Leading anti-tick vaccine targets are variably conserved in cattle fever ticks

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Busch, Nathan E. Stone, Grant L. Pemberton, Mackenzie L. Roberts, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4844765/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Apr, 2025 Read the published version in Parasites & Vectors → Version 1 posted 9 You are reading this latest preprint version Abstract Background Rhipicephalus ( Boophilus ) microplus causes significant cattle production losses worldwide because it transmits Babesia bovis and B. bigemina causative agents of bovine babesiosis. Control of these ticks primarily has relied on treatment of cattle with chemical acaricides, but frequent use, exacerbated by the one-host life cycle of these ticks, has led to high-level resistance to multiple classes of acaricides. Consequently, new approaches for control, such as anti-tick vaccines, are critically important. Key to this approach is targeting highly conserved antigenic epitopes to reduce the risk of vaccine escape in heterologous tick populations. Methods We evaluated amino acid conservation within 14 tick proteins across 167 R. microplus collected from geographically diverse locations in the Americas and Pakistan using PCR amplicon sequencing and in silico translation of exons. Results We found that amino acid conservation varied considerably across these proteins. Only one target, the voltage-dependent anion channel, was fully conserved in all 167 R. microplus samples (protein similarity 1.0). Five other proteins were highly conserved: the aquaporin RmAQP1 (0.989), vitellogenin receptor (0.985), serpin-1 (0.985), and subolesin (0.981). In contrast, the glycoprotein protease Bm86 was one of the least conserved (0.889). The Bm86 sequence used in the original Australian TickGARD vaccine carried many amino acid replacements compared to the R. microplus populations examined here, supporting the hypothesis that this vaccine target is not optimal for use in the Americas. By mapping amino acid replacements onto predicted 3D protein models, we also identified amino acid changes within several small peptide vaccines targeting portions of the aquaporin RmAQP2, chitinase, and Bm86. Conclusions These findings emphasize the importance of thoroughly analyzing protein variation within anti-tick vaccine targets across diverse tick populations before selecting candidate vaccine antigens. When considering protein conservation alone, RmAQP1, vitellogenin receptor, serpin-1, subolesin, and especially the voltage-dependent anion channel rank as high priority anti-tick vaccine candidates for use in the Americas and perhaps globally. Rhipicephalus microplus R. annulatus anti-tick vaccine conserved targets surface-exposed epitopes Figures Figure 1 Figure 2 Figure 3 BACKGROUND Ticks are the most important vectors of animal diseases worldwide and an important public health concern ( 1 , 2 ). Species such as the Asian longhorn tick ( Haemaphysalis longicornis ) and southern cattle tick ( Rhipicephalus [ Boophilus ] microplus ) have become global problems for livestock production due to their invasiveness, use of multiple hosts, and ability to transmit disease-causing pathogens ( 3 ). R. microplus and R. annulatus both transmit Babesia bovis and B. bigemina , which cause severe bovine babesiosis in naïve adult cattle, as well as the bacterium Anaplasma marginale that causes bovine anaplasmosis ( 4 ). Approximately one billion bovines are at risk of infestation by R. microplus ( 5 ) and the global economic impact on the cattle industry due to this species alone is estimated to be at least USD $ 13.9–18.7 billion per year ( 6 ). Management of this issue is based primarily on chemical control of ticks on infested hosts, and acaricides have been used on R. microplus populations for over one century, which has led to human-mediated selection for resistance to multiple chemical classes ( 7 – 11 ). Frequent treatment of cattle herds means that these one-host ticks experience repeated selection pressure that rapidly selects for high-level resistance; this can lead to resistance for as many as six chemical classes in certain R. microplus populations ( 12 ). Alternative control methods, such as anti-tick vaccines and plant-based compounds, are increasingly being evaluated as tools for tick control ( 13 – 15 ). Because cattle fever ticks feed on a single host animal throughout their development from larvae to adult, their life cycle lends itself to vaccination-based control. The strategy for anti-tick vaccines involves immunizing a host with one or more tick proteins that stimulate a strong IgG antibody response directed at those proteins within the tick. Once ticks attach and begin to blood feed, the host IgG antibodies bind to these target proteins in situ and disrupt tick feeding or physiology, leading to mortality or greatly reduced tick fitness. The nature and mechanism of the disruption caused is dependent on the functional role of the target antigen used in the vaccine ( 16 ). Two main categories of antigens used in anti-tick vaccines are secreted salivary proteins that naturally interact with the host immune system ( 17 ), and concealed antigens ( 18 ) within the tick that are not normally exposed to the host immune system, but nonetheless can be targeted by host antibodies delivered via the blood meal ( 19 ). Antibodies targeting secreted salivary proteins will substantially impact the attachment process and feeding interaction, whereas antibodies binding to concealed antigens will not – instead, they interfere with the function of tick proteins responsible for critical physiological roles within the tick. The first anti-tick vaccination test in the 1930s used homogenates of midgut and salivary gland from American dog tick, Dermacentor variabilis , to raise a polyclonal antibody response in guinea pigs ( Cavia porcellus ) that clearly impacted ticks upon blood feeding ( 20 , 21 ). In the mid-1970s, Galun ( 22 ) proposed the idea of controlling ticks by using IgG antibodies against tick juvenile hormones to interrupt normal tick development. Although not formally tested, her hypothesis is one of the earliest examples of the concealed antigen concept. The seminal work by Willadsen and co-workers ( 23 ) in the late 1980s identified a glycoprotein protease (Bm86) as a protective concealed antigen and set the stage for all future work on concealed antigens for vaccine development. Since these early studies, > 50 tick proteins have been tested in various host models, especially rabbits ( 24 , 25 ), and numerous review articles have discussed the successes