values and significance values are listed in Table 1.

14

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 3. Dose-Response curves of tyramine GPCRs from P. dumerilii and S. kowalevskii treated with varying concentrations of ligand. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3). EC50 values and significance values are listed in Table 1.

15

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 4. Dose-Response curves of octopamine GPCRs from P. dumerilii and S. kowalevskii treated with varying concentrations of ligand. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3). EC50 values and significance values are listed in Table 1.

16

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Figure 5. Evolution of adrenergic, octopamine, and tyramine signaling in bilaterians. (A) Phylogenetic tree of major clades of bilaterian animals with the presence/loss of specific GPCR families indicated. (B) Phyletic distribution of adrenergic, octopamine, and tyramine GPCR families across major bilaterian clades. Half squares mean losses in a large number of species in a phylum.

17

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Additional files Msti_m.26298

97

α2-adrenergic receptors

Xboc_m.19526 42 82 91

XP_013383118_Lingula XP_014681069.1_Priapulus_AA2 APC23843.1_Platynereis_AA2 XP_013096124.1_Biomphalaria_glabrata_Biomphalaria 100 XP_012943687.1_Aplysia_californica_Aplysia XP_011433775.1_Crassostrea_gigas_Crassostrea XP_014767930.1_Octopus_bimaculoides_Octopus EFX76926_Daphnia KF460458.1_Chilo_suppressalis_Oa 100 100 XP_006609424_Apis 100 XP_008198464.2_Tribolium_castaneum 100 XP_014242340.1_Cimex_lectularius_Cimex 94

