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Insects have about 50 neuropeptide genes and about 70 genes, coding for neuropeptide G protein-coupled receptors (GPCRs). An important, but small family of evolutionarily related insect neuropeptides consists of adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP). Normally, insects have one specific GPCR for each of these neuropeptides. The tick Ixodes scapularis is not an insect, but belongs to the subphylum Chelicerata, which comprises ticks, scorpions, mites, spiders, and horseshoe crabs. Many of the neuropeptides and neuropeptide GPCRs occurring in insects, also occur in chelicerates, illustrating that insects and chelicerates are evolutionarily closely related. The tick I. scapularis is an ectoparasite and health risk for humans, because it infects its human host with dangerous pathogens during a blood meal. Understanding the biology of ticks will help researchers to prevent tick-borne diseases. By annotating the I. scapularis genome sequence, we previously found that ticks contain as many as five genes, coding for presumed ACP receptors. In the current paper, we cloned these receptors and expressed each of them in Chinese Hamster Ovary (CHO) cells. Each expressed receptor was activated by nanomolar concentrations of ACP, demonstrating that all five receptors were functional ACP receptors. Phylogenetic tree analyses showed that the cloned tick ACP receptors were mostly related to insect ACP receptors and, next, to insect AKH receptors, suggesting that ACP receptor genes and AKH receptor genes originated by gene duplications from a common ancestor. Similar duplications have probably occurred for the ligand genes, during a process of ligand/receptor co-evolution. Interestingly, chelicerates, in contrast to all other arthropods, do not have AKH or AKH receptor genes. Therefore, the ancestor of chelicerates might have lost AKH and AKH receptor genes and functionally replaced them by ACP and ACP receptor genes. For the small family of AKH, ACP, and corazonin receptors and their ligands, gene losses and gene gains occur frequently between the various ecdysozoan clades. Tardigrades, for example, which are well known for their survival in extreme environments, have as many as ten corazonin receptor genes and six corazonin peptide genes, while insects only have one of each, or none., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Cornelis Grimmelikhuijzen reports financial support was provided by Carlsberg Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Many insects produce the cyclic neuropeptide inotocin (CLITNCPRGamide), which is the insect orthologue of the mammalian neuropeptides oxytocin and vasopressin. These insects also have one inotocin G protein-coupled receptor (GPCR), which is the orthologue of the mammalian oxytocin and vasopressin receptors. The tick Ixodes scapularis belongs to the subphylum Chelicerata, an arthropod taxon different from insects, to which also spiders, scorpions, and mites belong. I. scapularis is an ectoparasite and a health risk for humans, because it transfers pathogenic microorganisms to its human host during a blood meal, thereby causing serious neurological diseases, among them Lyme disease and tick-borne encephalitis (TBE). By annotating the genomic sequence of I. scapularis, we previously found one presumed tick inotocin preprohormone gene and, in contrast to insects, three genes coding for presumed inotocin GPCRs. We now find that these GPCR genes cluster on one genomic contig, suggesting that they originated by recent gene duplications. Closely located on the same contig are also four adipokinetic hormone/corazonin-related peptide (ACP) GPCR genes, and one crustacean cardioactive peptide (CCAP) GPCR gene, suggesting evolutionary relationships. These evolutionary relationships are confirmed by phylogenetic tree analyses of their gene products. We also cloned the tick inotocin preprohormone, which has a structural organization closely resembling mammalian oxytocin and vasopressin preprohormones, including the presence of a conserved neurophysin sequence, having seven cystine bridges. This neurophysin sequence has two cystine-knot domains, but in contrast to mammalian neurophysins, the tick neurophysin contains a canonical prohormone convertase cleavage signal and a peptide C-terminal amidation sequence (GKR), suggesting cleavage into two biologically active cystine-knot peptides. This cleavage/amidation sequence occurs in neurophysins from most hard tick species, but not in other chelicerates. Mature tick inotocin is different from insect inotocin and has the sequence CFITNCPPGamide. Finally, we cloned and stably expressed the three tick inotocin receptors in Chinese Hamster Ovary cells and found that each of them was activated by nanomolar concentrations of tick inotocin (EC 50 for ITR1 = 1.6 × 10 -8  M; EC 50 for ITR2 = 5.8 × 10 -9  M; EC 50 for ITR3 = 9.3 × 10 -9  M), thereby establishing that they are genuine tick inotocin receptors., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Cornelis Grimmelikhuijzen reports financial support was provided by Carlsberg Foundation., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

We have identified a corazonin G protein-coupled receptor (GPCR) gene in the tick Ixodes scapularis, which likely plays a central role in the physiology and behavior of this ectoparasite. This receptor gene is unusually large (1.133 Mb) and yields two corazonin (CRZ) receptor splice variants, where nearly half of the coding regions are exchanged: CRZ-Ra (containing exon 2, exon 3, and exon 4 of the gene) and CRZ-Rb (containing exon 1, exon 3, and exon 4 of the gene). CRZ-Ra codes for a GPCR with a canonical DRF sequence at the border of the third transmembrane helix and the second intracellular loop. The positively-charged R residue from the DRF sequence is important for coupling of G proteins after activation of a GPCR. CRZ-Rb, in contrast, codes for a GPCR with an unusual DQL sequence at this position, still retaining a negatively-charged D residue, but lacking a positively-charged R residue, suggesting different G protein coupling. Another difference between the two splice variants