and challenges of reducing tick burdens using specific tick antigens ( 24 , 26 – 42 ). Several studies have revealed that an important challenge for anti-vector vaccines is to raise a robust, long-lasting, and specific IgG antibody response that is protective against the arthropod pest, which has been difficult to achieve in real world settings ( 43 , 44 ). Another strategy is to use transmission-blocking vaccines to reduce a pathogen’s ability to successfully infect a tick vector, rather than killing the ticks themselves ( 45 – 48 ). All of the vaccines against R. microplus (and R. annulatus ) that have advanced to commercial development have been based on the glycoprotein protease Bm86, which is expressed in midgut epithelial cells ( 23 ). A recombinant rBm86 vaccine (TickGARD-PLUS) was developed from the Yeerongpilly strain of R. australis from Queensland, Australia ( 49 ); this species was previously considered to be R. microplus but is now formally recognized as a distinct species ( 50 ). However, it has been observed that Bm86 protein sequences of R. microplus populations in the Americas have diverged significantly from the R. australis Yeerongpilly strain (91–99% protein similarity) and sequence divergence correlates with variable vaccine efficacy (0–91%) ( 51 , 52 ). To counteract this problem, geographically appropriate protein alleles have been developed for a number of rBm86 vaccines employed in different countries ( 53 ), including GAVAC® in Cuba (Concord strain Bm95 allele AF150891.2), Tick Vac® and Go Tick® in Colombia and Brazil (proprietary alleles by Limor de Colombia, Bogotá, CO), and Bovimune ixovac® in Mexico (proprietary allele by Lapisa S.A., Michoacán, MX). An important issue for all full-length rBm86 formulations is that the specific surface-exposed epitopes contributing to a protective IgG response remain largely unknown ( 37 , 54 ). But directing a more specific antibody response has been made possible using the rSBm7462® formulation, which targets short chimeric peptides developed from three epitopes encoded in exons 1, 4 and 11 of the Bm86 gene ( 55 , 56 ); additional Bm86 epitopes are being evaluated in R. microplus populations in Mexico (Martinez-Alzate et al. 2019) and India ( 57 ). Protein variation in Bm86 has been characterized in R. microplus and R. annulatus ticks from diverse geographic locations in the Americas ( 51 , 58 , 59 ), India ( 57 ), and Africa and Thailand ( 60 ). Collectively, this work has uncovered extensive amino acid (aa) diversity in the full-length Bm86 protein, which is hypothesized to be the cause of decreased effectiveness in essentially all Bm86 vaccine formulations. The study of protein conservation in anti-tick vaccine targets other than Bm86 remains limited and only two studies have performed large-scale surveys of multiple R. microplus populations. The first investigated DNA and protein variation within subolesin (Sub) and voltage-dependent anion channel (VDAC) in populations from Mexico ( 61 ), and the second focused on Sub and tropomyosin (TPM) in R. microplus throughout India ( 62 ). Other studies have sampled a smaller number of populations to evaluate conservation in subolesin (Sub), vitellogenin receptor (VgR), and the aquaporins of R. microplus (RmAQP1 and RmAQP2) ( 63 – 65 ). To advance the development of next-generation anti-tick vaccines, we evaluated the level of conservation in Bm86 (as a control protein) and 13 other tick proteins, many of which were the focus of the Cattle Vaccine (CatVac) Consortium ( 66 ) and have been considered for vaccine development by a number of research groups (see Table 1 ). METHODS Tick Samples Rhipicephalus microplus: To evaluate global aa conservation within 14 anti-tick vaccine candidates, we examined individual R. microplus (n=167) collected from geographically diverse locations in North America, South America, and Pakistan ( Additional File 1 ). The majority of the samples were collected in the field from Bos taurus cattle in Mexico (n=57 from 14 states) and the US (n=88 from Texas, five counties). The five R. microplus samples collected in Pakistan for a previous study (67) were included to represent a population of R. microplus from Asia. All US field samples of R. microplus were collected from cattle; in certain instances, ticks were sampled on stray cattle from northern Mexico that were apprehended in Texas. The full methods for tick field collection and DNA extraction are described in Additional File 2 . Laboratory colonies of R. microplus : In addition to field collections, we made use of 21 ticks from seven R. microplus lab colonies. The first four colonies are maintained by the United States Department of Agriculture Cattle, Agricultural Research Service, Fever Tick Research Laboratory (USDA-ARS-CFTRL) and originated in Texas (Deutsch genome strain), Brazil (Santa Luiza), Colombia (Arauca), and Puerto Rico (Yabucoa). The others are two lab colonies (Porto Alegre [POA], and SLF) maintained by the Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF) in Eldorado do Sul, Brazil and a naturally occurring field population (IPV) at the IPVDF pastures. Other Rhipicephalus species: To evaluate protein diversity in a wider set of species in the genus Rhipicephalus we analyzed DNA samples from R. annulatus and R. appendiculatus . Field collections of R. annulatus ticks were made by APHIS or TAHC field inspectors from cattle and introduced red deer ( Cervus elaphus ) and processed as described above. We also sampled the Vega lab colony of R. annulatus (Texas) maintained at the USDA-ARS-CFTRL and the Muguga lab colony of R. appendiculatus , originally collected in Kenya and maintained for >20 years at the Roslin institute in Scotland, then at the USDA-ARS-Animal Disease Research Unit in Pullman, WA since 2013. Amplicon Sequencing We used amplicon sequencing (AmpSeq) on an Illumina™ short read platform (MiSeq™) to obtain exon DNA sequences encoding 14 anti-tick vaccine candidates for this study ( Table 1 ). Each protein has been used in cattle vaccination trials with published mRNA sequences that served as reference homologs. We chose exon sequencing instead of mRNA sequencing because our archive of field samples (>10,000 R. microplus ) consists entirely of DNA extractions. Gene-specific primers (n=176 for R. microplus were designed for 88 exon targets from 14 genes, and exon assays were divided into four multiplexed PCRs that maximized primer compatibility ( Additional File 3 ). Most priming sites were located at the ends of each exon, and therefore our data comprise partial length sequences for each gene/protein, with the exception of RmAQP2, which is full length. The