98

91

XP_002734932.1_Saccoglossus_kowalevskii_AA2

65 100

XP_002595000_Branchiostoma XP_003217630_Anolis 100 XP_007420380_Python 100 XP_004912925_Xenopus 100 ADRA2_CARAU_Carassius 100 CDQ85392_Oncorhynchus 100 100 XP_010895107_Esox 96 XP_015718435_Coturnix XP_006011954_Latimeria XP_002663705_Danio 100 CDQ90138_Oncorhynchus 100 CDQ73934_Oncorhynchus 100 100 81 XP_014026328 XP_007234270_Astyanax 100 94 XP_015262427_Gekko 100 XP_010350316_Saimiri 70 92 90 XP_005235799_Falco XP_007905605_Callorhinchus 97 XP_014059545_Salmo 84 91 XP_013986063_Salmo 99 XP_015463451_Astyanax 100 XP_005983264_Pantholops 100 ERE78076 100 95 100XP_015350355_Marmota XP_015262234_Gekko XP_013875010_Austrofundulus 100 93 XP_012697346_Clupea XP_007903477_Callorhinchus 100 XP_010886433_Esox 100 XP_005813740_Xiphophorus XP_007236033_Astyanax XP_002605761_Branchiostoma 100 CAL36767_Branchiostoma 100 XP_002605760.1 XP_002742354.2_Saccoglossus_T1-2 96 Msti_m.20273 tyramine type-1 receptors 98 87 Xboc_m.13939 99 Xboc_m.10057 XP_006822832_Saccoglossus XP_014776496.1_Octopus_bimaculoides XP_014672793_Priapulus 100 AKQ63052.1_Platynereis_dumerilii_T1 100 ELT94407.1_Capitella_teleta 78 NP_001024335.1_Caenorhabitis_elegans_T1 99 94 XP_011451309.1_Crassostrea_gigas XP_001944003.2_octopamine 97 CAA64865.1_Bombyx_mori_T1 KNC24708.1_Lucilia_cuprina 21 99 96 XP_011202418.1_Bactrocera_dorsalis 100 XP_002068046.2_Drosophila_willistoni 98 KFB51134.1_Anopheles_sinensis 100 ETN58983.1_Anopheles_darlingi 91 21 100 XP_312420.3_Anopheles_gambiae_str._PEST XP_001863342.1_Culex_quinquefasciatus XP_002426131.1_Pediculus_humanus_corporis 94 XP_008472990.1_Diaphorina_citri 35 91 83 ALM55746.1_Sitophilus_oryzae 95 XP_014278335.1_Halyomorpha_halys 96 KOB71731.1_Operophtera_brumata 100 XP_011562493.1_Plutella_xylostella NP_001164311.1_Tribolium_castaneum 65 KDR16544.1_Zootermopsis_nevadensis 97 KDR16545.1_Zootermopsis_nevadensis 65 XP_014481134.1_Dinoponera_quadriceps 68 100 XP_014235818.1_Trichogramma_pretiosum 99 99 XP_008206976.1_Nasonia_vitripennis Q25321.1_Locusta_migratoria_T1 100 CAQ48240.1_Periplaneta_americana_T1 ABY71758.1_Macrobrachium_rosenbergii 99 XP_015917655.1_Parasteatoda 81 XP_002415939.1_Ixodes_scapularis 81 XP_013784920.1_Limulus_polyphemus 100 100 XP_013791379_Limulus EF490687.1_Rhipicephalus_microplus_T1 Xboc_m.21892 XP_014678717.1_Priapulus_caudatus 77 XP_006812999.1_Saccoglossus_kowalevskii_T2B 100 XP_002734062.1_Saccoglossus_kowalevskii_T2A tyramine type-2 receptors 96 XP_014778476.1_Octopus_bimaculoides 96 ELU14919.1_Capitella_teleta 90 KU715093_Platynereis_dumerilii_T2 XP_004529356.1_Ceratitis_capitata XP_002096110.1_Drosophila_yakuba 82 87 82 KNC22330.1_Lucilia_cuprina 99 100 84 XP_011293189.1_Musca_domestica XP_013112135.1_Stomoxys_calcitrans 99 XP_001998427.2_Drosophila_mojavensis ETN64745.1_Anopheles_darlingi XP_004529353.2_Ceratitis_capitata 72 72 XP_001998428.2_Drosophila_mojavensis 93 XP_004529355.2_Ceratitis_capitata 100 XP_001954230.1_Drosophila_ananassae 100 100 XP_001359694.4_Drosophila_pseudoobscura_pseudoobscura XP_013112132.1_Stomoxys_calcitrans 100 100 68 KNC22325.1_Lucilia_cuprina KFB41294.1_Anopheles_sinensis 99 ETN64509.1_Anopheles_darlingi 85 XP_014207104.1_Copidosoma_floridanum 74 XP_014235895.1_Trichogramma_pretiosum 93 81 XP_011500363.1_Ceratosolen_solmsi_marchali_5HTR KOX76145.1_Melipona_quadrifasciata 100 100 KOC70478.1_Habropoda_laboriosa 93 XP_012257334.1_Athalia_rosae 91 XP_013142077.1_Papilio_polytes 93 ERL83362.1_Dendroctonus_ponderosae 99 XP_008197781.1_Tribolium_castaneum 100 100 DAA64505.1_Tribolium_castaneum 91 AHN85846.1_Nicrophorus_vespilloides XP_002429476.1_Pediculus_humanus_corporis 93 100 KDR18992.1_Zootermopsis_nevadensis XP_008475339.1_Diaphorina_citri 100 XP_015368899 XP_013786964.1_Limulus_polyphemus Msti_m.26958 100 Msti_m.21331 α1-adrenergic receptors ALR88680.1_Saccoglossus_AA1-1 100 XP_003726237_Strongylocentrotus XP_014662992.1_Priapulus_caudatus_AA1 100 XP_014663408_Priapulus 85 XP_015598849_Cephus 100 99 EFX64650_Daphnia 100 XP_003748403_Metaseiulus 85 99 100 