Neuropeptides in Rhipicephalus microplus and other hard ticks

Abstract

The synganglion is the central nervous system of ticks and, as such, controls tick physiology. It does so through the production and release of signaling molecules, many of which are neuropeptides. These peptides can function as neurotransmitters, neuromodulators and/or neurohormones, although in most cases their functions remain to be established. We identified and performed in silico characterization of neuropeptides present in different life stages and organs of Rhipicephalus microplus, generating transcriptomes from ovary, salivary glands, fat body, midgut and embryo. Annotation of synganglion transcripts led to the identification of 32 functional categories of proteins, of which the most abundant were: secreted, energetic metabolism and oxidant metabolism/detoxification. Neuropeptide precursors are among the sequences over-represented in R. microplus synganglion, with at least 5-fold higher transcription compared with other stages/organs. A total of 52 neuropeptide precursors were identified: ACP, achatin, allatostatins A, CC and CCC, allatotropin, bursicon A/B, calcitonin A and B, CCAP, CCHamide, CCRFamide, CCH/ITP, corazonin, DH31, DH44, eclosion hormone, EFLamide, EFLGGPamide, elevenin, ETH, FMRFamide myosuppressin-like, glycoprotein A2/B5, gonadulin, IGF, inotocin, insulin-like peptides, iPTH, leucokinin, myoinhibitory peptide, NPF 1 and 2, orcokinin, proctolin, pyrokinin/periviscerokinin, relaxin, RYamide, SIFamide, sNPF, sulfakinin, tachykinin and trissin. Several of these neuropeptides have not been previously reported in ticks, as the presence of ETH that was first clearly identified in Parasitiformes, which include ticks and mites. Prediction of the mature neuropeptides from precursor sequences was performed using available information about these peptides from other species, conserved domains and motifs. Almost all neuropeptides identified are also present in other tick species. Characterizing the role of neuropeptides and their respective receptors in tick physiology can aid the evaluation of their potential as drug targets.

1. Introduction

The synganglion is the central nervous system (CNS) of ticks, a highly condensed and fused nerve mass, localized in the anterior ventral region of the body (Sonenshine and Roe, 2013). Among Prostriata, Metastriata and Argasidae ticks, the synganglion presents the same basic arrangement (Lees and Bowman, 2007). In addition, it is not strongly modified by blood acquisition, having nearly the same size in unfed and fed females. On the other hand, the synganglion is impacted by the drop-off from the host, when apoptosis is observed 72 h post-detachment (Freitas et al., 2007; Lees and Bowman, 2007). The synganglion produces numerous neuropeptides that control various internal organs and physiological processes, acting either as hormones or through peripheral innervation (Lees and Bowman, 2007; Šimo et al., 2013b; Sonenshine and Roe, 2013). These neuropeptides act as neurotransmitters, neuromodulators and/or neurohormones (Burbach, 2011).

In insects, neuropeptides affect social behavior, feeding, reproduction, stress and addiction, circadian rhythms, learning and memory (Schoofs et al., 2017). When axonal delivery of neuropeptides occurs, the same peptide may affect different organs and have distinct effects. For instance, in Rhodnius prolixus (Lange et al., 2012), myoinhibitory peptide inhibits hindgut contraction, as well as salivary gland contraction and saliva secretion, thus possibly regulating the digestive process, while a role in the secretion and transport through the ejaculatory duct in males has also been suggested (Lange et al., 2012). Glycoprotein hormone A2/B5 (GPA2/GPB5) is released into the hemolymph after a blood meal in the mosquito Aedes aegypti and regulates the activity of V-type H⁺-ATPase and P-type Na⁺/K⁺-ATPase transporters to balance the Na⁺/K⁺ levels (Paluzzi et al., 2014). Meanwhile, in ticks, knowledge about physiological roles played by neuropeptides is still very limited, since few peptides have been identified or functionally characterized. In Ixodes scapularis, myoinhibitory peptide inhibits and SIFamide stimulates hindgut (Šimo and Park, 2014). In addition, both these neuropeptides and elevenin innervate the salivary glands, possibly controlling salivary secretion (Šimo et al., 2013a, 2009; Kim et al., 2018).