full details of the PCR and AmpSeq methods are provided in Additional File 2 , and the success rate of each exon across all 167 R. microplus is shown in Additional File 4 . Bioinformatic Analysis Our bioinformatic methods follow that of a recent publication (68) and the full details are described in Additional File 2 . To set up standardized reference sequences for downstream analyses, we downloaded mRNA sequences of all 14 homologs from the first whole genome sequence of R. microplus based on the USDA Deutsch lab colony from Texas (GenBank WOVZ00000000.1; Bioprojects PRJNA412317 and PRJNA312025) (69, 70). We note that exon sequences could not be concatenated because the relationship of exons in heterozygotes to their source allele (their “phase”) was unknown (see DNA sequence in Additional File 5 ; https://github.com/GrantPem/Busch_etal_2024). Each partial exon sequence was then translated in silico using BioEdit (71) to obtain predicted aa sequences that were aligned against the 14 Deutsch references (aa sequence in Additional File 6 ). Protein similarity to the Deutsch reference was calculated as 1 – (#aa replacements / total aa positions assayed). The location of aa replacements within each 10-aa window across the full-length proteins was visualized as a heat map of conserved versus variable peptides. To evaluate protein conservation at predicted surface epitopes, we also mapped the specific location of each aa replacement onto predicted 3D protein structures using the AlphaFold website (https://alphafold.ebi.ac.uk/) (72, 73). We chose to visualize eight of the 14 proteins: the five most conserved proteins, two proteins with published short peptide vaccines, and Bm86 because it is used in commercially produced vaccines. Table 1. List of 14 published anti-tick vaccine targets used in our evaluation of protein conservation in Rhipicephalus ( Boophilus ) microplus . Abbreviation Protein Full length (aa) Physiological target mRNA Reference RmAQP1 Aquaporin-1 316 Water balance; salivary glands KJ626366.1 RmAQP2 Aquaporin-2 294 Water balance; salivary glands, gut, ovaries KP406519.1 Bm86 Glycoprotein (Bm86/Bm95) 650 Intestinal lining of midgut M29321.1 Chit Chitinase-1 436 Cell structure and exoskeleton GBBR01000100.1 COX3 Cytochrome oxidase III 259 Mitochondria KP143546.1 GST Glutathione S-transferase 216 Detoxification KF784792.1 MP4 Metalloprotease 4 (reprolysin) 559 Salivary gland and digestive tract DQ118970.1 RmS-1 Serine protease inhibitor 1 380 Innate immune response; development KC990100.1 RmS-5 Serine protease inhibitor 5 404 Innate immune response; development KC990104.1 RmS-11 Serine protease inhibitor 11 380 Innate immune response; development KC990110.1 Sub Subolesin 161 Gene expression and regulation KM115651.1 VDAC Voltage-dependent anion channel 273 Mitochondria GU994210.1 VgR Vitellogenin receptor 1799 Egg development KY781176.1 Vora Voraxin p 139 Reproduction JX502818.1 The column “Physiological target” broadly summarizes the focus of each vaccine as described in the available literature. Each reference sequence (based on mRNA) was chosen from a single published vaccine trial to ensure our data match previously described homologs. We compared each dataset to the Deutsch genome sequence (WOVZ00000000.1) to evaluate protein similarity. p Partial cds for Voraxin in Rhipicephalus ( Boophilus ) microplus (see GenBank annotation page). RESULTS We found varying levels of conservation among the 14 proteins examined in this study (Fig. 1 ). Because we used the Deutsch genome as a standardized reference, we were able to make a direct comparison of conservation across all 14 proteins in this dataset. The highest protein similarity within our sample of 167 R. microplus ticks was observed in VDAC, which displayed no aa replacements in any of the sampled populations (Fig. 1 ). The DNA alignment for VDAC reveals 20 single nucleotide polymorphisms (SNPs) within R. microplus but all are synonymous, equating to a K A / K S ratio of zero ( Additional File 5 ). Four other proteins were highly conserved (RmAQP1, VgR, RmS-1, and Sub), with protein similarity values > 0.98 (Fig. 1 ). The majority of SNPs in these four genes were synonymous and yielded K A / K S estimates of 0.09, 0.07, 0.09, and 0.16, respectively. Intermediate levels of protein conservation were found in seven proteins (COX3, RmAQP2, Chit, GST, RmS-11, RmS-5, and Vora, Table 1 ) and ranged from 0.904–0.979; the lowest levels were found in Bm86 (0.889) and MP4 (0.802). Although we did not calculate protein similarity values for other Rhipicephalus species due to low sample size, we note that protein conservation appears to decrease when other Rhipicephalus species are included in the comparison ( Additional File 6 ). As an extreme example, Bm86 exhibited twice the number of aa replacements across seven Rhipicephalus species as compared to within R. microplus alone. The small number of aa changes in highly conserved proteins were typically spaced far apart. As observed in Fig. 2 , more variable proteins exhibited evidence of mutational hotspots, with aa changes clustered in multiple short segments of the protein. Despite the high density of changes in some of these proteins, short stretches of highly conserved peptides can also be found in each protein. One caveat for identifying conserved peptide regions is that our sampling design supports the detection of rare aa replacements in North America, but not in other locations due to smaller sample sizes. Another caveat is that the true amount of protein variation is probably underestimated in our dataset, because: 1) not all aa positions were queried due to the location of priming sites inside exons, 2) not all exons amplified equally well, and 3) tick populations from certain regions (Brazil and Pakistan) tended to fail more often than ticks from North America. However, 60 of the 88 exons had success rates > 90% across our R. microplus samples ( Additional File 4 ) and these provide high confidence for estimating conservation at these exons, especially in North America. The predicted 3D structural model for R. microplus VDAC ( 74 ) is shown in Fig. 3 A. In addition to being completely conserved in R. microplus , the VDAC protein was conserved in all 12 R. annulatus from Texas, which were identical to the R. microplus allele ( Additional File 6 ). In the DNA sequences, R. annulatus individuals were variable at the same 20 nucleotide positions as R. microplus , and all SNPs were synonymous ( Additional File 5 ). In contrast, our samples of R. appendiculatus displayed nine aa replacements in VDAC, and two publicly available R. sanguineus sequences (XP_037498097.1 