XP_013787844_Limulus XP_014777674_Octopus 100 98 APC23842.1_Platynereis_AA1 100 XP_009050255_Lottia XP_011422115_Crassostrea 99 XP_012936806_Aplysia AAA35496.1_Homo_sapiens_AA1A 98 XP_009993014_Chaetura 99 XP_010795024_Notothenia 100 100 CDQ72256_Oncorhynchus 100 XP_014051077_Salmo 100 100 KTG32062_Cyprinus XP_007896221_Callorhinchus 87 XP_003782039_Otolemur 99 KTF87842_Cyprinus 84 99 XP_011478573_Oryzias 99 82 XP_01090054_Esox 100XP_012697555_Clupea 100 KTG31243_Cyprinus XP_002933012_Xenopus 100 XP_004665892_Jaculus 100 XP_006625863_Lepisosteus 100 XP_008334310_Cynoglossus 100 100 XP_015827734.1_Nothobranchius_furzeri XP_006010360_Latimeria XP_006823182.1_Saccoglossus_kowalevskii_Oa octopamine α receptors XM_013530280.1_Lingula_anatina 90 KU530199_Platynereis_dumerilii_Oa 100 XP_014675454.1_Priapulus_caudatus 93 XP_014772693.1_Octopus_bimaculoides 97 93 XP_009058244.1_Lottia_gigantea 100 U62771.1_Lymnaea_stagnalis_Oa 100 XP_012937854.1_Aplysia_californica XP_013790352_Limulus 91 100 XP_013772246_Limulus GU074418.1_Balanus_improvisus_Oa XP_014291339.1_Halyomorpha_halys 56 XP_014253839.1_Cimex_lectularius 100 XP_002429940.1_Pediculus_humanus_corporis 31 94 XP_014225936.1_Trichogramma_pretiosum 100 XP_011303410.1_Fopius_arisanus 100 83 100XP_011702891.1_Wasmannia_auropunctata 99 CAD67999.1_Apis_mellifera_Oa AAP93817.1_Periplaneta_americana_Oa XP_011185898.1_Bactrocera_cucurbitae 99 73 XP_011185894.1_Bactrocera_cucurbitae 92 XP_012156222.1_Ceratitis_capitata 77 XP_002096452.2_Drosophila_yakuba 100 99 XP_013110682.1_Stomoxys_calcitrans 100 XP_011291813.1_Musca_domestica 93 AAC17442.1_Drosophila_melanogaster_Oa 100 99 XP_001994470.1_Drosophila_grimshawi XP_311113.4_Anopheles_gambiae_str._PEST 100EGK96547.1_Anopheles_gambiae_Oa 88 100 XP_001869401.1_Culex_quinquefasciatus 100 XP_001648244.2_Aedes_aegypti ERL88449.1_Dendroctonus_ponderosae 100 48 97 AHN85844.1_Nicrophorus_vespilloides NP_001280520.1_Tribolium_castaneum EHJ74631.1_Danaus_plexippus Xboc_m.20509 XP_002733926.1_Saccoglossus_kowalevskii_Ob octopamine β receptors KU886229_Platynereis_dumerilii 100 ELT86969.1_Capitella_teleta XP_011448928.1_Crassostrea_gigas 98 100 AY055377.1_Spisula_solidissima_Ob 100 XP_014771877.1_Octopus_bimaculoides 99 AF117654.1_Aplysia_kurodai_Ob 100 99 FC563777.1_Lottia_gigantea XP_013389322.1_Lingula_anatina 100 GU074422.1_Balanus_improvisus_Ob AEO17899.1_Drosophila_melanogaster 97 100KNC25005.1_Lucilia_cuprina 89 XP_014088159.1_Bactrocera_oleae XP_001947781.1_Acyrthosiphon_pisum 98 XP_012269188.1_Athalia_rosae 98 100 100 XP_011349656.1_Cerapachys_biroi 98 NP_001280514.1_Tribolium_castaneum 9899 XP_011557485.1_Plutella_xylostella 93 95 100 XP_002422997.1_Pediculus_humanus_corporis XP_014241423.1_Cimex_lectularius GU074421.1_Balanus_improvisus_Ob XP_001948521.2_Acyrthosiphon_pisum 96 87 NP_001280505.1_Tribolium_castaneum 97 97 HF548211.1_Apis_mellifera_Ob 86 100 HF548212.1_Apis_mellifera_Ob GU074419.1_Balanus_improvisus_Ob 100 GU074420.1_Balanus_improvisus_Ob 100 EFX87996.1_Daphnia_pulex JN620367.1_Chilo_suppressalis_Ob 92 Q4LBB9.2_Drosophila_melanogaster_Ob 42 100 KNC25006.1_Lucilia_cuprina 100 100 100 XP_011294329.1_Musca_domestica AHN85842.1_Nicrophorus_vespilloides 92 XP_008207151 100 XP_012343074 XP_014671143.1_Priapulus_caudatus_Ecdysozoa Msti_m.23488 CAL36658.1_Branchiostoma_floridae Dopamine receptors 98 XP_006825733.1_Saccoglossus XP_013419306.1_Lingula_anatina 98 XP_011417061.1_Crassostrea_gigas 99 100 XP_013063747.1_Biomphalaria_glabrata 100 100 99 XP_005099999.1_Aplysia_californica XP_014768037.1_Octopus_bimaculoides XP_014663547.1_Priapulus_caudatus 99 NP_001155849.1_Nasonia_vitripennis 100 XP_014233405.1_dopamine 91 XP_014297585.1_Microplitis_demolitor 89 XP_013186105.1_Amyelois_transitella 92 BAM15635.1_Gryllus_bimaculatus 99 90 98 44 AJF34871.1_Culex_quinquefasciatus 100 XP_014260659.1_Cimex_lectularius XP_008178377.1_Acyrthosiphon_pisum 100 AFC88980.1_Rhipicephalus_microplus 100 XP_013783833.1_Limulus_polyphemus 100 100 XP_013774755.1_Limulus_polyphemus XP_015791409_Tetranychus_urticae 66