Rhipicephalus microplus parasitizes livestock and wild ruminants from subtropical and tropical regions (McCoy et al., 2013). Apart from the damage caused by parasitism, such as anemia and reduced weight gain and milk production, this tick is also a vector of disease-causing Anaplasma bacteria and Babesia protozoa (Jonsson, 2006). Until now, R. microplus control strategies have been based on the use of chemical acaricides. Among the main targets are ion channels, like voltage-gated sodium channel (synthetic pyrethroids), GABA and glutamate-gated chloride channels (phenylpyrazoles and macrocyclic lactones) and acetylcholinesterase (organophosphates) (Baffi et al., 2007; Bloomquist, 2003, 1994, 1993; Kumar et al., 2020; Temeyer et al., 2010). In fact, the synganglion is the target of the majority of the currently used acaricides (Narahashi, 2002; Roma et al., 2014). However, R. microplus has become resistant to almost all of the available ectoparasiticide classes (Guerrero et al., 2012; Klafke et al., 2017). At this point, identification of new molecules with acaricidal activity and/or new biochemical targets are required. In arthropods, G protein-coupled receptors (GPCRs) have been suggested as a new target for control (Guerrero et al., 2016; Ngai and McDowell, 2017; Xiong et al., 2020). Neuropeptides are the main ligands of GPCRs, which in ticks are found both in the CNS and the periphery (Guerrero et al., 2016; Veenstra, 2016a). Thus, the understanding of tick physiology and neurobiology may be helpful to find new targets that interfere in salivation, digestion, elimination of sodium after a blood meal, or reproduction. Consequently, these targets could be useful to promote the development of alternative methods or strategies for tick control (Bendena, 2010; Caers et al., 2012; Gough et al., 2017).

This study aimed to identify and characterize neuropeptides in R. microplus and other tick species by an in silico approach using tick transcriptome and genome databases. Tick neuropeptides were identified based on comparison with coding regions, protein domains and similarity with neuropeptide sequences from other arthropods. Transcripts of 52 neuropeptide precursors were identified: ACP, achatin, allatostatins A, CC and CCC, allatotropin, bursicon A/B, calcitonins A and B, CCAP, CCHamide, CCRFamide, CCH/ITP, corazonin, DH31, DH44, eclosion hormone, EFLamide, EFLGGPamide, elevenin, ETH, FMRFamide myosuppressin-like, glycoprotein A2/B5, gonadulin, IGF, inotocin, two insulin-like peptides, iPTH, leucokinin, myoinhibitory peptide, NPF 1 and 2, orcokinin, proctolin, pyrokinin/periviscerokinin, relaxin, RYamide, SIFamide, sNPF, sulfakinin, tachykinin and trissin. Mature peptides were predicted based on conserved domains, motifs and post-translational modifications, characteristics of each neuropeptide.

2. Materials and methods

2.1. Ethics statement

This work was handled in accordance with the ethic and methodological guidance, in agreement with the International and National Directives and Norms by the Animal Experimentation Ethics Committee of Universidade Federal do Rio Grande do Sul (UFRGS) (project 14403).

2.2. Animals

Rhipicephalus microplus ticks from a laboratory colony (Porto Alegre strain, Porto Alegre, Brazil) were used to infest a Hereford calf, which was brought from a naturally tick-free area. The calf, maintained in an insulated pen, was infested with about 20,000 10-day-old R. microplus larvae (Reck et al., 2009). After 21 days, 20 ticks that were manually (partially engorged female) or naturally (fully engorged female) detached from the calf were collected for dissection.

2.3. Synganglion RNA extraction, cDNA library construction and sequencing

Initially, ticks were washed with 70% ethanol and then the synganglion was dissected using a scalpel blade and fine-tipped forceps. Synganglia were washed in ice-cold phosphate-buffered saline pH 7.2 and then immersed in TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was extracted following TRIzol manufacturer’s instructions. RNA purity and integrity were checked by PicoGreen^® dsDNA Quantitation Reagent and Kits (Invitrogen, Carlsbad, CA, USA). A total of 10 μg of RNA were used to prepare the cDNA library, using the Illumina TruSeq^™ RNA Sample Preparation Kit (Illumina, San Diego, USA) according to the manufacturer’s recommendations. The current synganglion RNA-seq was performed on Illumina HiSeq 1000, at the same time as other R. microplus organs/stages (Tirloni et al., 2020).

2.4. Synganglion transcriptome analysis

Synganglion transcriptome analysis was performed as described previously (Karim et al., 2011; Ribeiro et al., 2014). The raw reads were first quality-filtered by removing Illumina adaptor sequences and low-quality bases and then assembled with Abyss software (k-values vary from 50 to 95 at five-fold intervals). Since Abyss software may miss highly expressed contigs, the Trinity assembler was also used on the raw data. The resulting assemblies were merged by an iterative BLAST and CAP3 assembler (Karim et al., 2011).