and UFQ89927.1) contained eight replacements, five of which were shared with R. appendiculatus ( Additional File 6 ). Interestingly, only one of these replacements (L136V) was located on an external surface loop ( Additional File 7A ) predicted in a 3D structural model for VDAC ( 74 ). The other aa changes from R. appendiculatus and R. sanguineus were located within the transmembrane barrel and internal loops that extend into the cytoplasm. Aquaporin-1 was the second most conserved protein across R. microplus from the Americas and Pakistan (Fig. 1 , Fig. 3 B). It is important to note that ticks carry multiple genes in the aquaporin family (18 reported from Ixodes scapularis ) ( 65 ) and their annotation has not been standardized across tick species. For instance, RmAQP1 was shown to be a homolog of IsAQP9 in a recent gene tree reconstruction ( 65 ). In our dataset, the RmAQP1 protein contained just three changes (I264V, L286I, and T294V) across the full-length protein (316 aa) within R. microplus ( Additional File 6 ). These changes were found in a small number of ticks in lab colonies from Brazil (IPV, POA, and Santa Luiza) and three individual ticks from Texas (Cameron County); no ticks from Mexico, Colombia, or Puerto Rico carried these changes. Ticks from Pakistan all failed to amplify the exon assay (AQP1_E01502) containing these three residues ( Additional File 4 ), as did all 12 R. annulatus samples (data not shown). In the publicly available R. annulatus genome sequence (WOVY00000000.1; Bioproject PRJNA593711), three replacements occur in AQP1 (T223S, M266V, and T294V). The T223S replacement is the most important because it is located in an external loop of the 3D protein model ( Additional File 7B ). Our R. appendiculatus samples contained 10 other replacements and two GenBank R. sanguineus AQP9 sequences (XP_037510823.1 and KAH7963214.1) contained 17 replacements, but only one was at the same aa position as R. microplus (I264S). The third most conserved locus was VgR (Fig. 1 , Fig. 3 C), with the caveat that we assayed only 434 positions of 1799 in the full-length protein, and three of the eight exon assays had lower success rates (80–86%) ( Additional File 4 ). Because VgR is a large protein, we focused on two ligand-binding domains (LBDs) encoded by exons 2–3 (105 aa) and 14–20 (329 aa). We found three aa replacements across R. microplus from the Americas, which represented one inter-class change (N19T) and two intra-class changes (R1193H and T1216S). None of the three replacements were found in the five ticks from Pakistan, which carried changes at three other positions (T56I, P1167Q, and A1184G) for a total of six VgR replacements in our overall R. microplus dataset. R1193H and T1216S are probably linked because they co-occurred in all 60 R. microplus individuals from the Americas that carried them, as well as the 12 R. annulatus samples ( Additional File 6 ). Furthermore, they were found in all North and South American countries that we sampled ( Additional File 1 ). Every R. annulatus tick from Texas carried four replacements observed in R. microplus (N19T, R1193H, A1184V, and T1216S), as well as two others (S63N and A921S). The R. appendiculatus ticks in our study amplified at only two of eight assays (exons 16 and 19), but the data from exon 16 alone identified 11 polymorphic aa residues. A similar level of variation (13 replacements in a 187 aa peptide) is also evident in the partial VgR of R. appendiculatus Muguga strain from Kenya (ATP60167.1) ( Additional File 6 ). Likewise, R. sanguineus (XP_037521270.1) contained 135 replacements across the entire protein, 45 of which overlap with the two LBDs that we screened in R. microplus . The T1216S replacement in R. sanguineus is shared with R. microplus and R. annulatus , and R. sanguineus had an intra-class replacement at position 19 (N19D). RmS-1 was well conserved across the R. microplus populations we sampled, with a protein similarity of 0.985 across the 336 aa positions that we assayed of 380 in the full-length protein (Fig. 1 ). Five aa replacements (F101L, E140A, E306K, M337I, and I354V) were observed, all of which occurred in ticks from North America; the E140A change was also found in ticks from South America and Pakistan ( Additional File 6 ). The first four are inter-class changes and I354V is an intra-class replacement. Two replacements (F101L and E306K) were found in surface loops of the protein (Fig. 3 D). M337I and I354V appear to be linked, because they both were present in every tick that carried them (seven Texas locations). The R. annulatus ticks sampled in Texas had replacements at four other positions (K52E, E275K, I280M, and L286M), and none carried the E140A replacement that was common in R. microplus . Seventeen aa replacements were present in R. appendiculatus ticks in three of the four assays. Assay A0101 failed in our 10 samples of R. appendiculatus , but a full-length sequence from GenBank (AAK61375.1) shows > 20 replacements in this section of the protein alone (positions 1-100) ( Additional File 6 ). Likewise, R. sanguineus (XP_037521270.1) contains 46 replacements across the entire protein. The other two members of the serpin family that we investigated (RmS-5 and RmS-11) showed much more variation in R. microplus ; RmS-5 had 27 replacements in the 360 positions that we assayed (similarity = 0.925) and RmS-11 had 19 replacements in 340 assayed positions (similarity = 0.944). Subolesin was highly conserved in R. microplus from the Americas (Fig. 1 , Fig. 3 E) with only a single aa change (I41V) in the 52 positions that we assayed (of 162 in the full-length protein). Our R. microplus samples from Colombia and Pakistan are missing data at the assay that covers position 41, due to failed amplification. The I41V replacement is an intra-class change (isoleucine and valine are both aliphatic acids) and it is possible that the valine replacement would not significantly impact IgG reactivity, but this remains unknown. We found this replacement to be rare but widespread in Texas and Mexico (states of Tamaulipas, Zacatecas, and Campeche) yet absent in our samples from southern Brazil. It was also present in 10 of 12 R. annulatus from Texas. We were unable to obtain data for positions 53–161 in our R. microplus samples because two AmpSeq assays failed to amplify (Fig. 2 ), despite multiple attempts at designing new forward and reverse primer pairs. Our primer sets for Sub did not amplify any of the R. appendiculatus individuals in our sample set. However, R. appendiculatus GenBank accession QKY58555.1 has one inter-class replacement (N62S), and a second sample (ABA62331.1) has one