98

100

91

Msti_m.14301 XP_002122923.1_Ciona_intestinalis_beta-2_adrenergic deuterostomia_gi_724933078_ref_XP_010383600.1_ 100 XP_007260201_beta-1_adrenergic 100 XP_006000436 β adrenergic receptors XP_007903898_beta-1_adrenergic NP_000675.1_Homo_sapiens XP_013918020_beta-1_adrenergic XP_015818613.1_Nothobranchius_furzeri_beta-1_adrenergic XP_006002873_beta-1_adrenergic XP_00790621_Callorhinchus 99 XP_013873112_beta-2_adrenergic 100 XP_007553424_beta-2_adrenergic 100 XP_006784977_beta-2_adrenergic 100 100 100 XP_010869486_beta-2_adrenergic 81 100 XP_007231763-beta-2-adrenergic XP_015204860_Lepisosteus 63 KPP77020_Scleropages 61 XP_014054344_Salmo XP_004073575_Oryzias 100 10062 95 XP_013870150_Austrofundulus 94 100 XP_015232667_beta-2_adrenergic 98 KPP58190_beta-2-adrenergic XP_015262106_Gekko 100 NP_001085791_Xenopus CAA06539.1_Myxine_glutinosa CAA06540.1_Petromyzon_marinus 100 CAA06541.1_Petromyzon_marinus XP_013397587.1_5HTR_1A_Lingula XP_011674268.1_5HTR_1D_Strongylocentrotus XP_012695048.1_5HTR_7_Clupea 76 XP_007244750.1_5HTR_7_Astyanax 100 100 92 XP_007568067.2_5HTR_7_Poecilia 69 XP_006643246.1_5HTR_7_Lepisosteus 76 XP_005997495.1_5HTR_7_Latimeria 100 XP_018584957.1_5HTR_7_Scleropages XP_014096646.1_5HTR_1_Bactrocera 100 XP_003436743.1_Anopheles 100 100 99 XP_001866051.1_Culex XP_014281330.1_5HTR_1 100 XP_014678386.1_5HTR_1D_Priapulus 100 XP_014663879.1_5HTR_1B_Priapulus 100 XP_014788935.1_5HTR_1_Octopus 100 XP_009020628.1 66 96 XP_013378759.1_5HTR_1A_Lingula 100 XP_009066515.1_Lottia 100 53 XP_012940601.1_5HTR_1A_Aplysia XP_006820691.1_5HTR_1A_Saccoglossus XP_002601117.1_Branchiostoma XP_013074022.1_5HTR_1D_Biomphalaria 57 XP_009028850.1_Helobdella 99 XP_009066850.1_Lottia 98 XP_011455576.1_Crassostrea XP_015905348.1_5HTR_2B_Parasteatoda XP_003742827.1_5HTR_2B_Metaseiulus 100 XP_002404998.1_Ixodes 90 XP_015924596.1_5HTR_2A_Parasteatoda 97 XP_002405023.1_Ixodes 99 74 XP_013789173.1_5HTR_2B_Limulus 100 XP_013789181.1_5HTR_2A_Limulus 100 100 XP_013791278.1_5-hydroxytryptamine 79 97 XP_013789239.1_Limulus XP_013781760.1_5HTR_2A_Limulus 95 98 XP_013785454.1_5HTR_2A_Limulus 100 100 XP_013775446.1_5HTR_2A_Limulus 100 XP_013777810.1_5HTR_2A_Limulus XP_013115395.1_5HTR_2B_Stomoxys 100 XP_014294529.1_5HTR_2A_Halyomorpha 82 100 XP_015113987.1_5HTR_2A_Diachasma 61 100 XP_015523714.1_Neodiprion 99 100 XP_011347974.1_5HTR_2A_Cerapachys XP_012282343.1_Orussus 100 XP_015838795.1_5HTR_isoform XP_014679417.1_Priapulus XP_008322888.1_5HTR_1A-beta_Cynoglossus 95 XP_010731295.1_5HTR_1A_Larimichthys 100 XP_017322775.1_5HTR_1A_Ictalurus XP_008401851.1_5HTR_1F_Poecilia 96 XP_016341876.1_5HTR_1F_Sinocyclocheilus 98 XP_018584792.1_5HTR_1F_Scleropages 82 100 99 XP_016089563.1_5HTR_1F_Sinocyclocheilus 100 XP_012688903.1_5HTR_1F_Clupea 100 XP_018598909.1_5HTR_1F_Scleropages XP_009090906.1_5HTR_1F_Serinus XP_012678014.1_5HTR_1E_Clupea 100 100 XP_010877910.1_5HTR_1E_Esox 74 XP_017307091.1_5HTR_1E_Ictalurus 91 XP_016370122.1_5HTR_1E_Sinocyclocheilus 100 100 XP_006626209.2_5HTR_1E_Lepisosteus 81 93 XP_007892053.1_5HTR_1E_Callorhinchus XP_007893173.1_5HTR_1D_Callorhinchus 100 XP_017330977.1_5HTR_1D_Ictalurus 87 100 XP_007886351.1_5HTR_1B_Callorhinchus 100 XP_018590111.1_5HTR_1B_Scleropages 76 100 XP_018599065.1_5HTR_1B_Scleropages XP_002606172.1_Branchiostoma 100 XP_002595699.1_Branchiostoma Msti_m.21580 XP_006814563.1_5HTR_1A-alpha_Saccoglossus 22

96

99

78 42

39

Additional file 1. Maximum likelihood tree of adrenergic, octopamine, and tyramine receptors. Bootstrap support values are shown. This tree containing all investigated GPCRs. The tree was rooted on 5HT receptor sequences. Subtrees are shown in Additional files 2-8. 0.5

18

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

α1-adrenergic receptors

100

100

85 99

Msti_m.26958 Msti_m.21331

ALR88680.1_Saccoglossus_AA1-1 XP_003726237_Strongylocentrotus XP_014662992.1_Priapulus_caudatus_AA1 100 XP_014663408_Priapulus XP_015598849_Cephus

Xenacoelomorpha Hemichordata Priapulida

100

EFX64650_Daphnia XP_003748403_Metaseiulus XP_013787844_Limulus XP_014777674_Octopus Annelida 100 APC23842.1_Platynereis_AA1 100 XP_009050255_Lottia XP_011422115_Crassostrea 99 XP_012936806_Aplysia AAA35496.1_Homo_sapiens_AA1A 98 XP_009993014_Chaetura XP_010795024_Notothenia 100 100 CDQ72256_Oncorhynchus 100 XP_014051077_Salmo 100 100 KTG32062_Cyprinus XP_007896221_Callorhinchus 87 XP_003782039_Otolemur KTF87842_Cyprinus 84 99 XP_011478573_Oryzias 99 82 XP_01090054_Esox 100XP_012697555_Clupea 100 KTG31243_Cyprinus XP_002933012_Xenopus 100 XP_004665892_Jaculus 100 XP_006625863_Lepisosteus 100 XP_008334310_Cynoglossus 100 100 XP_015827734.1_Nothobranchius XP_006010360_Latimeria 100

85

100

98

99

99

Additional file 2. Maximum likelihood tree of α1-adrenergic receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. 19