To extract the coding sequences (CDS), we used an automated pipeline that is based on (i) sequence similarity to known proteins, or (ii) the identification of the larger open reading frame (ORF) containing a signal peptide from each contig. The presence of a signal peptide was evaluated by SignalP software version 5.0 (Almagro Armenteros et al., 2019). These results were combined and redundant sequences were removed. The subsequent CDS and their protein sequences were mapped into a hyperlinked Excel spreadsheet. Other protein features, such as transmembrane domains, furin cleavage and glycosylation sites were determined using the tools from the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/). To automate a functional annotation of proteins, we used the transcripts’ matches to several databases, e.g., Gene Ontology, Pfam, Swissprot, KOG, SMART, Refseq-invertebrates and Acari [organism] protein sequences, which were obtained from GenBank. Manual annotation was performed as detailed previously (Karim et al., 2011). In addition, tick genomes (Barrero, et al., 2017; Jia et al., 2020) were used as reference to estimate the assembly quality transcriptome completeness using BUSCO v. 4.1.3 (Seppey et al., 2019). Transcript abundance was estimated by mapping the reads back into the CDS, using BLASTn with a word size of 25 (-W 25 switch); a maximum of two gaps and a minimum score equal to the best score were established as cut-off. From this result we obtained the number of reads for each CDS, considering the synganglion library as well as the other R. microplus organs/stages libraries. Additionally, the average, maximum and minimum CDS read coverage were determined for each CDS. The chi-square test was used for statistical analysis (using the number of reads per CDS). Significance was assigned if p < 0.05 and the minimal expected read value for the CDS had five or more reads. Reads mapping and RPKM values were included in an Excel spreadsheet. The RPKM values were used to represent relative expression. Values were normalized calculating Z-score and used to generate heatmaps using the heatmap2 function from the ggplot2 library in R.

2.5. Neuropeptide predictions in R. microplus and other tick species

Initially, neuropeptide precursors from other arthropods (sequences from GenBank or FlyBase databases) (Supplementary Table 1) were used to search for putative R. microplus neuropeptides in the R. microplus transcriptome, including ovary, fat body and synganglion (from partially and fully fed females); salivary glands and digestive cells (from partially fed females); digestive cells (from fully fed females); and embryos. Searches were performed using BLASTx, tBLASTx or tBLASTn tools.

The majority of the R. microplus neuropeptide precursors were identified using the following parameters: E-value ≤1 × 10 e⁻¹⁰ and the presence of conserved domains or motifs (analyzed by InterProScan, Prosite and NCBI-CDD) characteristic of neuropeptides. However, for very small neuropeptide sequences, less stringent conditions were used.

In addition, the remaining sequences, i.e. those absent in aforementioned annotated databases, were identified using Sequence Read Archives (SRAs; SRR1186998, SRR1187005, SRR1187007, SRR1187010, SRR1187012, SRR1187013, SRR1187017, SRR7754368, SRR7754369, SRR7754370, SRR7754371, SRR7876048, SRR7876049, SRR13614645, SRR13614646, SRR13614647 and SRR13614648). The SRA files were downloaded using the SRA toolkit package (https://www.ncbi.nlm.nih.gov/sra/docs/toolkitsoft/) and searches were performed via the tBLASTn tool using tick and other arthropod sequences. The carboxyl-terminal amidation at glycine residues was predicted by homology to known arthropod neuropeptide precursors. Sequence alignments and similarity were assessed using the ClustalW tool with default parameters in BioEdit 7.2.5 software (Hall, 1999) and the presence of signal peptide was also verified.

In other arthropods, genes coding CCH/ITP (Dircksen, 2009), calcitonin A and B (Veenstra, 2014), EFLamide and EFLGPPamide (Veenstra et al., 2012) are alternatively spliced. Therefore, we performed manual searches in homologous tick sequences to identify alternative spliced transcripts.

Finally, the neuropeptide sequences identified in the R. microplus transcriptome were used to search for the gene sequences in different tick species, namely R. microplus, Rhipicephalus sanguineus sensu lato, Ixodes persulcatus, Dermacentor silvarum, Haemaphysalis longicornis and Hyalomma asiaticum (Jia et al., 2020), as well as I. scapularis (Gulia-Nuss et al., 2016). tBLASTn tool (E-value ≤1 × 10 e⁻⁵) was used to search for the transcripts in the genome databases.