intra-class (H95R) and two inter-class (A90T and P82A) replacements ( Additional File 6 ). An R. sanguineus sequence from GenBank (XP_037520396.1) carries H95R, plus three different replacements (S84C, A80T and H86P). It is worth noting that RmAQP2 (homolog of IsAQP1 and RsAQP7) stands out as being well conserved in R. microplus from the Americas but not Pakistan (Fig. 3 F). A total of seven aa replacements were found in the full-length protein (294 aa) across all of our R. microplus samples, but three of these (R8H, A136T, G175V) were only found in R. microplus from the Americas ( Additional File 6 ). These aa changes were rare; R8H and G175V were found in just one tick each from Mexico and Colombia. The A136T change was also rare, found only in Brazil and Pakistan. Therefore, AQP2 is more conserved in R. microplus from the Americas than the S-1 protein. The other four replacements (V249L, L254I, D275H, and E276G) occurred only in ticks from Pakistan ( Additional File 6 ). This disproportionate number of changes compared to ticks from the Americas is consistent with the long-term spatial and temporal separation of populations from Asia and the Americas. One change, A136T, sits on an extracellular loop in the middle of published vaccine peptide 2 (residues 125–156) ( 75 ). Other Rhipicephalus species contained greater variation within RmAQP2, including R. annulatus (eight changes) and R. appendiculatus (12 changes). The RmAQP2 homolog in R. sanguineus is RsAQP7 (XP_037518224.1), which had 21 replacements and one indel. In all other proteins, we found decreasing levels of conservation within R. microplus , with MP4 being the least conserved (Fig. 1 ). The Bm86 protein was the second least conserved protein in our samples from the Americas and Pakistan, with 53 replacements ( Additional File 7H ) in the 476 positions (of 650 total) that we assayed (Fig. 3 H). Many segments of the protein show evidence of mutational hotspots with clusters of aa changes (Fig. 2 ); 70% of the 10-aa sliding windows contain 1–5 replacements. The only highly conserved region occurs at aa positions 400–480. This region is encoded by Bm86 exons 11 and 12, both of which had a high success rate (96%) in R. microplus and yielded data for all ticks from Brazil and Colombia, as well as three of the five ticks from Pakistan. Therefore, this conserved region was assayed with high confidence. Understanding the specific location of aa replacements is important for evaluating the risk of vaccine escape from short peptide vaccines developed from proteins such as Sub ( 64 ), RmAQP2 ( 75 ), Chit ( 76 ), and Bm86 ( 56 ). In our 167 R. microplus samples, four of 12 (33%) published short peptides contained at least one aa replacement (Fig. 3 F, 3 G, and 3 H). When all GenBank entries are included, the number rises to eight of 12 (67%), and each of these four proteins carries at least one aa replacement in at least one short peptide target ( Additional File 6 ). The SBm7462® construct for Bm86 ( 55 ) has multiple replacements within each short peptide, although some may be restricted to certain regions of the world. DISCUSSION In this study we provide insights into the conservation of 14 protein candidates for anti-tick vaccines and compare them to Bm86 protein used in commercially available vaccines for cattle. We found that conservation varies across these proteins, with the greatest levels observed in VDAC, AQP1, VgR, RmS-1, and Sub across R. microplus samples from the Americas and Pakistan. When considering protein conservation alone, these five proteins each rank as high priority vaccine candidates. In DNA sequences, the d N / d S ratios (estimated by K A / K S ) of these five genes were close to (or at) zero and consistent with a signature of past purifying selection ( 77 ). We propose that screening d N / d S ratios of gene sequences will be a useful filtering step for identifying conserved vaccine targets. The top five proteins do not have mutational hotspots that are found in other proteins, and the small number of aa replacements could readily be incorporated into, or avoided in, future vaccine formulations. These proteins display a fraction of the number of Bm86 replacements observed within R. microplus alone. Despite the density of changes we observed in the less conserved proteins, we also note that short, conserved stretches exist in each protein and could potentially serve as targets for future vaccines based on specific epitopes, even if there is a large amount of variation across the rest of the protein sequence. For the top five conserved proteins and three others (RmAQP2, Chit, and Bm86), we provide coordinates of replacements observed in our dataset and illustrate their locations using 3D predictive models of each protein (Fig. 3 ). The ideal vaccine target would be a highly conserved functional epitope on a protein with a critical biological activity that is exposed on the surface where it is available for antibody binding. Ideally, antibody binding to this epitope would abrogate a critical biological function that will result in tick mortality or reproductive failure. Screening tick populations for genetic variation at potential vaccine targets and other population genetic markers ( 68 ) has become an important goal for vaccine development, and the use of AmpSeq has great utility for rapidly screening large numbers of individuals, especially when RNA is not available. We chose to employ exon sequencing because it allowed us to survey diverse R. microplus samples from a large DNA collection representing > 10,000 field-collected ticks. Exon sequences provide information on coding regions that are important for vaccine development without the need for whole genome sequences in multiple tick populations. Once the DNA sequence is obtained, it is straightforward to find non-synonymous mutations that lead to aa changes. The AmpSeq method will be especially efficient for investigating conservation within short epitopes that are known to be highly protective against R. microplus (and other tick species) in experimental trials. In our sample set, we found amino acid changes in one-third of the existing published short peptides for RmAQP2, Chit, and Bm86. That said, one potential limitation of the AmpSeq approach is that priming sites will typically need to be located within exons due to the high density of intronic SNPs, resulting in a small amount of missing data from each exon. Fortunately, this is not a problem if the research goal is to examine linear IgG epitopes of 8–12 aa or vaccines based on short peptides (< 30 aa). Conserved Proteins Voltage-dependent anion channel VDAC stands out in our