Putative acetylcholinesterase genes from a one-host tick species, the winter tick (Dermacentor albipictus)

Abstract

Tick control is heavily reliant on chemical acaricides; however, acaricide resistance and potentially toxic environmental effects underscore the need to develop new tick control strategies. Tick acetylcholinesterase is the target of organophosphate and carbamate acaricides, and the protein has been studied to develop novel anticholinesterase compounds that may exhibit significant improvements in arthropod specificity and mammalian safety. In support of these efforts, we mined the chromosome-level genome assemblies of Dermacentor albipictus and Rhipicephalus microplus to identify genes encoding acetylcholinesterase and evaluated transcript expression using publicly available D. albipictus transcriptome datasets. These one-host ixodid tick species are of significance to wildlife and livestock health, and genomic comparisons between them are highly relevant given their shared lifecycle. Our in silico search identified 73 and 52 putative AChE-like protein sequences in the D. albipictus and R. microplus genomes, respectively, and these encode conserved domains and features consistent with enzymatically active cholinesterases. AChE1 is one of the major AChEs involved in neuronal transmission in insects, and a clade of AChE1-like sequences that included representatives from D. albipictus and R. microplus was observed, including the BmAChE1 protein that has been enzymatically characterized. Alternative splicing and gene duplication events were evident, including a conserved AChE gene duplication event that appears across five tick species, indicating a role for these processes in AChE diversification. Different expression patterns of AChE were observed, with indication that its expression was coincident with the expression of receptors for acetylcholine, the substrate for AChE. The notable number of AChE-encoding genes supports the diversification and specialization of this gene family in one-host tick species, emphasizing the complex role that AChEs play in tick physiology and development.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13071-025-07205-z.

Ticks transmit disease-causing pathogens that are of consequence to human and animal health. Integrated tick management strategies include habitat modification, repellents, and biological control methods, yet tick control, particularly on livestock, has largely relied on chemical acaricides [1, 2]. The widespread use of acaricides has led to the development of tick resistance to these chemicals. Most significantly, this has been reported worldwide for the southern cattle tick, Rhipicephalus microplus, that transmits the pathogens causing bovine babesiosis and bovine anaplasmosis and is the most important tick species affecting livestock globally [3]. The one-host biology of this species leads to increased exposure of all its lifestages to acaricide pressure, increasing the likelihood of resistance development [4].

Acetylcholinesterase (AChE) is an enzyme that has a critical role in the nervous system, as it hydrolyzes the neurotransmitter acetylcholine to terminate nerve transmission [5]. Organophosphate and carbamate compounds were first developed as nerve agents that were subsequently commercialized as pesticides because of their AChE inhibition properties, resulting in excessive acetylcholine buildup that was lethal towards arthropod pests [6]. Recently, AChE has been the intended target for the development of novel arthropod-specific acaricides with the goal of identifying inhibitors that have limited toxicity towards human and animal hosts [7–9]. In support of these efforts, it is critical to identify and enzymatically characterize AChEs from tick species of importance.

Ixodid tick and arachnid genomes comprise multiple genes encoding AChEs [10, 11], which is in contrast to many insects that have only one or two genes [12]. Three different AChEs from R. microplus (BmAChE1, BmAChE2, and BmAChE3) were extensively characterized by Temeyer et al. [13], confirming differences in their biological, genetic, and biochemical properties, including evidence for their redundancy as a compensatory mechanism for survival [14]. Within Ixodidae, few tick species exhibit a one-host tick lifecycle. To enable genomic comparisons between representative one-host species, our group recently produced chromosome-level genome assemblies for R. microplus [15] and for the winter tick, Dermacentor albipictus, a one-host, ixodid tick that is widely distributed across North America [16]. Dermacentor albipictus predominantly infest wild and domestic ungulates, particularly moose, elk, caribou, and deer in extremely high numbers, sometimes resulting in death [17, 18]. This species is a reported vector of Anaplasma marginale and Babesia duncani, and there is evidence that it harbors Rickettsia felis [19–21]. Cattle are readily parasitized by D. albipictus [22], and acaricidal pressure may have similar consequences on this species as it does for R. microplus. Here, we mined the D. albipictus genome assembly for genes encoding putative AChEs, and we evaluated the phylogenetic relationship of these sequences between these one-host species. We highlight a remarkable number of tick proteins in the AChE gene family and describe evidence for alternative splicing and gene duplication that underscore the complexity of tick acetylcholinesterases.