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

α2-adrenergic receptors

Msti_m.26298

97

Xenacoelomorpha

Xboc_m.19526 42 82 91 91

Priapulida XP_013383118_Lingula XP_014681069.1_Priapulus_AA2b Annelida APC23843.1_Platynereis_AA2 XP_013096124.1_Biomphalaria_glabrata 100 XP_012943687.1_Aplysia_californica 94 Mollusca XP_011433775.1_Crassostrea_gigas_Crassostrea 98 XP_014767930.1_Octopus_bimaculoides_Octopus EFX76926_Daphnia KF460458.1_Chilo_suppressalis_Oa 100 100 XP_006609424_Apis 100 XP_008198464.2_Tribolium_castaneum 100 Hemichordata XP_014242340.1_Cimex_lectularius_Cimex XP_002734932.1_Saccoglossus_kowalevskii_AA2

65 100

66

94 70

100

XP_002595000_Branchiostoma XP_003217630_Anolis 100 XP_007420380_Python 100 XP_004912925_Xenopus 100 ADRA2_CARAU_Carassius 100 CDQ85392_Oncorhynchus 100 100 XP_010895107_Esox 96 XP_015718435_Coturnix XP_006011954_Latimeria XP_002663705_Danio 100 CDQ90138_Oncorhynchus 100 CDQ73934_Oncorhynchus 100 100 81 XP_014026328 XP_007234270_Astyanax 100 XP_015262427_Gekko 100 XP_010350316_Saimiri 92 90 XP_005235799_Falco XP_007905605_Callorhinchus 97 XP_014059545_Salmo 84 91 XP_013986063_Salmo 99 XP_015463451_Astyanax 100 XP_005983264_Pantholops 100 ERE78076 100 95 100XP_015350355_Marmota XP_015262234_Gekko XP_013875010_Austrofundulus 100 93 XP_012697346_Clupea XP_007903477_Callorhinchus 100 XP_010886433_Esox 100 XP_005813740_Xiphophorus XP_007236033_Astyanax XP_002605761_Branchiostoma CAL36767_Branchiostoma 100 XP_002605760.1

Cephalochordata

Additional file 3. Maximum likelihood tree of α2-adrenergic receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. 20

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

β-adrenergic receptors

96

99

100

100 98

100

Msti_m.14301 XP_002122923.1_Ciona_intestinalis XP_010383600.1_Rhinopithecus 100 XP_007260201_Astyanax 100 XP_006000436_Latimeria 22 XP_007903898 99 NP_000675.1_Homo_sapiens 78 XP_013918020 42 39 XP_015818613.1_Nothobranchius_furzeri XP_006002873 XP_00790621_Callorhinchus XP_013873112 100 XP_007553424 100 XP_006784977 100 100 XP_010869486 81 100 XP_007231763 XP_015204860_Lepisosteus 63 KPP77020_Scleropages 61 XP_014054344_Salmo XP_004073575_Oryzias 100 10062 95 XP_013870150_Austrofundulus 94 XP_015232667 KPP58190 XP_015262106_Gekko 100 NP_001085791_Xenopus CAA06539.1_Myxine_glutinosa CAA06540.1_Petromyzon_marinus CAA06541.1_Petromyzon_marinus

Xenacoelomorpha

Additional file 4. Maximum likelihood tree of β-adrenergic receptors. Bootstrap support values are shown for some nodes of interest. This tree is part of a larger tree containing all investigated GPCRs. 21

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Tyramine-1 receptors

Hemichordata 96 87

98

99

100

78 99

96

83

99

XP_002742354.2_Saccoglossus_T1-2 Msti_m.20273 Xenacoelomorpha Xboc_m.13939 Xboc_m.10057 XP_006822832_Saccoglossus XP_014776496.1_Octopus_bimaculoides Priapulida XP_014672793_Priapulus AKQ63052.1_Platynereis_dumerilii_T1 100 ELT94407.1_Capitella_teleta NP_001024335.1_Caenorhabitis_elegans_T1 94 XP_011451309.1_Crassostrea_gigas XP_001944003.2_octopamine 97 CAA64865.1_Bombyx_mori_T1 KNC24708.1_Lucilia_cuprina 21 99 XP_011202418.1_Bactrocera_dorsalis 100 XP_002068046.2_Drosophila_willistoni 98 KFB51134.1_Anopheles_sinensis 100 ETN58983.1_Anopheles_darlingi 91 21 100 XP_312420.3_Anopheles_gambiae_str._PEST XP_001863342.1_Culex_quinquefasciatus XP_002426131.1_Pediculus_humanus_corporis 94 XP_008472990.1_Diaphorina_citri 35 91 ALM55746.1_Sitophilus_oryzae 95 XP_014278335.1_Halyomorpha_halys 96 KOB71731.1_Operophtera_brumata 100 XP_011562493.1_Plutella_xylostella NP_001164311.1_Tribolium_castaneum 65 KDR16544.1_Zootermopsis_nevadensis 97 KDR16545.1_Zootermopsis_nevadensis 65 XP_014481134.1_Dinoponera_quadriceps 68 100 XP_014235818.1_Trichogramma_pretiosum 99 99 XP_008206976.1_Nasonia_vitripennis Q25321.1_Locusta_migratoria_T1 100 CAQ48240.1_Periplaneta_americana_T1 ABY71758.1_Macrobrachium_rosenbergii XP_015917655.1_Parasteatoda 81 XP_002415939.1_Ixodes_scapularis 81 XP_013784920.1_Limulus_polyphemus 100 100 XP_013791379_Limulus EF490687.1_Rhipicephalus_microplus_T1

Annelida Nematoda

Additional file 5. Maximum likelihood tree of Tyramine-1 receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. The identifiers of deorphanized tyramine receptors were tagged with _T1.