study as being the highest priority vaccine target for global populations of R. microplus , based on the complete absence of aa replacements in the ticks we surveyed in this study. The small number of publicly available VDAC sequences are also fully conserved ( Additional File 6 ), one of which is from a lab strain from China (Rmic-2018) used for genome sequencing ( 78 ). We did not find the three VDAC replacements that have been reported previously in R. microplus from Mexico ( 61 ) (K27G in Jalisco, P133L in Tabasco, and N238P in Sinaloa) in our North American R. microplus sample set (n = 139), which might suggest they are either rare or possibly artifacts from PCR and cloning prior to Sanger sequencing. Thus, our findings suggest that VDAC is likely to have a very low risk of vaccine failure due to protein variation. This anion channel is the most abundant protein in the outer mitochondrial membrane of eukaryotic cells ( 74 ) and has a central role in apoptotic machinery ( 79 ). In vaccination stall trials, VDAC showed an 82% efficacy for reducing R. microplus on vaccinated cattle ( 80 ). Surprisingly, VDAC appears to be targeted by Babesia during the infection of tick midgut cells, and infected ticks experience increased expression of this mRNA and redistribution of VDAC protein compared to uninfected ticks ( 81 , 82 ); therefore, it also is being investigated for its potential as a transmission-blocking vaccine ( 80 ). The R. annulatus in our sample set were also fully conserved at VDAC, suggesting that this target could also be effective against the R. annulatus population from northern Mexico. The shared protein sequence in both R. microplus and R. annulatus is ideal for the development of a future vaccine that could be used by tick control programs against both species. Conservation in the predicted external loops of VDAC was also very high in R. appendiculatus and R. sanguineus ( Additional File 7A ), which potentially means that a VDAC vaccine targeted at these peptides will be useful against multiple Rhipicephalus species. However, other aa replacements in the transmembrane barrel and internal cytoplasmic loops could potentially reduce IgG antibody reactivity for a vaccine based on full-length protein, and these species-specific changes would need to be incorporated before vaccinating against other tick species. Aquaporins The aquaporins are an important family of osmoregulatory proteins for diverse organisms, including animals, plants, and bacteria ( 83 ). To maintain water balance, ticks secrete excess water and ions from bloodmeals back into the host ( 84 ). For example, IsAQP1 in I. scapularis (a homolog of RmAQP2) is expressed at high levels in salivary glands during blood feeding but decreases once ticks are engorged ( 65 ). Due to their metabolic importance, aquaporins are being considered as a target for anti-tick vaccines ( 85 ) and have been the focus of in-silico analyses to identify potential epitopes ( 86 ). We found the RmAQP1 protein to be highly conserved in R. microplus , with only three aa changes in our samples from North and South America. None of these replacements sit in the extracellular loops of the predicted 3D protein structure model (Fig. 3 B). Therefore, RmAQP1 ranks as another high priority vaccine target for global populations of R. microplus . However, wider survey of protein conservation is needed to determine if RmAQP1 could be protective against other closely related species, such as R. annulatus . A potentially significant aa replacement (T223S) found in the R. annulatus genome sequence is located in one of the external loops of the 3D model, emphasizing the need to characterize additional populations of R. annulatus and other tick species of interest for a future vaccine. The RmAQP2 protein is also well conserved in our tick samples from the Americas, and the three aa replacements we detected were rare. The A136T change is probably the most important of these because it sits on an extracellular loop in the middle of vaccine peptide 2 ( 75 ) and could potentially reduce IgG reactivity (Fig. 3 F). Other than the presence of A136T in two Brazilian lab colonies (IPV and POA), all aa positions within the three published peptides were fully conserved in R. microplus from North America. We also note that A136T sits at the end of a short, predicted epitope (M8; positions 124–136), which a modeling study ( 86 ) predicts will be highly immunogenic in IsAQP1, a homolog of RmAQP2. Other Rhipicephalus species display greater variation within AQP2, including R. annulatus , which carries an A126T replacement in peptide 2. Fortunately, conservation was much higher in peptide 1 (only an A60G in R. sanguineus ) and peptide 3 (S241A/D in R. sanguineus and R. appendiculatus , respectively). Our RmAQP2 findings further demonstrate the utility of screening exons with AmpSeq to detect any aa changes in short peptide vaccines. This information can then be used to tailor vaccine formulations to insure effectiveness against targeted tick populations. Vitellogenin receptor Vitellogenin receptor regulates the absorption of yolk proteins such as vitellin, the most abundant lipoglycoprotein in tick eggs ( 87 ). It stands out as a valuable vaccine candidate because vitellogenin (the precursor molecule to vitellin) is manufactured in the fat bodies and midgut of females and transported to oocytes via hemolymph ( 88 ); therefore, disruption of this receptor is expected to reduce the acquisition of vitellogenin essential to building egg mass and decrease tick fitness ( 63 ). We report the first in-depth survey of variation in the two ligand-binding domains (LBDs) of this protein, which had just three aa changes in R. microplus from the Americas. Of these, the most important is probably R1193H because it is located inside a predicted low-density lipid (LDL) binding region of the protein ( Additional File 6 ) ( 87 ). This widespread replacement was found in all countries that we sampled in North and South America and should be taken into consideration for any future vaccines that include this aa position. Due to the high level of conservation in R. microplus (and perhaps R. annulatus ), VgR ranks as a high priority anti-tick vaccine candidate. The level of conservation in VgR quickly decreases in alignments that include R. appendiculatus and R. sanguineus ( Additional File 6 ), and future vaccine formulations targeting multiple tick species would need to account for this extensive cross-species variation. Another potential solution might be to focus on shorter, highly conserved peptides within the two LBD domains that occur in all Rhipicephalus species that we