The tick Ixodes scapularis has five different GPCRs specifically activated by ACP (adipokinetic hormone/corazonin-related peptide)

The tick Ixodes scapularis has five different GPCRs specifically activated by ACP (adipokinetic hormone/corazonin-related peptide)


Introduction
Adipokinetic hormone/corazonin-related peptide (ACP) is member of a small family of insect neuropeptides, to which also adipokinetic hormone (AKH), and corazonin belong [1].Not only are these neuropeptide sequences structurally related, but also are the sequences of their G protein-coupled receptors (GPCRs) [1,2].From these findings, we and other researchers previously concluded that these neuropeptide/receptors couples originated by gene duplications from a single ligand gene and a single receptor gene followed by co-evolution of their ligands and receptors [1][2][3][4][5].Although insect ACP, AKH, and corazonin peptides are closely related, their receptors are specific for their cognate ligands, i.e., ACP receptors are not activated by AKH, or corazonin.Likewise, AKH receptors are not activated by ACP or corazonin, and corazonin receptors are not activated by ACP or AKH [1,5].We assume that this is due to an evolutionary pressure to keep the three signalling systems physiologically separated.
Insects are the largest animal taxon (class) with between 1.3 million and 6 million different species [6].When inspecting the sequenced genomes from a large number of insect species, we found that the AKH gene and its receptor gene are always present in insects.However, the presence of ACP and its receptor strongly varies.They occur in beetles (order Coleoptera) and in mosquitoes, wasps, butterflies and moths, but are absent in Drosophila melanogaster and bees [1,2].Corazonin and its receptor are present in all insects with the exception of beetles and aphids [2,5,7,8].
The tick Ixodes scapilaris is not an insect, but belongs to the subphylum Chelicerata, which comprises also horshoe crabs, spiders, mites and scorpions.I. scapularis is a health risk for humans, because it is a vector for serious human diseases, such as Lyme disease and tick-borne encephalitis (TBE).Understanding the biology of ticks might enable researchers to reduce tick populations and, thus, prevent tick-borne human diseases.
We have previously been part of an international consortium to sequence and annotate the newly sequenced genome from I. scapularis and found that the that ticks shad five presumed ACP receptor genes, while insects had usually only one [9].We hypothesized that these large numbers of tick ACP receptor genes might suggest that they must be important for ticks and, thus, should be excellent drug targets [10].In the current report, we wanted to clone the five presumed receptors, express them in Chinese Hamster Ovary (CHO) cells, and functionally characterize them.We also wished to clarify the evolutionary relationships of chelicerate ACP receptors with other ecdysozoan ACP, AKH, and corazonin receptors.

Materials and methods
No experiments with live animals have been carried out and our investigations comply with the ARRIVE guidelines for animal experiments.RNA purification and cDNA synthesis was done as described previously [11].PCR cloning was carried out as described in Ref. [12].All PCR primers are given in Supplementary file 1.The PCR products were ligated into the vector pCR4-TOPO and sequenced as described before [12].The coding sequences of the receptors were transferred into the expression vector pIRES2-EGFP as described in Ref. [12].
Growth and transfection of Chinese Hamster Ovary (CHO) cells as well as the bioassay were carried out as described earlier [13].Tick ACP was from Genemed Synthesis.
MUSCLE and MEGA 11 software were used for protein alignments and phylogenetic tree analyses.Neighbor-Joining trees were bootstrapped using 1000 replicates.Accession numbers of all used protein sequences are given in Supplementary file 2. SignalP 6.0 software [14] was used to predict the presence of a signal peptide.Deep TMHMM software [15] was used to predict transmembrane helices [11].Dose-response curves and EC50 values were calculated as in Ref. [12].

Cloning of the five tick ACP receptors
We cloned the five presumed tick ACP receptors (ACPR1 to ACPR5), using tick mRNA, standard PCR cloning techniques, and PCR primers based on their genomic sequences (Fig. 1; Supplementary file 1).All five receptors are family-A GPCRs and closely related to each other as well as to the first cloned insect ACP receptor from the mosquito Anopheles gambiae [1].The five tick ACP receptor genes have also two common introns with the mosquito and Tribolium castaneum ACP receptor (Fig. 1).Each ACP.receptor has seven transmembrane helices (TM1 to TM7) and the canonical DRY sequence at the border of TM3 and the second intracellular loop (Fig. 1).The sequences of the five presumed tick ACP receptors have been submitted to GenBank (Supplementary file 2).