The chromosome-level genomes of R. microplus (USDA_Rmic, GCA_043290135) and D. albipictus (USDA_Dalb.pri_finalv2, GCA_038994185) were annotated by the NCBI using the Eukaryotic Genome Annotation Pipeline. A gene list was populated by downloading those sequences annotated as “acetylcholinesterase,” then evaluating the protein sequences using the NCBI Conserved Domain analysis tool [23]. Three well-characterized AChE genes from R. microplus (GenBank Accessions USH09479 (BmAChE1), AAC18857 (BmAChE2), and AAP92139 (BmAChE3)), an Amblyomma americanum cholinesterase (WDY73567; [24]), and an R. microplus carboxylesterase (WMQ58240; [25]) were used as positive controls for conserved domains since their enzymatic activity has been confirmed experimentally. As such, amino acid sequences mined from the genomes were included in the analysis if they encoded the “Ab (alpha–beta) hydrolase superfamily” (cl21494), “Carboxylesterase (COesterase)” (pfam00135), “Esterase_lipase” (cd00312), and “PnbA” (carboxylesterase type B; COG2272) domains, as well as their encoding “catalytic triad” and “substrate binding” features. Putative signal peptides and subcellular localizations were predicted using SignalP 6.0 and DeepLoc, respectively [26]. A separate gene list was populated by searching the D. albipictus genome for sequences annotated as “choline acyltransferase” or “acetylcholine receptor.” These, too, were submitted to the NCBI Conserved Domain analysis tool for identification of domains and features. Putative nicotinic or muscarinic acetylcholine receptors were included in the analysis if they encoded “ligand gated ion channel, extracellular domain” (cl28912), “ligand-gated ion channel, transmembrane” (cd19051), “7-transmembrane muscarinic acetylcholine receptor” (cd15049; DM1 and M1 like), or “neurotransmitter-gated ion-channel ligand binding domain” (cl47783) conserved domains. Further, choline acyltransferase sequences were included if they encoded a “Choline/Carnitine o-acyltransferase” (cl08288) domain.

The AChE amino acid sequences were aligned using MUSCLE v5.3 with the -super5 option [27], and Drosophila melanogaster esterase (AAP21002) was used as an outgroup. All sequences are available in Supplementary File S1. A phylogenetic tree was then inferred from this alignment using IQ-Tree v3.0.1 with ModelFinder, SH-aLRT test (–alrt 1000), and ultrafast bootstrap (–B 1000) options [28–30]. As part of the NCBI annotation pipeline, gene models encoding each protein sequence were available in the NCBI Sequence Viewer. These were examined for each transcript to validate exon–intron structure and identify evidence of alternative splicing, and these instances were recorded (Supplementary File S1). Based on phylogenetic inference, a set of duplicated AChE genes in the D. albipictus and R. microplus genomes occupied the same clade, possibly suggestive of a conserved duplication. To identify whether this may be present in other tick species, the BLAST algorithm was used to search chromosome-level genome assemblies of Dermacentor variabilis (American dog tick; ASM5094787v1, NC_134572), Dermacentor andersoni (Rocky Mountain wood tick; qqDerAnde1, NC_092818.1), and Rhipicephalus sanguineus (brown dog tick; BIME_Rsan_1.4, NC_051180) for the presence of sequences homologous to the duplicated set. Transcript expression profiles were obtained for sequences on both gene lists using available D. albipictus RNA-seq datasets [31] representing unfed, newly molted adult females (uf_Female), unfed newly molted adult males (uf_Male), 0–6 h embryos (embr_06), ovaries from preoviposition females (ov_preov), and reproductive systems from fed, unmated males (MRS_fum). These reads were quantified at the transcript level and quasi-mapped to the reference GCA_038994185 using Salmon [32]. Bias models were enabled, and sample-specific fragment length distributions were estimated during processing. Salmon produced raw transcript per million counts (TPMs), while transcript estimates to genes were performed with tximport in R [33] using a transcript-to-gene map. Gene-level data were normalized (size-factor normalization) and processed using DESeq2 [34]. Visualization of normalized counts were transformed to row-wise z-scores calculated through R’s heatmap function. Data are accessible in SRA Accessions SRX24226854-SRX24226858. The inferred AChE phylogenetic tree was visualized and annotated using ggtree [35], and heat maps were produced to represent the expression profiles for select genes using ggplot2 in R [36].