22

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Tyramine-2 receptors

Xboc_m.21892

Xenacoelomorpha

Priapulida 77 100 96 96 90

99

68

100

XP_014678717.1_Priapulus_caudatus XP_006812999.1_Saccoglossus_kowalevskii_T2B Hemichordata XP_002734062.1_Saccoglossus_kowalevskii_T2A XP_014778476.1_Octopus_bimaculoides ELU14919.1_Capitella_teleta Annelida KU715093_Platynereis_dumerilii_T2 XP_004529356.1_Ceratitis_capitata XP_002096110.1_Drosophila_yakuba 82 87 82 KNC22330.1_Lucilia_cuprina 100 84 XP_011293189.1_Musca_domestica XP_013112135.1_Stomoxys_calcitrans 99 XP_001998427.2_Drosophila_mojavensis ETN64745.1_Anopheles_darlingi XP_004529353.2_Ceratitis_capitata 72 72 XP_001998428.2_Drosophila_mojavensis 93 XP_004529355.2_Ceratitis_capitata 100 XP_001954230.1_Drosophila_ananassae 100 100 XP_001359694.4_Drosophila_pseudoobscura XP_013112132.1_Stomoxys_calcitrans 100 100 KNC22325.1_Lucilia_cuprina KFB41294.1_Anopheles_sinensis 99 ETN64509.1_Anopheles_darlingi 85 XP_014207104.1_Copidosoma_floridanum 74 XP_014235895.1_Trichogramma_pretiosum 93 81 XP_011500363.1_Ceratosolen_solmsi KOX76145.1_Melipona_quadrifasciata 100 100 KOC70478.1_Habropoda_laboriosa 93 XP_012257334.1_Athalia_rosae 91 XP_013142077.1_Papilio_polytes 93 ERL83362.1_Dendroctonus_ponderosae 99 XP_008197781.1_Tribolium_castaneum 100 100 DAA64505.1_Tribolium_castaneum 91 AHN85846.1_Nicrophorus_vespilloides XP_002429476.1_Pediculus_humanus_corporis 93 100 KDR18992.1_Zootermopsis_nevadensis XP_008475339.1_Diaphorina_citri XP_015368899 XP_013786964.1_Limulus_polyphemus

Additional file 6. Maximum likelihood tree of Tyramine-2 receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. The identifiers of deorphanized tyramine receptors were tagged with _T2. 23

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Octopamin-α receptors Hemichordata XP_006823182.1_Saccoglossus_kowalevskii_Oa XM_013530280.1_Lingula_anatina KU530199_Platynereis_dumerilii_Oa XP_014675454.1_Priapulus_caudatus 93 XP_014772693.1_Octopus_bimaculoides 93 XP_009058244.1_Lottia_gigantea 100 U62771.1_Lymnaea_stagnalis_Oa 100 XP_012937854.1_Aplysia_californica XP_013790352_Limulus 91 100 XP_013772246_Limulus GU074418.1_Balanus_improvisus_Oa XP_014291339.1_Halyomorpha_halys 56 XP_014253839.1_Cimex_lectularius 100 XP_002429940.1_Pediculus_humanus_corporis 31 94 XP_014225936.1_Trichogramma_pretiosum 100 XP_011303410.1_Fopius_arisanus 100 83 100XP_011702891.1_Wasmannia_auropunctata 99 CAD67999.1_Apis_mellifera_Oa AAP93817.1_Periplaneta_americana_Oa XP_011185898.1_Bactrocera_cucurbitae 73 XP_011185894.1_Bactrocera_cucurbitae 92 XP_012156222.1_Ceratitis_capitata 77 XP_002096452.2_Drosophila_yakuba 100 99 XP_013110682.1_Stomoxys_calcitrans 100 XP_011291813.1_Musca_domestica 93 AAC17442.1_Drosophila_melanogaster_Oa 100 99 XP_001994470.1_Drosophila_grimshawi XP_311113.4_Anopheles_gambiae_str._PEST 100EGK96547.1_Anopheles_gambiae_Oa 88 100 XP_001869401.1_Culex_quinquefasciatus 100 XP_001648244.2_Aedes_aegypti ERL88449.1_Dendroctonus_ponderosae 100 48 97 AHN85844.1_Nicrophorus_vespilloides NP_001280520.1_Tribolium_castaneum EHJ74631.1_Danaus_plexippus

90 100

97

Annelida Priapulida Mollusca

Additional file 7. Maximum likelihood tree of Octopamine-α receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. The identifiers of deorphanized octopamine receptors were tagged with _Oa.