2.6. Data availability

Raw reads were deposited in the NCBI Sequence Read Archive (Bio-sample SAMN02463642 and Bioproject PRJNA232001). Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GHWJ00000000 (the version described is GHWJ01000000 and TSA Database is SRR1187012). Neuropeptide sequences were deposited in NCBI BankIt, under the accession numbers described :ACP OK001352; Achatin BK059528; Allatostatin-A MT506377; Allatostatin-CC OK001353; Allatostatin-CCC OK001354; Allatotropin MT506374; Bursicon_A MT506355; Bursicon_B MT506364; Calcitonin-A_spliced-variant OK001355; Calcitonin-B_spliced-variant OK001356; CCAP OK001357; CCHamide MT506358; CCRFamide OK001358; Corazonin MT506366; DH31–1 MT506372; DH31–2 OK001371; DH44 MT506356; Eclosion_hormone OK001372; EFLamide_EFLamide_spliced-variant OK001359; EFLamide_EFLGGPamide_spliced-variant OK001373; Elevenin MT506367; ETH OK001374; FMRFamide_Myosuppressin-like OK001360; Glycoprotein_A2 MT506375; Glycoprotein_B5 OK001375; Gonadulin OK001361; I-CHH/ITP MT506363; II-CHH/ITP MT506371; III-CHH/ITP MT506370; II-Insulin-like MT506368; I-Insulin-like MT506369; IGF MT506376; iPTH OK001362; Inotocin/Vasopressin MT506361; IV-CHH/ITP_spliced-variant OK001363; IV-CHH/ITP MT506373; Leucokinin OK001364; Myoinhibitory-peptide MT506354; NPF1 OK001365; NPF2 OK001366; Orcokinin1 OK001367; Orcokinin2 MT506362; Orcokinin3 OK001368; Proctolin OK001369; Pyrokinin/Periviscerokinin OK001376; Relaxin OK001370; RYamide OK001377; SIFamide MT506357; sNPF MT506359; Sulfakinin MT506360; Tachykinin OK001378; Trissin MT506365.

3. Results and discussion

A total of 26,309,385 reads were obtained from R. microplus synganglion transcriptome sequencing. The assembly generated 94,813 contigs with a minimum length of 150 bp and an average length of 697 bp. After analyzing the primary sequences of the gene fragments, 18,004 CDS were further functionally annotated (Supplementary Table 2) and reads were mapped back into the assembled transcriptome giving a view of expression level for each gene in terms of RPKM. A BUSCO analysis of the predicted proteome of this tick indicated a 51.8% of completeness.

The CDS were annotated based on public databases, as described above, to provide functional information for each sequence. Sequences were classified into 32 categories, of which the most abundant included secreted (29%), unknown (24%), energetic metabolism (8%), followed by unknown conserved, protein synthesis machinery and oxidant metabolism/detoxification (6% each) . Similarly, in the R. sanguineus sensu lato synganglion, most CDS were classified as related to cell growth, division and RNA synthesis (27%) and metabolism (15%), unknown CDS corresponding to 34% (Lees et al., 2009).

In a comparison among different R. microplus tissues and stages, similar transcription profiles are observed between digestive cells from partially engorged females and fat body and between digestive cells from fully engorged females and salivary glands, while embryo, synganglion and ovary presented distinct profiles (data not shown). Moreover, 166 transcripts were at least five-fold more abundant in the synganglion than in the other organs/stages combined (Fig. 1A). These transcripts highly expressed in synganglion were classified in 17 categories; most of them are related to secreted (36%), unknown conserved (25%) and unknown (12%), followed by neuropeptide (6%) and oxidant metabolism/detoxification (4%). Accordingly, the female Dermacentor variabilis synganglion transcriptome showed that, depending on the stage (unfed, partially fed or fully engorged ticks), the transcriptional profile changed, but functional categories remained similar. The main biological functions were related to cellular and metabolic processes, localization and biological regulation (Bissinger et al., 2011).

Fig. 1. Rhipicephalus microplus neuropeptides transcriptome analysis.

A) Pie-chart from CDS that are at least five-fold more abundant in synganglion than in the other organs/stages combined (ovary (OV), synganglion (SYN) and fat body (FB) from partially and fully engorged females; salivary glands (SG) from fully engorged females; embryo (EMB); midgut from partially engorged females (DIG.P) and from fully engorged females (DIG.F)). B) Heatmap comparing neuropeptides transcripts among R. microplus organs/stages. The RPKMs of expressed genes in the tissues/stages is shown inside the boxes.

Similar transcriptional profiles were observed in insect central nervous system. For instance, from the kissing bug (R. prolixus), most transcripts were classified as protein modification, signal transduction, DNA and amino acid metabolic processes, transmembrane transport, ion binding, oxidoreductase and kinase activity (Ons et al., 2016). In the locust Schistocerca gregaria, most transcripts were related to primary metabolic process, cellular metabolic process, regulation of cellular process and cellular component organization (Badisco et al., 2011).

Neuronal tissues and endocrine cells are responsible for the synthesis and secretion of signaling molecules such as neuropeptides (Nässel, 1996). In agreement with this conceptualization, data presented here show that neuropeptides represent 0.12% of the total RPKM in R. microplus synganglion transcriptome and are a meaningful portion of over-represented synganglion CDS (6%), although they are also present in other tissues (Fig. 1B). This work provides an updated list of R. microplus neuropeptides. Searches in a previously published transcriptome (Tirloni et al., 2020) allowed to identify the expression levels of 28 neuropeptide precursors in different tick organs. Additionally, a manual search in the respective raw data (SRA files) led to the identification of 24 additional neuropeptide sequences (Supplementary Table 3 and Figure 2).