The sequence of tick ACP and its preprohormone
We annotated the tick ACP preprohormone from the tick genomic sequence [10], using T. castaneum ACP as a query [1] and cloned the protein, using tick mRNA and PCR primers based on the genomic sequence of the preprohormone (Supplementary file 1).A cartoon of the tick ACP gene structure is shown in Fig. 2A and of its mRNA in Fig. 2B.The complete amino acid sequence of the tick ACP preprohormone is presented in Fig. 2C.The structure of immature tick ACP is QITFSKNWQPGKR (Fig. 2C, first line) and this sequence is cleaved from the preprohormone by a signal peptidase (cleaving N-terminally of the first Q residue) and subsequently from the prohormone by a Prohormone Convertase (PC1/PC3, which cleaves at the C-terminus of the KR sequence).After removal of two basic C-terminal residues (KR) by a carboxypeptidase and conversion of the C-terminal G residue into a C-terminal amide group, the N-terminal Q residue gets converted into a pyroGlu (pQ) group [16].The final, mature structure of tick ACP, therefore, is pQITFSKNWQPamide.This sequence is slightly different from the ACP sequences occurring in insects [1,17], but identical to ACP sequences from other hard ticks (Fig. 2C).Arthropod ACP sequences are always rather well conserved, only having few amino acid residue exchanges (Fig. 2C) [1].Finally, Fig. 2C  several common amino acid residues.Mature ACPs are always ten amino residues long, corazonins are always eleven amino acid residues long, while AKHs are varying between eight and ten residues.The sequences of vertebrate gonadotropin-releasing hormones (GnRHs) are also included in Supplementary file 3, but these sequences showed no significant similarities with ACP, corazonin, and AKH.Thus, although the vertebrate GnRH receptors are clearly members of the AKH/ACP/corazonin receptor family [2,4], GnRH itself is not structurally related to the AKH/ACP/corazonin neuropeptide family.

Deorphanization of the five tick ACP receptors
We expressed the five presumed ACP receptors in pre-treated CHO cells as described before [1,[9][10][11].Activation of the receptors in these cells can be measured as a strong bioluminescence response and quantified by a plate reader.Fig. 3A-E show that all five presumed tick ACP For insects, it has been established that ACP receptors can only be activated by ACP and not by the structurally related AKH, or corazonin peptides [1].Fig. 3G-K show that the five tick ACP receptors could not significantly be activated by tick corazonin as well.In contrast to all insects, we found that ticks do not have AKH or AKH receptors [10].In Fig. 3G-K, therefore, we tested two insect AKHs and found that the five tick ACP receptors could also not significantly be activated by these insect AKHs.

The presence of ACP signalling in chelicerata
The phylogenetic tree from Fig. 4 shows that all investigated chelicerate species with a sequenced genome contain between one and five genes, coding for ACP receptors.Furthermore, all these chelicerates have ACP preprohormone genes, which can vary from one ACP gene in ticks to five ACP genes in the horseshoe crab L. polyphemus (Fig. 2C).Thus, ACP signalling appears to be both ubiquitous and abundant in Fig. 4. Phylogenetic tree of ACP receptors (blue background) coded for by the genomes from a broad selection of chelicerate species with a sequenced genome (each chelicerate species highlighted by a red dot); from a small selection of sequenced insect species (highlighted by blue dots); and from a single sequenced tardigrade species (highlighted by black dots).Included in the tree are also AKH receptors (green background), corazonin receptors (pink background), and GnRH receptors (yellow background and yellow dots).There is a fifth group of receptors identified in a few chelicerate species that are structurally related to the AKH and corazonin receptors, but that are different from the ACP receptors (no background color).These orphan receptors are preliminary named ACP receptor like receptors (ACPRLs).It can be seen that the vertebrate GnRH receptors are structurally closely related to the arthropod ACP, AKH, and corazonin receptors.For further details, see text.