24

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Octopamin-β receptors Hemichordata XP_002733926.1_Saccoglossus_kowalevskii_Ob KU886229_Platynereis_dumerilii Annelida ELT86969.1_Capitella_teleta XP_011448928.1_Crassostrea_gigas 98 100 AY055377.1_Spisula_solidissima_Ob 100 XP_014771877.1_Octopus_bimaculoides Mollusca 99 AF117654.1_Aplysia_kurodai_Ob 100 99 FC563777.1_Lottia_gigantea XP_013389322.1_Lingula_anatina GU074422.1_Balanus_improvisus_Ob AEO17899.1_Drosophila_melanogaster 97 100KNC25005.1_Lucilia_cuprina 89 XP_014088159.1_Bactrocera_oleae XP_001947781.1_Acyrthosiphon_pisum 98 XP_012269188.1_Athalia_rosae 100 100 XP_011349656.1_Cerapachys_biroi 98 NP_001280514.1_Tribolium_castaneum 9899 XP_011557485.1_Plutella_xylostella 95 100 XP_002422997.1_Pediculus_humanus_corporis XP_014241423.1_Cimex_lectularius GU074421.1_Balanus_improvisus_Ob XP_001948521.2_Acyrthosiphon_pisum 96 87 NP_001280505.1_Tribolium_castaneum 97 97 HF548211.1_Apis_mellifera_Ob 86 100 HF548212.1_Apis_mellifera_Ob GU074419.1_Balanus_improvisus_Ob 100 GU074420.1_Balanus_improvisus_Ob 100 EFX87996.1_Daphnia_pulex JN620367.1_Chilo_suppressalis_Ob 92 Q4LBB9.2_Drosophila_melanogaster_Ob 100 KNC25006.1_Lucilia_cuprina 100 100 XP_011294329.1_Musca_domestica AHN85842.1_Nicrophorus_vespilloides 92 XP_008207151 100 XP_012343074 Priapulida XP_014671143.1_Priapulus_caudatus 100

100

93

100

Additional file 8. Maximum likelihood tree of Octopamine-β receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. The identifiers of deorphanized octopamine receptors were tagged with _Ob. 25