Fig. 2. Predicted structures of neuropeptide precursors.

Rhipicephalus microplus neuropeptide sequences were predicted and the 52 precursors are presented here alphabetically. Protein identifiers are provided in parenthesis next to the protein name. Signal peptides are shown in yellow, while predicted mature neuropeptides are in dark blue. Processing sites for mature peptides are shaded in red and putative glycine-derived C-terminal amidation sites in turquoise. Cysteines are shaded in pink.

Genome analysis of seven tick species led to the identification of neuropeptide precursors gene sequences in R. microplus, R. sanguineus sensu lato, I. persulcatus, D. silvarum, H. longicornis and H. asiaticum (Jia et al., 2020), as well as those present in I. scapularis (Gulia-Nuss et al., 2016) (Supplementary Table 4). The majority of the neuropeptides were identified in all genomes, which indicates that neuropeptides are conserved among the tick species analyzed. However, it was not possible to identify all neuropeptide sequences in the published genome assemblies (Gulia-Nuss et al., 2016; Jia et al., 2020): allatostatin CC and CCC and ACP were not identified in the R. microplus assembly, even though transcripts for these peptides were found. Similarly, allatostatin CC and ACP were not identified in H. asiaticum and R. sanguineus sensu lato, respectively. On the other hand, the absence of sNPF-2 from R. microplus transcriptome is not unexpected, since sNPF-2 seems to be a gene duplication specific to a few tick species.

In insects, metamorphosis and reproduction are controlled by juvenile hormones (JH). Synthesis, function and regulation of these hormones, including the participation of neuropeptides, are better understood in these arthropods than in others (Weaver and Audsley, 2009). In some insects, allatostatin A inhibits the biosynthesis and release of JH (Bendena et al., 2020). Although ticks do not synthesize JH (Neese et al., 2000), elements of JH pathway were identified (Zhu et al., 2016). In contrast, allatotropin may be involved in the positive regulation of JH pathway (Egekwu et al., 2016). Moreover, allatostatin A was hypothesized to have a myoinhibitory activity in ticks, like already reported for insects (Šimo and Park, 2014).

In ticks and in many insects, myoinhibitory peptide (MIP), often called allatostatin B (Coast and Schooley, 2011), acts by inhibiting the contraction of hindgut and visceral muscles (Lange et al., 2012; Šimo and Park, 2014), while SIFamide stimulates their motility (Šimo and Park, 2014). Both neuropeptides innervate salivary gland acini (type II and type III) and were proposed to play a role in controlling the secretion of salivary components, which is analogous to the activity on the hindgut (Šimo et al., 2013a, 2009). Elevenin was also suggested to be involved in saliva secretion during rapid engorgement phase in I. scapularis females (Kim et al., 2018). In parallel, similar to MIP, a myoinhibitory activity of allatostatin C was described in the moth Lacanobia oleracea (Matthews et al., 2007). In arthropods, this somato-statin ortholog went through a gene triplication, codifying for three peptides, allatostatin C, allatostatin CC and allatostatin CCC (Veenstra, 2016b). In ticks, only the last two precursors were found, suggesting the loss of allatostatin C.

Proctolin is known to act as a co-transmitter stimulating the contraction of skeletal, visceral and cardiac muscles in insects (Orchard et al., 2011; Ormerod et al., 2016). The role of proctolin in ticks remains unknown. Myoactivity was also suggested for FMRFamide, since it was detected in muscles of different tick species, like Ornithodoros parkeri and D. variabilis (Zhu et al., 1995).