Chelicerata.
A high number of ACP receptor genes, such as the five receptor genes in ticks (abbreviated Isca-ACPR1 to Isca-ACPR5 in Fig. 4), appears not to be confined to ticks, because other chelicerates, such as the horseshoe crab L. polyphemus, also have five of these genes (abbreviated Lpol-ACPR-1, Lpol-ACPR2 and Lpol-ACPRL1 to Lpol-ACPR3 in Fig. 4).The same holds for the herbivore mite T. urticae (not shown).It appears, therefore, that a large number of ACP receptor genes is not a characteristic property of ticks, but rather a common feature for many chelicerates, which discriminates them from insects, which normally have one ACP receptor gene.ACP signaling, therefore, has probably nothing to do with tick-specific physiological processes, such as a massive diuresis after a blood meal, which was a theory that we proposed in an earlier publication [8].

Do all chelicerates lack AKH and AKH receptors?
The subphylum Chelicerata consists of three classes: Pygnogonida (sea spiders), Xiphosura (horseshoe crabs), and Arachnida (ticks, mites, spiders, and scorpions) [18].We checked the sequenced genomes from representatives of Xiphosura and Arachnida (the same species as mentioned in Fig. 4) for the presence of AKH genes or AKH receptor genes, but obtained only negative results.We did not screen representatives of Pygnogonida, because no sequenced genomes are available for these species.With this restriction, we might conclude that AKH and AKH receptors are absent in all chelicerates.For I. scapularis and the mite T. urticae, it was earlier observed that AKH genes were lacking [8,19].Except for chelicerates, all other arthropods and even lophotrochozoans have AKH and AKH receptors which play essential roles in carbohydrate mobilization and metabolism [20,21].It is, therefore, remarkable that a whole subphylum can survive without AKH signaling.

Evolutionary implications of the lack of AKH signaling in chelicerates
We have previously proposed a model for the evolution of AKH and ACP signalling [2,4].In this model, the ACP receptor and AKH receptor genes arose from a common ancestor gene by gene duplication and the same happened for their ligand genes in a process of receptor/ligand co-evolution.The general absence of AKH and AKH receptor genes in chelicerates would suggest that the common ancestor of chelicerates would have lost AKH and the AKH receptor genes and functionally replaced them by ACP and a high number of ACP receptor genes.Alternatively, the evolution of ACP signaling might have preceded the evolution of AKH signaling.This last possibility would position the Chelicerata at the roots of ecdysozoan evolution, because AKH signaling is not lacking in any other ecdysozoan clade.For such a basal position of Chelicerata, however, no arguments exist [18].Furthermore, Lophotrochozoa, which are a sister taxon to Ecdysozoa, have AKH and AKH receptor genes [18], which again would argue against an evolutionary origin of AKH signaling within Ecdysozoa.

Novel orphan receptors in chelicerates
The phylogenetic tree from Fig. 4 shows a small cluster of orphan GPCRs from the horseshoe crab L. polyphemus, the scorpion Centruroides sculpturatus, and the spider Stegodyphus dumicola, which we preliminary named ACP receptor-like receptors (ACPRLs), because they are positioned in a cluster located between the chelicerate ACP and corazonin receptors (not highlighted by a background color in Fig. 4).The ligands for these receptors are unknown and could be either ACP-like, AKH-like, or corazonin-like peptides.