is that exon 2 from CRZ-Ra codes for an N-terminal signal sequence. Normally, GPCRs do not have N-terminal signal sequences, although a few mammalian GPCRs have. In the tick CRZ-Ra, the signal sequence probably assists with inserting the receptor correctly into the RER membrane. We stably transfected Chinese Hamster Ovary cells with each of the two splice variants and carried out bioluminescence bioassays that also included the use of the human promiscuous G protein G 16 . CRZ-Ra turned out to be selective for I. scapularis corazonin (EC 50  = 10 -8  M) and could not be activated by related neuropeptides like adipokinetic hormone (AKH) and AKH/corazonin-related peptide (ACP). Similarly, also CRZ-Rb could only be activated by corazonin, although about 4-fold higher concentrations were needed to activate it (EC 50  = 4 x 10 -8  M). The genomic organization of the tick corazonin GPCR gene is similar to that of the insect AKH and ACP receptor genes. This similar genomic organization can also be found in the human gonadotropin-releasing hormone (GnRH) receptor gene, confirming previous conclusions that the corazonin, AKH, and ACP receptor genes are the true arthropod orthologues of the human GnRH receptor gene., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Cornelis Grimmelikhuijzen reports financial support was provided by Carlsberg Foundation., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Bilateria have bilateral symmetry and are subdivided into Deuterostomia (animals like vertebrates) and Protostomia (animals like insects and mollusks). Neuropeptides occur in both Proto- and Deuterostomia and they are frequently structurally related across these two lineages. For example, peptides belonging to the oxytocin/vasopressin family exist in both clades. The same is true for the G protein-coupled receptors (GPCRs) of these peptides. These observations suggest that these neuropeptides and their GPCRs were already present in the common ancestor of Proto- and Deuterostomia, which lived about 700 million years ago (MYA). Furthermore, neuropeptides and their GPCRs occur in two early-branching phyla that diverged before the emergence of Bilateria: Cnidaria (animals like corals and sea anemones), and Placozoa (small disk-like animals, feeding on algae). The sequences of these neuropeptides and their GPCRs, however, are not closely related to those from Bilateria. In addition, cnidarian neuropeptides and their receptors are not closely related to those from Placozoa. We propose that the divergence times between Cnidaria, Placozoa, and Bilateria might be too long for recognizing sequence identities. Leucine-rich repeats-containing GPCRs (LGRs) are a special class of GPCRs that are characterized by a long N-terminus containing 10-20 leucine-rich domains, which are used for ligand binding. Among the ligands for LGRs are dimeric glycoprotein hormones, and insulin-like peptides, such as relaxin. LGRs have been found not only in Proto- and Deuterostomia, but also in early emerging phyla, such as Cnidaria and Placozoa. Humans have eight LGRs. In our current review, we have revisited the annotations of LGRs from the sea anemone Nematostella vectensis and the placozoan Trichoplax adhaerens . We identified 13 sea anemone LGRs and no less than 46 LGRs from T . adhaerens . All eight human LGRs appear to have orthologues in sea anemones and placozoans. LGRs and their ligands, therefore, have a long evolutionary history, going back to the common ancestor of Cnidaria and Placozoa., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hauser, Koch and Grimmelikhuijzen.)

Background: The animal phylum Cnidaria consists of six classes or subphyla: Hydrozoa, Scyphozoa, Cubozoa, Staurozoa, Anthozoa, and Endocnidozoa. Cnidarians have an early evolutionary origin, diverging before the emergence of the Bilateria. Extant members from this phylum, therefore, are important resources for understanding the evolution of the nervous system. Cnidarian nervous systems are strongly peptidergic. Using genomics, we have recently shown that three neuropeptide families (the X 1 PRX 2 amides, GRFamides, and GLWamides) are wide-spread in four (Scyphozoa, Cubozoa, Staurozoa, Anthozoa) out of six cnidarian classes or subphyla, suggesting that these three neuropeptide families emerged in the common cnidarian ancestor. In the current paper, we analyze the remaining cnidarian class, Hydrozoa, and the subphylum Endocnidozoa, to make firm conclusions about the evolution of neuropeptide genes in Cnidaria., Results: We analyzed sixteen hydrozoan species with a sequenced genome or transcriptome, using a recently developed software program for discovering neuropeptide genes. These species belonged to various hydrozoan subclasses and orders, among them the laboratory models Hydra, Hydractinia, and Clytia. We found that each species contained three to five neuropeptide families. A common feature for all hydrozoans was that they contained genes coding for (i) X 1 PRX 2 amide peptides, (ii) GRFamide peptides, and (iii) GLWamide peptides. These results support our previous conclusions that these three neuropeptide families evolved early in evolution. In addition to these three neuropeptide families, hydrozoans expressed up to two other neuropeptide gene families, which, however, were only occurring in certain animal groups. Endocnidozoa (Myxozoa) are microscopically small endoparasites, which are strongly reduced. For long, it was unknown to which phylum these parasites belonged, but recently they have been associated with cnidarians. We analyzed nine endocnidozoan species and found that two of them (Polypodium hydriforme and Buddenbrockia plumatellae) expressed neuropeptide genes. These genes coded for neuropeptides belonging to the GRFamide and GLWamide families with structures closely resembling them from hydrozoans., Conclusions: We found X 1 PRX 2 amide, GRFamide, and GLWamide peptides in all species belonging to the Hydrozoa, confirming that these peptides originated in the common cnidarian ancestor. In addition, we discovered GRFamide and GLWamide peptide genes in some members of the Endocnidozoa, thereby linking these parasites to Hydrozoa., (© 2021. The Author(s).)