Eclosion hormone is a neuropeptide already described in D. variabilis (Donohue et al., 2010), and induces the neuropeptide cascade that leads to pre-ecdysis and ecdysis behavior with the release of ETH and CCAP in arthropods (Gammie and Truman, 1997; Park et al., 2002; Žitňan et al., 1996). In a Drosophila model, ETH and CCAP knockout insects showed impaired ecdysis behavior and, consequent lethality, mainly in larval and pupal stage, respectively (Park et al., 2003, 2002). ETH has already been described in several arthropod species, such as A. aegypti (Dai and Adams, 2009), Drosophila melanogaster (Park et al., 1999), Manduca sexta (Žitňan et al., 1996), Anopheles gambiae (Holt et al., 2002) and Tribolium castaneum (Amare and Sweedler, 2007). Although, a genomic DNA fragment has already been identified in I. scapularis (Roller et al., 2010), this is the first report of a clear presence of ETH in parasitiformes. Furthermore, it is important to highlight that the motif (FFJKXXKXVPRX-NH₂) (Roller et al., 2010) is well conserved among arthropod ETH sequences, where only a few differences in mature peptide sequences were identified (Supplementary Figure 1). Bursicon is a cystine knot glycoprotein responsible for cuticle tanning and slcerotization (post-ecdysis) and is co-localized with CCAP (Dewey et al., 2004; Park et al., 2003). In addition to mimicking aspects of the gregarious phase of migratory locusts (Tawfik et al., 1999), corazonin was also shown to initiate ecdysis behavior in Lepidoptera (Kim et al., 2004). Here, all four neuropeptides (eclosion hormone, ETH, CCAP, bursicon and corazonin) were identified in R. microplus. It is interesting to note that bursicon was detected in adult ticks, despite the fact that ticks do not molt again once the adult phase is reached. Although the physiological effects of these neuropeptides have not yet been determined in ticks, it is a reasonable hypothesis that they are associated with cuticle expansion and development (synthesis and sclerotization) during and/or post blood feeding (Bissinger et al., 2011).

Besides ecdysis, CCAP has also been reported to interact with NPF. Both peptides affected feeding behavior in Drosophila and CCAP RNAi induced a reduction in NPF signaling (Williams et al., 2020). On the other hand, in cockroaches, CCAP is produced by enteroendocrine cells and triggers the release of both α-amylase and protease (Sakai et al., 2006, 2004).

Various other peptides and hormones that also affect food intake and energy metabolism in insects act as anorexigenic (inhibits appetite) or orexigenic (stimulates appetite) factors. A relationship between nutritional availability and the peptides sulfakinin, corazonin and periviscerokinin was suggested in I. scapularis and Amblyomma maculatum (Adamson et al., 2013). Indeed, sulfakinin has been suggested to function as a satiety factor (Meyering-Vos and Müller, 2007). Accordingly, sulfakinin transcription is upregulated in fed D. variabilis when compared with the unfed tick (Bissinger et al., 2011). Another neuropeptide involved in feeding regulation is NPF, although its role may be either orexigenic or anorexigenic, depending on the invertebrate species or the type of meal (Fadda et al., 2019). The sNPF neuropeptide is an example of an orexigenic factor. Injection of sNPF in cockroach starved nymphs led to a very significant increase in weight 24 h later, which can only be explained by increased food intake (Zeng et al., 2021). Also, Sudhakar et al. (2020) proposed a positive feedback model between insulin-producing cells and sNPF neurons during short periods of starvation. Whereas ticks have only one CCHamide gene, insects have typically two such genes coding similar peptides, each with its own receptor. CCHamide-2 was described in D. melanogaster as a growth regulator depending on nutritional availability (Sano et al., 2015). Feeding induces the release of CCHamide-2 into the circulation, which subsequently modifies feeding behavior (Li et al., 2013) and leads to the release of insulin-like peptides (ILPs) (Sano et al., 2015). Indeed, CCHamide-2 gene knockout led to reduced food intake, locomotion and development, as well as decreased ILP transcription (Ren et al., 2015). Accordingly, transcription of ILP genes is higher in all unfed life stages of I. scapularis ticks (Sharma et al., 2019). In addition to its established function as a diuretic peptide (Terhzaz et al., 1999), leucokinin is also related to ILP signaling in Drosophila. Increased transcriptional and protein levels of ILP were observed in flies when the leucokinin precursor gene was knocked out, as well as when the leucokinin receptor gene was absent or inhibited (Zandawala et al., 2018b). As a result, leucokinin mutant flies present a higher resistance to starvation. Furthermore, in Drosophila, the inactivation of leucokinin neurons led to an increased abdominal size due to water volume retained in the hemolymph, thus inducing a high frequency of small meals (Al-Anzi et al., 2010; Liu et al., 2015).