ACP, AKH, and corazonin receptors: gene gains and gene losses
Within the small family of ACP, AKH, corazonin and their receptors, there are frequent and sometimes dramatic gene gains and gene losses in between the different ecdysozoan phyla, subphyla, classes and other clades.Fig. 4 illustrates this for ACP, AKH, and corazonin receptors in Insecta, Chelicerata, and Tardigrada.ACP receptors and their ligands are present in Chelicerata (sometimes in high copy numbers), Tardigrada, and some insects such as beetles, but absent in other insects, such as bees and flies.AKH receptors and their ligands are present in all insects with a sequenced genome, but absent in Chelicerata.Corazonin receptors and their ligands are present in all Chelicerata, Tardigrada and in insects, except for beetles and aphids.Especially in Tardigrada high corazonin and corazonin receptor gene copy numbers can be found.The tardigrade Hypsibius exemplaris, for example, has as many as ten corazonin receptor genes (Fig. 4) and six corazonin genes (Supplementary file 3), while most insects have only one of each, or none.Tardigrades are renowned for their resilience against environmental stressors like very low temperatures, high radiation, and drought through a process of extreme desiccation [22].It would be interesting to find out, whether corazonin is involved in this desiccation, (or subsequent rehydration), because corazonin was recently found to control water homeostasis in Drosophila [23].

Fig. 1 .
Fig. 1.Alignment of the amino acid sequences of ACPR1, -2, -3, -4, and -5 from I. scapularis.The GenBank accession numbers for these sequences are given in Supplementary file 1.For comparison, the single ACP receptor from the beetle T. castaneum is also given [1].The boxed residues indicate the intron positions of the receptor genes.Residues that are identical in more than three receptors are highlighted in yellow.Abbreviations: Isca-ACPR1 to Isca-ACPR5, I. scapularis ACP receptor R1 to R5; Tcas-ACPR.T. castaneum ACP receptor.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2 .Fig. 3 .
Fig. 2. Cartoon of the I. scapularis ACP gene, its transcript, and its preprohormone.(A) The gene consists of two exons.Exon 1 codes for the signal peptide (highlighted in orange), the N-terminal part (highlighted in red) and the middle part (highlighted in light blue) of the ACP preprohormone.Exon 2 codes for the Cterminal part of the preprohormone (highlighted in light blue).(B).The I. scapularis ACP gene transcript.(C) The I. scapularis ACP preprohormone aligned with a selection of other chelicerate ACP prohormones.The immature ACP sequence is highlighted in red.Amino acid residues in common with other immature chelicerate ACP sequences are highlighted as well in red.Two cysteine residues, forming a cysteine bridge are highlighted in yellow.For further details, see text.Abbreviations: Cscu, Centruroides sculpturatus; Dand, Dermacentor andersoni; Lpol, Limulus polyphemus; Sdum, Stegodyphus dumicola; Vdes, Varroa destructor.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig.4.Phylogenetic tree of ACP receptors (blue background) coded for by the genomes from a broad selection of chelicerate species with a sequenced genome (each chelicerate species highlighted by a red dot); from a small selection of sequenced insect species (highlighted by blue dots); and from a single sequenced tardigrade species (highlighted by black dots).Included in the tree are also AKH receptors (green background), corazonin receptors (pink background), and GnRH receptors (yellow background and yellow dots).There is a fifth group of receptors identified in a few chelicerate species that are structurally related to the AKH and corazonin receptors, but that are different from the ACP receptors (no background color).These orphan receptors are preliminary named ACP receptor like receptors (ACPRLs).It can be seen that the vertebrate GnRH receptors are structurally closely related to the arthropod ACP, AKH, and corazonin receptors.For further details, see text.Protein abbreviations: ACP-R, ACP receptor; AKH-R, AKH receptor; CRZ-R, corazonin receptor; GnRH-R, gonadotropin releasing hormone (GnRH) receptor.Species abbreviations: Agam, Anopheles gambiae (mosquito); Cscu, Centruroides sculpturatus (scorpion); Dand, Dermacentor andersoni (tick); Dmel, Drosophila melanogaster (fruitfly); Drer, Danio rerio (zebra fish); Ggal, Gallus gallus (chicken); Hsap, Homo sapiens (human); Hexe, Hypsibius exemplaris (water bear); Isca, Ixodes scapularis (tick); Lpol, Limulus polyphemus (horseshoe crab); Mmus, Mus musculus (mouse); Nvit, Nasonia vitripennis (parasitic wasp); Rmic, Rhipicephalus microplus (tick); Sdum, Stegodyphus dumicola (social spider); Tcas, Tribolium castaneum (beetle); Turt, Tetranychus urticae (herbivorous mite); Vdes, Varroa destructor (ectoparasitic mite).(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)