A query of the D. albipictus and R. microplus genomes identified 156 and 91 protein-coding sequences, respectively, that were annotated as “acetylcholinesterase” or “acetylcholinesterase-like.” Identical proteins (33 in D. albipictus, 17 in R. microplus), resulting from sequence variation in the 5′ or 3′ untranslated regions of a locus, were removed. We further curated this dataset to eliminate proteins that lacked features consistent with a functional AChE enzyme. The alpha–beta hydrolase protein superfamily, to which acetylcholinesterase belongs, comprises enzymes that act on carboxylic esters, and these reactions involve a triad of amino acid residues (“catalytic triad”) that are essential for catalyzing enzymatic activity. Specifically, AChE is a serine hydrolase with a distinct catalytic triad comprising serine, glutamic acid, and histidine, which are required for the hydrolysis of acetylcholine (Supplementary File S1; [37]). As such, sequences were included if they encoded the catalytic triad Ser/Glu/His residues that define a functional AChE enzyme. This resulted in the further removal of 50 D. albipictus and 22 R. microplus proteins. We confirmed the presence of BmAChE1 and BmAChE2 sequences in this dataset, while BmAChE3 was identified within locus LOC119171776, a fusion of two gene models, and was added to the gene list. After curation, the dataset comprised 73 D. albipictus and 52 R. microplus proteins, and the phylogenetic relationship of these sequences was inferred (Fig. 1, Supplementary File S1), reflecting highly diverse AChEs in these one-host tick species. Of note is the distinct clade of AChE1-like proteins (Fig. 1, purple box) with representative sequences obtained from GenBank for Tetranychus urticae (two-spotted spider mite) and Blatella germanica (German cockroach, insect ace-1 and ace-2). In insects, AChE1 is primarily associated with neuronal functions, and it has the closest relationship to the neuronal AChE in vertebrates [12]. The R. microplus BmAChE1 and A. americanum cholinesterase are also present within this clade, and their enzymatic activity and substrate preference for acetylcholine has been described [13, 24]. The current analysis identified five homologous AChE1-like sequences in D. albipictus, and it also highlighted two additional paralogs in R. microplus that are AChE1-like. Proteins within the sister clade to AChE-1 like proteins exhibit all hallmarks of AChE and are labeled “non-AChE1” (Fig. 1).

Fig. 1.

A Inferred phylogeny of putative acetylcholinesterase (AChE) proteins identified from the chromosome-level genome assemblies of the winter tick, Dermacentor albipictus (Dalb), and the cattle tick, Rhipicephalus microplus (Rmic). Amino acid sequences were aligned using MUSCLE, and the phylogenetic tree was inferred using IQTree (best-fit substitution model, branch support assessed with 1000 replicates of UFBoot bootstrap approximation; boostrap percentage reflected). A Drosophila melanogaster (Dmel) esterase (AAP21002) was used as an outgroup, and AChE sequences from Tetranychus urticae (Turt; ADK12702) and Blatella germanica (Bger; ABB89946, ABB89947) are identified. All sequences are available in Supplementary File S1. Predicted cell localizations are represented by a vertical bar (see legend in figure), and a heat map of corresponding D. albipictus AChE transcripts mined from existing RNA-seq data is presented [31]. Filled circles on taxa labels identify those isoforms produced by alternative splicing (Supplementary File S1) and taxa highlighted in yellow identify those R. microplus and Amblyomma americanum (Aam) sequences that have experimental evidence of enzymatic activity towards acetylthiocholine, the preferred substrate for AChE. A purple box identifies a clade of AChE1-like sequences, and a sister clade comprising non-AChE1-like sequences is labeled. A blue box identifies a clade of non-AChE1-like sequences that are predicted to be localized to the cell membrane. An orange box identifies a putatively conserved AChE gene duplication (see text). B Inferred phylogeny of AChE proteins from Rhipicephalus sanguineus (Rsang), D. variabilis (Dvar), and D. andersoni (Dand) that are homologous to duplicated AChEs from D. albipictus and R. microplus. Clades I, II, and III identify duplicated sets (see text), and representative gene organizations and distances between duplications within the respective genomes are depicted. Filled circles on taxa identify those isoforms produced by alternative splicing