Roles in arthropod feeding regulation and digestion were also suggested for RYamide (Mekata et al., 2017; Roller et al., 2016a), and a function in water balance was hypothesized in Drosophila (Veenstra and Khammassi, 2017). Trissin is another neuropeptide hypothesized to act regulating gut contractions and food intake in Bombyx mori (Roller et al., 2016b). However, its precise role remains to be elucidated. ACP is already known from other arthropods, but is being described for the first time in ticks. In females of the crustacean Macrobrachium rosenbergii, the injection of ACP peptide caused an increase in total hemolymph lipid content and a reduction in oocyte proliferation (Suwansa-ard et al., 2016). Interestingly, in the cricket Gryllus bimaculatus, researchers observed that AKH and ACP knockdown significantly increased the ingestion of food, but hemolymph lipid level was not affected. Conversely, the administration of ACP induced an increase in lipid and carbohydrates levels (Zhou et al., 2018). More recently, it was shown that ACP is involved in Locusta migratoria long distance flight, ACP knockout locusts did not fly as long or as far as the wild type, while the capacity for flight was enhanced by ACP (Hou et al., 2021). Ion balance and water transport are regulated by neurohormones with diuretic and antidiuretic activity. In the mosquito A. gambiae, DH44 has a nonspecific role in sodium/potassium transport (diuretic effect), while DH31 stimulates sodium transepithelial transport (natriuretic and diuretic effect) (Coast, 2005). Other effects associated with diuretic hormones were also reported. Drosophila DH44 decreases desiccation tolerance, as indicated by reduction of DH44 leading to increased survival during this stress condition (Cannell et al., 2016). DH44 is co-expressed with leucokinin, both neuropeptides modulate diuretic pathways, with effects on fluid secretion. RNAi inactivation of these neuropeptide precursors showed an increase in desiccation and starvation resistance (Zandawala et al., 2018a). In addition, leucokinin was shown to stimulate fluid secretion by the Malpighian tubules (Terhzaz et al., 1999) and a knockdown of this neuropeptide receptor in ticks showed delayed oviposition, egg hatching and reduced egg masses, indicating a role in tick reproduction (Brock et al., 2019). In the green shore crab, DH31 has a myoactive activity, which is related to rhythmic coordination (Alexander et al., 2018). Benguettat et al. (2018) showed that, in the Drosophila intestinal lumen, the presence of opportunistic bacteria leads to an increased formation of reactive oxygen species (ROS), which bind to transient receptor potential A1 channel (TRPA) receptors, favoring DH31 release. Then, DH31 binds to receptors in neighboring muscular cells, causing muscular contractions and, consequently, driving a quick expulsion of the bacteria.

GPA2/GPB5 is another cystine knot glycoprotein hormone and regulates hydromineral balance (Paluzzi et al., 2014). Similarly, to what was found in other species, genes coding the two subunits of these neuropeptides are located next to one another in R. microplus genome (Hsu et al., 2002; Roller et al., 2008; Sudo et al., 2005). In decapods, the crustacean hyperglycemic hormone (CHH) is best known for increasing hemolymph glucose concentrations, while its insect ortholog, ion transport peptide (ITP), has antidiuretic effects (Chung et al., 2010; Gáliková et al., 2018; Webster et al., 2012). However, CHH has now also been shown to have an indirect role in coordinating ion transport in decapods, since it regulates the expression of Na⁺/K⁺-ATPase and carbonic anhydrase, enzymes involved in osmotic pressure regulation in Portunus trituberculatus gills (Sun et al., 2019). In insects, ITP genes are alternatively spliced into two different forms (Dircksen, 2009). In the R. microplus genome, four CHH/ITP genes were identified. Because one of these genes is alternatively spliced, there are in total five different transcripts. Inotocin is a vasopressin ortholog commonly present in insects, but notably absent in flies and bees. This neuropeptide was identified in Y-organs (responsible for ecdysteroid synthesis) during the molting phases of the crab Carcinus maenas (Oliphant et al., 2018). In addition, periviscerokinin, the first neurohormone identified in ticks (Neupert et al., 2005), is known to play a role in diuresis, and as a myotropic agent in insects (Wegener et al., 2002).

In insects, a variety of effects have been attributed to orcokinin, e.g. regulation of pigmentation (Wang et al., 2019), vitellogenin transcription (Ons et al., 2015) and circadian rhythmicity (Hofer, 2006; Jiang et al., 2015), but no effects are known in ticks. Orcokinin immunoreactivity was detected in synganglion, hindgut and salivary glands of I. scapularis, but LC-MS/MS analysis identified the peptide only in synganglion and hindgut (Roller et al., 2015). In R. microplus, orcokinin transcripts were detected not only in synganglion, but also in ovary, salivary glands, fat body, midgut and embryo. Insect tachykinins were initially identified in L. migratoria and characterized as neuropeptides that stimulate gut contractions (Schoofs et al., 1990; Siviter et al., 2000). This peptide is expressed in the gut, where in Drosophila it regulates lipid biosynthesis (Song et al., 2014). In neurons in the brain, it plays a role in male aggressive behavior (Asahina et al., 2014). Moreover, a tachykinin-like, named natalisin, involved in insect reproduction has been described in D. melanogaster, T. castaneum and B. mori (Jiang et al., 2013), being observed only in arthropods so far. Both neuropeptide precursors present very similar motif sequences, but only tachykinin was identified in R. microplus transcriptome evaluated here (Jiang et al., 2013; Mateos-Hernández et al., 2021). However, these peptides were detected in genome assemblies of tick species (Jia et al., 2020; Gulia-Nuss et al., 2016; Mateos-Hernández et al., 2021), while transcripts of natalisin were not found in the in silico analysis of I. scapularis embryonic cells (Mateos-Hernández et al., 2021). Also, NPF has many physiological roles and can influence feeding, metabolism, reproduction and stress response (Nässel and Wegener, 2011).