evaluated. A surprising feature of VgR is that B. bovis parasites likely access developing oocytes by hitchhiking on vitellogenin molecules as they pass through the VgR ( 89 , 90 ). Thus, blocking the VgR could potentially serve a dual role that decreases egg quality and blocks the entry of B. bovis to any eggs and hatched larvae, effectively disrupting the Babesia life cycle by blocking transmission. Because male ticks do not transmit B. bovis in cattle, only females need to be impacted by a VgR vaccine. Serine Protease Inhibitor-1 Proteins in the serpin family are involved with diverse physiological functions in eukaryotes ( 91 ). In ticks, serpins modulate the host interaction during bloodfeeding, but also play a role in development and reproduction ( 92 ). Twenty-four serpins have been described in R. microplus and are hypothesized to be functional in the extracellular environment ( 93 , 94 ). Due to their importance in gene regulation, serpins have been investigated as anti-tick vaccine candidates against multiple tick species ( 95 – 97 ), and vaccination with recombinant H. longicornis serpin-2 (rHLS2) provided rabbits partial protection against H. longicornis ticks ( 95 ). We found RmS-1 to be well conserved in R. microplus from the Americas and Pakistan. The two most significant replacements are likely F101L and E306K because of their position in surface loops of the protein (Fig. 3 D), which may have the potential to impact IgG reactivity if epitopes exist on these loops. E306K was common in Texas, but F101L was very rare and we only detected it in Mexico (n = 2) and Colombia (n = 2). These replacements should be taken into account to reduce the risk of vaccine escape in this candidate. Because RmS-1 is expressed in salivary gland, midgut, and ovary ( 93 ), it might have the potential to simultaneously affect multiple physiological functions in ticks. Subolesin Subolesin has been frequently investigated as a vaccine candidate and is one of the leading targets for a universal vaccine against ticks and other arthropod disease vectors ( 64 , 98 – 100 ). It plays a broad role in gene regulation and affects the expression of tick reproduction and aspects of the innate immune system ( 101 – 103 ). In an experimental field trial in Mexico, a Sub vaccine provided 67% efficacy against R. microplus in calves grazing on infested pastures ( 104 ). A field trial in Uganda is also being planned to evaluate the efficacy of Sub to protect cattle against R. appendiculatus and R. decoloratus ( 105 ). Sub is one of the few vaccine candidates that has been surveyed for genetic variation in R. microplus populations from Mexico, and the study by Pérez Soria et al. ( 61 ) reported just one aa replacement (S19T) from a single R. microplus in Nayarit, Mexico. We did not find this change in our 139 R. microplus samples from North America (including three R. microplus from Nayarit), which may indicate it is rare. In our R. microplus samples, we found just one intra-class replacement (I41V) in the N-terminal half of Sub. It was shared by R. microplus and R. annulatus in Mexico and Texas but not observed outside of North America. This aa change maintains aliphatic residues (isoleucine and valine) that may be less likely to impact IgG reactivity than inter-class changes. Position 41 does not occur within the linear epitopes designed previously from tick Sub and insect akirin sequences in the Q38 chimera vaccine ( 64 ) and, thus, is not expected to impact the efficacy of this engineered vaccine. Sub is relatively less conserved in R. microplus sequences from India ( 57 ), where most ticks carry 1–2 aa replacements compared to the Deutsch reference ( Additional File 6 ). Other GenBank sequences of R. microplus from Mexico reveal Sub replacements between aa positions 98–122 (L100P, K115R, and I121M), which lie within published linear epitope #1 of the Q38 Sub/akirin chimera sequence ( 64 ). The geographically widespread diversity of Sub has implications for epitope #1 that could reduce its global effectiveness. In contrast, linear epitope #2 of the Q38 chimera is completely conserved in all publicly available sequences for R. microplus and seven other Rhipicephalus species; this epitope is based on Sub positions 130–139 (STKLAEQYDT). However, the published Q38 chimera sequence reports an alanine in position 131, rather than the threonine found in all other sequences. Other than this synthetic change in Q38, linear epitope #2 is one of the most highly conserved vaccine peptides yet reported within the genus Rhipicephalus . Less conserved proteins The proteins that exhibited intermediate levels of conservation (0.904–0.979) have each shown promise as anti-tick vaccine candidates in published cattle trials; however, their effectiveness in field settings will need to factor in any existing aa variation within the tick populations being targeted for control. Additional diversity is likely present in other globally distributed populations of R. microplus , and future surveys of genetic variation would be recommended before using any of the less conserved vaccine candidates. One potential solution is to focus on epitopes that are both highly antigenic and highly conserved. For instance, a recent vaccine trial used epitope prediction in Chit to develop four small peptide candidates (Soria-Perez et al. 2024), one of which (chitinase 3) had 71% efficacy against R. microplus in an experimental cattle trial. This peptide is fully conserved in R. microplus from the Americas ( Additional File 8 ) and would likely be appropriate for use against populations in Mexico and Texas. However, aa replacements do occur in R. microplus sequences from Brazil and China, and the use of chitinase 3 in these regions would need to account for these changes (and perhaps others). Likewise, GST has shown promise in past studies of tick control ( 106 – 108 ) but is only moderately conserved in R. microplus (0.95). Future investigation will need to account for GST variation in R. microplus (eight positions) and R. annulatus (five positions). In the RmS-11 protein, we found that many R. microplus individuals have a premature stop codon at residue 141; this is a significant aa change because the full length RmS-11 is 380 aa. It remains unknown whether this severely truncated protein would be functional, but if so, any epitopes in the downstream half of the protein would be missing and could significantly decrease the efficacy of an RmS-11 vaccine based on a full-length protein. The Bm86 protein has been the basis of all commercially available vaccine formulations against R. microplus and R. annulatus . As such, it is the most