Cholinesterases have evolved to serve different functions, which can account for the multiple genes that code for AChE in invertebrate genomes [10, 11]. Alternative splicing is one mechanism that can contribute to an increase in protein diversity and alternative tissue expression [38, 39], and there is evidence of this occurring in the genes encoding AChE in D. albipictus and R. microplus. Specifically, gene models for 10 loci in D. albipictus are putatively alternatively spliced to produce 25 AChE-like isoforms, with variability in the first exon being encountered most frequently (Supplementary File S1). In R. microplus, different exon usage at four loci produced eight isoforms. Furthermore, alternative splicing of tick AChEs has been reported from R. australis (nee R. microplus), R. sanguineus, and R. annulatus [40, 41]. In the latter, Temeyer et al. [42] observed comparable biochemical properties between BmAChE1 and three alternatively spliced AChE1-like isoforms from R. annulatus, further supporting a compensatory role for these isoforms. Gene duplication is also a mechanism that can contribute to the emergence of genes with diverse functions, and we observed evidence of this at two sites within the D. albipictus genome. In these instances, the genes were located within 19 kb and 11.8 kb of each other on chromosomes 4 and 10, respectively, and they encoded AChE proteins that were 60% and 90% identical (Supplementary File S1), respectively. Similarly, gene duplication of putative AChEs was evident in R. microplus with genes located 0.25 kb and 21 kb apart and 10.6 kb apart on chromosome 4 and an unplaced scaffold, respectively, exhibiting 83%, 96%, and 57% amino acid identity (Supplementary File S1). One D. albipictus gene duplication event and two R. microplus events are on the same branch (Fig. 1, orange box), suggesting the duplications may be conserved. To address this, homologues of these representative duplications were identified from the chromosome-level genome assemblies of D. variabilis, D. andersoni, and R. sanguineus. The homologues encoded hallmarks of AChEs, as confirmed using the Conserved Domain database, and they were used in phylogenetic comparison with the D. albipictus and R. microplus duplications (Fig. 1B). Clade I includes two genes encoding R. sanguineus AChEs that are 13 kb apart and 83% identical, sharing a lineage with an R. microplus duplication. In clade II, which includes the D. albipictus duplication, two genes encoding D. variabilis AChE are separated by 36 kb and are 61% identical. In addition, this clade included two D. andersoni AChE isoforms (XP_050034532 and XP_050034533) resulting from alternative splicing at a single locus (LOC126531156). Lastly, in clade III, which includes an R. microplus duplication, four genes encoding R. sanguineus AChE are arranged in tandem, separated by 21–42 kb, and exhibit 58–80% identity (Supplementary File S3). Collectively, these results support conservation of this duplication event across several tick species.

In silico predictions of protein localization and membrane associations using DeepLoc 2.1 identified 52 D. albipictus and 36 R. microplus putative AChE sequences as extracellular (Fig. 1), all of which were predicted to be soluble proteins (Supplementary File S2). Within this set, there were two D. albipictus AChEs that had comparable probabilities of extracellular or lyososomal/vacuole localization, and these are reflected in Fig. 1. Of the collective 30 sequences predicted to be localized to the cell membrane, 23 had signals of being lipid anchored and 7 of being transmembrane (Supplementary File S2). As above, there were two sequences with similar probabilities of cell membrane or lysosomal/vacuole localization (Fig. 1). These differences in localization are consistent with the varied tissue distribution and functions of AChEs. Interestingly, the clades comprising R. microplus AChEs for which enzymatic activity has been validated are not predominantly extracellular/soluble (Fig. 1, taxa highlighted in yellow). For example, the lineage where R. microplus AChE3 resides includes proteins predicted to be extracellular, as well as localized to the cell membrane and/or lysosome/vacuoles. Further, within the AChE1-like lineage, there are sister clades comprising paralogs predicted to be either extracellular (includes BmAChE1) or cell membrane-bound (Fig. 1). Of the 30 AChEs predicted to be localized to the cell membrane, more than half (n = 19) are on a separate clade of non-AChE1 like proteins (Fig. 1, blue box). The D. albipictus genes within this clade are predominantly expressed in newly molted males and reproductive systems of fed males, with limited expression detected in embryos or females/female tissues (Fig. 1). The differential expression pattern of AChEs in all tissues and lifestages, though, is evident in genes outside this clade. Choline acyltransferase (ChAT) is an essential enzyme that catalyzes the transfer of an acetyl group from coenzyme acetyl-CoA to choline, yielding acetylcholine. Acetylcholine receptors (AChR) bind acetylcholine and mediate physiological responses that differ in speed and mechanism, which is reflected in the two types of AChRs, nicotinic (nAChR) and muscarinic (mAChR)  [43]. The nAChRs are ligand-gated ion channels that enable fast synaptic transmission at neuromuscular junctions, while mAChRs are G-protein coupled receptors that initiate second messenger pathways to regulate arthropod physiology. In the D. albipictus RNA-seq, three ChAT transcripts were identified, as were 25 nAChR and three mAChRs (Supplementary File S4). Given the expression profile of D. albipictus AChEs, we examined whether there was any correlation with the expression of transcripts encoding ChAT and AChRs (Fig. 2). Interestingly, a cluster of transcripts had a biased expression in embryos and newly molted adult females, and the cluster included one ChAT, six nAChR, and all three mAChRs. Conversely, the other two ChAT transcripts and one nAChR exhibited biased expression in preoviposition ovaries and male reproductive systems. Coincident with biased AChE expression in newly molted adult males, 17 nAChRs had a comparable expression pattern and were elevated relative to the other tissues and lifestages examined, possibly reflecting an increased need for removal of the neurotransmitter. However, these data represent only one snapshot in time and lack information from neuronal tissues (i.e., tick synganglia). In R. microplus, the three verified AChEs are all expressed in synganglia, as well as other non-neuronal tissues [10]. Given the complex expression network underlying this pathway, these analyses would be strengthened by additional RNA-seq datasets for D. albipictus.