Calcitonin is another neuropeptide gene that produces two spliced variants (named A and B), as previously described in insects and decapods (Veenstra, 2016c, 2014). In R. microplus, the calcitonin A transcript is found in the synganglion, while the B transcript is present in digestive cells of partially and fully engorged females, and in fat body. This is similar to the expression of insect calcitonins A and B, that are found in the CNS and the gut, respectively (Veenstra, 2014).

Arthropods have a number of peptides that contain the typical insulin core sequences. Three of these peptides, gonadulin, insulin-growth factor (IGF) and relaxin, seem to have originated from an ancient gene triplication (Veenstra, 2020a), while other insulin-related peptides evolved later from IGF (Veenstra, 2021). Gonadulin is often expressed in the ovary and, at least in L. migratoria, silencing it by RNAi strongly diminishes vitellogenesis, while IGF functions as a growth hormone and the function of relaxin is not very clear (Veenstra, 2020a,b, 2021). In R. microplus, as in the locust (Veenstra et al., 2021), gonadulin transcripts were more abundant in the ovary than in the synganglion.

We found several R. microplus neuropeptides for which the function in arthropods is not known. Achatin was described for the first time in the snail Achatina fulica (Kamatani et al., 1989). It contains a d-amino acid residue, which makes it an unusual neuropeptide, but whether this is also the case in arthropod achatins is an open question. Achatin precursors were previously identified in other chelicerates (Stedodyphus mimosarum, Mesobuthus martensii, Symphilella vulgaris) (Veenstra, 2016a), as well as in I. scapularis ticks (Gulia-Nuss et al., 2016). Another intriguing neuropeptide gene is the EFLamide gene. Initially detected in the spider mite Tetranychus, it produces two spliced variants that encode two different peptides, EFLamide and EFLGGPamide (Veenstra et al., 2012). Such alternative transcripts are also produced from the tick EFLamide genes. This gene is abundantly expressed in decapod crustaceans, where only the EFLamide transcript is found (Veenstra, 2016c), but in insects, this gene has either been lost or has a very limited expression. Thus, in L. migratoria there are only two EFLamide expressing neurons, and in Pyrrhocoris apterus, null mutants for EFLamide seemed perfectly normal (Kotwica-Rolinska et al., 2020; Veenstra and Šimo, 2020). CCRFamide is a neuropeptide that was identified in silico only and little is known about its function, except that it is expressed in the nervous system and hypothesized to act either as a neuromodulator or a neurohormone in the lobster Homarus americanus (Hull et al., 2020). Lastly, a novel neuropeptide named iPTH was identified for the first time in ticks. This peptide was recently described in the beetle T. castaneum and other arthropods; it is hypothesized to function in the regulation of cuticle formation (Xie et al., 2020).

The characterization of tick metabolic pathways can support the identification of new physiologic targets to develop new methods for tick control. The development of new control strategies is essential, since the continuous use and misuse of acaricides has led to resistance to nearly all drugs used to date. However, most of the active principles act against only a few biological targets, including acetylcholinesterase, GABA-gated chloride channel, sodium channel and octopamine/tyramine (OCT/TYR) receptor, and the development of new molecules against these targets encounters technical and economic challenges (Guerrero et al., 2012; Jonsson, 2018). Therefore, the identification of new biological targets for acaricides is a promising approach to overcome the problem of resistance (Saramago et al., 2018; Yu et al., 2016). In this sense, transcriptome and proteome analysis, together with phylogenetic comparison among tick and insect genes, is a powerful tool to identify and characterize potential targets and to develop novel acaricides. This work describes 52 R. microplus neuropeptide precursors that were identified using other arthropods sequences in a transcriptomic approach. For the first time, ACP, allatostatin CCC, calcitonins A and B, CCAP, CCHamide, CCRFamide, EFLGGPamide, ETH, gonadulin, IGF, iPTH, NPF, RYamide and trissin were identified in tick tissues. Virtually all those neuropeptides seem to be ubiquitously present in ticks. The receptor(s) of one or more of these neuropeptides may constitute a good target for a novel generation of acaricides. Clearly, functional studies that characterize the role of these neuropeptides and respective receptors in tick physiology would provide useful information to evaluate and compare their potential as drug targets.