aromatic amino acid decarboxylase

XP_014769443.1_Octopus_bimaculoides XP_011441559.1_Crassostrea_gigas EKC37654.1_Crassostrea_gigas NP_001191536.1_Aplysia_californica XP_012940696.1_Aplysia_californica XP_013070812.1_Biomphalaria_glabrata BAQ94593.1_Ambigolimax_valentianus XP_013401543.1_Lingula_anatina XP_014666248.1_Priapulus_caudatus XP_013781988.1_Limulus_polyphemus XP_014245782.1_Cimex_lectularius XP_008202389.1_Nasonia_vitripennis XP_012274121.1_Orussus_abietinus XP_006570903.1_Apis_mellifera XP_006621116.1_Apis_dorsata KFB44891.1_Anopheles_sinensis XP_011290217.1_Musca_domestica ACJ13262.1_Drosophila_melanogaster XP_002731843.1_Saccoglossus_kowalevskii lcl|comp422145_c0_seq4_Platynereis_dumerilii ELU11164.1_Capitella_teleta XP_009023368.1_Helobdella_robusta 91 XP_014774406.1_Octopus_bimaculoides XP_009048193.1_Lottia_gigantea XP_005107794.1_Aplysia_californica XP_011449820.1_Crassostrea_gigas ADP08788.1_Azumapecten_farreri XP_013404119.1_Lingula_anatina 100 EFX81779.1_Daphnia_pulex AHN85839.1_Nicrophorus_vespilloides XP_308519.3_Anopheles_gambiae_str._PEST XP_001656857.1_Aedes_aegypti XP_001870804.1_Culex_quinquefasciatus XP_002050467.1_Drosophila_virilis XP_001360141.2_Drosophila_pseudoobscura_pseudoobscura XP_015054436.1_Drosophila_yakuba XP_002074575.1_Drosophila_willistoni XP_004526583.1_Ceratitis_capitata 100 XP_011196622.1_Bactrocera_cucurbitae KNC26593.1_Lucilia_cuprina XP_005180269.1_Musca_domestica XP_002426536.1_Pediculus_humanus_corporis XP_008198031.1_Tribolium_castaneum ENN79134.1_Dendroctonus_ponderosae XP_014241491.1_Cimex_lectularius XP_014291344.1_Halyomorpha_halys XP_014291341.1_Halyomorpha_halys BAO52000.1_Gryllus_bimaculatus XP_012265736.1_Athalia_rosae XP_003699867.1_Megachile_rotundata XP_012534953.1_Monomorium_pharaonis XP_011061414.1_Acromyrmex_echinatior XP_015122001.1_Diachasma_alloeum XP_011297268.1_Fopius_arisanus 96 XP_008548921.1_Microplitis_demolitor XP_972728.2_Tribolium_castaneum KRT84890.1_Oryctes_borbonicus KDR12158.1_Zootermopsis_nevadensis 100 XP_014227242.1_Trichogramma_pretiosum XP_012265735.1_Athalia_rosae XP_008548824.1_Microplitis_demolitor XP_011297267.1_Fopius_arisanus XP_014489121.1_Dinoponera_quadriceps XP_012055250.1_Atta_cephalotes XP_012272741.1_Orussus_abietinus XP_014605048.1_Polistes_canadensis XP_012147325.1_Megachile_rotundata KOX68436.1_Melipona_quadrifasciata XP_013789180.1_Limulus_polyphemus XP_003744997.1_Metaseiulus_occidentalis XP_002630249.1_Caenorhabditis_briggsae NP_495744.1_Caenorhabditis_elegans XP_001899866.1_Brugia_malayi EFO20782.2_Loa_loa KHN71251.1_Toxocara_canis CEF60746.1_Strongyloides_ratti XP_014671545.1_Priapulus_caudatus XP_005108337.1_Aplysia_californica XP_011443238.1_Crassostrea_gigas EKC25403.1_Crassostrea_gigas XP_002735383.1_Saccoglossus_kowalevskii XP_007895676.1_Callorhinchus_milii XP_006635648.1_Lepisosteus_oculatus XP_012680382.1_Clupea_harengus XP_010895280.1_Esox_lucius XP_005949669.1_Haplochromis_burtoni XP_015245016.1_Cyprinodon_variegatus XP_003966032.1_Takifugu_rubripes 95 KTG31909.1_Cyprinus_carpio XP_010138453.1_Buceros_rhinoceros_silvestris 100 XP_005434516.1_Falco_cherrug XP_009680213.1_Struthio_camelus_australis 52XP_010219192.1_Tinamus_guttatus XP_010022195.1_Nestor_notabilis XP_010006646.1_Chaetura_pelagica XP_008489540.1_Calypte_anna XP_015508789.1_Parus_major XP_007061422.1_Chelonia_mydas XP_005278593.1_Chrysemys_picta_bellii XP_003407186.1_Loxodonta_africana XP_004708097.1_Echinops_telfairi XP_006875343.1_Chrysochloris_asiatica XP_007942142.1_Orycteropus_afer_afer XP_004739444.1_Mustela_putorius_furo XP_004415117.1_Odobenus_rosmarus_divergens XP_848285.2_Canis_lupus_familiaris XP_014649145.1_Ceratotherium_simum_simum XP_012025784.1_Ovis_aries_musimon XP_004694560.1_Condylura_cristata XP_010339695.1_Saimiri_boliviensis_boliviensis XP_003896028.1_Papio_anubis ELV13924.1_Tupaia_chinensis XP_004582273.2_Ochotona_princeps XP_012879949.1_Dipodomys_ordii XP_008847491.1_Nannospalax_galili XP_008768473.1_Rattus_norvegicus XP_006973100.1_Peromyscus_maniculatus_bairdii XP_005396658.1_Chinchilla_lanigera XP_013000851.1_Cavia_porcellus XP_004630583.1_Octodon_degus XP_004839911.1_Heterocephalus_glaber XP_015345826.1_Marmota_marmota_marmota XP_003797782.1_Otolemur_garnettii XP_012506091.1_Propithecus_coquereli XP_003762585.1_Sarcophilus_harrisii XP_001379620.3_Monodelphis_domestica XP_006000049.1_Latimeria_chalumnae XP_014667849.1_Priapulus_caudatus XP_012942331.1_Aplysia_californica XP_012942336.1_Aplysia_californica XP_013088909.1_Biomphalaria_glabrata BAM35936.1_Lymnaea_stagnalis EKC41301.1_Crassostrea_gigas XP_014775466.1_Octopus_bimaculoides XP_009050823.1_Lottia_gigantea XP_013772406.1_Limulus_polyphemus XP_012226027.1_Linepithema_humile XP_002002172.1_Drosophila_mojavensis XP_001950555.2_Acyrthosiphon_pisum XP_002426339.1_Pediculus_humanus_corporis XP_008193582.1_Tribolium_castaneum XP_012262153.1_Athalia_rosae XP_008553493.1_Microplitis_demolitor XP_003704683.1_Megachile_rotundata XP_014230308.1_Trichogramma_pretiosum CAJ38793.1_Platynereis_dumerilii XP_009028665.1_Helobdella_robusta ELU05968.1_Capitella_teleta ELU12210.1_Capitella_teleta XP_013421253.1_Lingula_anatina XP_013419956.1_Lingula_anatina NP_001161568.1_Saccoglossus_kowalevskii XP_002597913.1_Branchiostoma_floridae XP_006030466.1_Alligator_sinensis XP_004010670.1_Ovis_aries AAI30528.1_Homo_sapiens NP_058712.2_Rattus_norvegicus XP_544676.3_Canis_lupus_familiaris BAP16218.1_Gallus_gallus ELU18925.1_Capitella_teleta lcl|Contig8616_Platynereis_dumerilii XP_009056307.1_Lottia_gigantea

tyrosine decarboxylase

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

0.2

Additional file 9. Maximum likelihood tree of tyrosine decarboxylase and aromatic amino acid decarboxylase enzymes. Bootstrap support values are shown for selected nodes. P. dumerilii. P. caudatus, and S. kowalevskii sequences are highlighted in color. The Caenorhabditis elegans tyrosine decarboxylase was experimentally shown to be required for tyramine biosynthesis [32].

26

bioRxiv preprint first posted online Jul. 13, 2016; doi: http://dx.doi.org/10.1101/063743. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Additional file 10. Dose-response curves of adrenergic, tyramine, and octopamine receptors from P. dumerilii, P. caudatus, and S. kowalevskii treated with varying concentrations of inhibitors. Data, representing luminescence units relative to the maximum of the fitted doseresponse curves, are shown as mean ± SEM (n = 3). IC50 values are listed in Table 1.

27