Fig. 2.

Heatmap representing the transcript expression profile of choline acyltransferase (ChAT) and acetylcholine receptors (AChR), either nicotinic (nAChR, neuronal AChR) or muscarinic, in available RNA-seq datasets from Dermacentor albipictus [31]. Using a heatmap, genes with similar expression patterns are clustered and normalized counts were transformed to row-wise z-scores

The expression of putatively cell membrane-bound AChE in non-neuronal reproductive tissues supports involvement in nonclassical roles, such as cell differentiation or apoptosis, as suggested by Silman [44]. These nonenzymatic roles further include AChE interaction with proteins to enable cell adhesion that supports cell signaling [45]. Rapid cell division and differentiation are occurring in reproductive tissues and, based on the expression data here, AChEs may have a role during these processes. AChEs have also been implicated in regulating expression of arthropod immune response pathways and stress tolerance [46–48], which are critical defenses for newly molted adult ticks. Of curiosity are the 50 D. albipictus and 22 R. microplus proteins that were annotated as acetylcholinesterase, but excluded from the gene list because they did not encode the Ser/Glu/His catalytic triad. These were predominantly predicted to be localized to the cell membrane and exhibited a transcript expression profile biased towards reproductive tissues (Olafson, unpublished), suggesting the importance of enzymes in the alpha-hydrolase superfamily to these one-host tick species.

Chromosome-level genome assemblies enable comparative analysis of gene families, and the increased numbers of AChEs identified from one-host ticks suggests diversification and specialization of this gene family. Indeed, recent evidence indicates AChE is secreted in tick saliva, possibly having a role in both pathogen transmission and host immunomodulation [49, 50]. Cholinergic systems play a key role in physiology and metabolism, likely reflecting the ancient emergence of acetylcholine [10]. Our in silico analysis identifies diverged, tick-specific AChE paralogs as candidate targets for new acaricide development, supporting additional biochemical characterization to verify enzymatic or nonenzymatic activities and define their roles in tick biology.

Author contributions

PUO: conceptualization, formal analysis, investigation, writing—original draft preparation; PS: data curation, formal analysis, visualization, writing—review & editing; DL: data curation, formal analysis, writing—review & editing; KHL: funding acquisition, writing—review & editing; KGS: writing—review & editing; KBT: conceptualization, supervision, writing—review & editing. All authors read and approved the final manuscript.

Funding

This work was funded by USDA appropriated funds (Project 3094-10400-002-000-D, Biology, Management, and Surveillance of Ticks of Veterinary Importance and Project 3094-10400-004-000-D, Exploiting Veterinary Pest Genomics to Improve Animal Health).

Data availability

The datasets analyzed during the current study are publicly available in NCBI, to include the chromosome-level genomes of *R. microplus* (USDA_Rmic, GCA_043290135) and *D. albipictus* (USDA_Dalb.pri_finalv2, GCA_038994185) and the *D. albipictus* RNA-seq datasets (Accessions SRX24226854-SRX24226858).

Declarations

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Competing interests

The authors declare no competing interests.