highly studied vaccine target and the current model for comparison for all vaccine candidates that have followed it ( 39 ). This protein makes up a very minor fraction of the midgut proteome ( 109 ) but was immunodominant in early trials and induced a strong enough IgG response to be highly protective against R. australis . Unfortunately, this protein is not well conserved globally ( 51 , 57 – 60 ) and studies of sequence variation were not performed until after the vaccine had been developed. Our results confirm the overall lack of conservation as we found 53 replacements (of 476 assayed positions) across examined R. microplus from the Americas and Pakistan. The use of AmpSeq proved to be a straightforward method for evaluating conservation in short Bm86 peptides such as SBm7462® ( 55 ), which appears to be appropriate for most Brazilian R. microplus populations in peptide 1, but peptides 2 and 3 each have single aa replacements in the four Brazilian lab colonies of R. microplus that we analyzed. None of the three peptides were fully conserved in ticks from North America or other countries. Likewise, four highly ranked Bm86 epitopes from a recent modeling study ( 57 ) each have multiple aa replacements, including aa positions 18–45 (2 changes), 97–129 (2 changes), 280–311 (5 changes), and 563–606 (5 changes). CONCLUSIONS It is important to note that protein conservation is only one of the factors that affect vaccine efficacy. Gene expression at specific life stages could also prevent effective protection ( 31 ), even when a well-designed vaccine with a high binding affinity to its target protein. The expression of redundant proteins coded by multi-gene families could also reduce the protectiveness of a vaccine, such as in the sialome ( 110 ). One way to address these limitations may be to use two or more antigens that are expressed at different parasitic life stages or in different tissue compartments of the tick, allowing host antibodies more than one chance at causing damage to ticks ( 111 ). Raising a strong antibody response to more than one antigen can be difficult to accomplish ( 112 ), but co-immunization at different body sites shows promise as a way to ramp up the IgG response against multiple antigens ( 62 , 113 ), as do vaccines delivered as DNA ( 114 ) and mRNA ( 115 ). Adjuvants differ in their ability to stimulate the bovine immune response ( 116 ), and can even favor specific IgG subtypes ( 117 ). A delivery platform that continuously presents antigens to the host immune system in an optimum way ( 118 ) could lead to improved protection against ticks. Information on specific aa changes in target proteins will complement the current advances in vaccine development and lead to more appropriate vaccine formulations with minimal risk of vaccine escape. An increasing focus of anti-tick vaccine studies is the use of protein modeling to predict B-cell epitopes that are highly immunogenic ( 119 ), and a number of R. microplus proteins (AQP1, Bm86, Chit, Sub) have been evaluated with Bepipred, IMGT® IEDB, Pepitope, VaxiJen and machine learning methods ( 57 , 76 , 86 , 120 ). Epitope prediction is valuable for prioritizing important peptides in a protein, but epitope choice will also need to be informed by a study of peptide sequence conservation. To provide an improved understanding of polyclonal antibody responses to a full-length protein vaccine, future studies of highly specific bovine immunological responses will benefit from the use of 10x Genomics sequencing of individual B-cells to identify IgG binding motifs and specific IgG subtypes (IgG1, IgG2, etc.) ( 121 ), and new tools enabled by liquid synthetic peptide arrays (PepSeq ™ platform) to identify specific linear epitopes ( 122 ). In combination, these different sources of information will provide a strong foundation for the development of the next generation of anti-tick vaccines. The importance of genetic surveys for specific vaccine targets is now recognized and an increasing number of large-scale surveys of variation are being published. However, finding globally useful targets will require diverse sampling sets from all continents where R. microplus has invaded. The same will be true for other highly invasive ticks that have dispersed globally, such as R. sanguineus sensu lato, H. longicornis , and Amblyomma variegatum (tropical bont tick). Abbreviations aa, amino acid; RmAQP1, aquaporin 1; RmAQP2, aquaporin 2; Chit, chitinase, S-1, serpin-1; Sub, subolesin; VDAC, voltage-dependent anion channel; VgR, vitellogenin receptor. Declarations Funding This work was supported by the following funding: USDA-NIFA-AFRI 2018-67015-28301 award to JDB, USDA-NIFA-AFRI 2015-67015-23047 award to DMW, USDA-APHIS-VS AP20VSSPRS00C117 award to JDB, USDA-ARS (NACA 2021 58-2090-1-037) award to JDB, a Northern Arizona University Hooper Undergraduate Research Award (HURA) to RET, and a Pakistan-US Science and Technology Cooperation program award (US Department of State #PGA-P21049) to SK. Authors statement The findings and conclusions in this report are those of the authorsand do not necessarily represent the views of the United States Department of Agriculture. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity provider and employer. Data availability The data supporting the conclusions of this article are included within the article and its additional files. All GenBank accessions are listed in Additional File 4 . Aligned DNA and protein sequences are available at https://github.com/GrantPem/Busch_etal_2024. CRediT authorship contribution statement All authors read and approved the final manuscript. Joseph D. Busch: Writing – original draft, Writing – review & editing, Conceptualization, Funding acquisition, Methodology, Investigation, Supervision, Project administration, Visualization. Nathan E. Stone: Writing – original draft, Writing – review & editing, Methodology, Investigation, Supervision, Formal analysis, Software, Data curation. Grant L. Pemberton: Writing – review & editing, Software, Investigation, Formal analysis, Data curation. Mackenzie L. Roberts: Investigation. Rebekah E. Turner: Investigation. Natalie Thornton: Investigation. Jason W. Sahl: Writing – review & editing, Software, Formal analysis, Data curation. Darrin Lemmer: Sofware, Formal analysis. Greta Buckmeier: Resources. Sara K. Davis: Resources. Roberto I. Guerrero Solorio: Resources. Shahid Karim: Writing – review & editing, Resources. Guilherme Klafke: Writing – review & editing, Resources. Donald B. Thomas: Writing – review & editing, Conceptualization, Resources. Pia U. Olafson: Writing – review & editing, Conceptualization, Resources. Massaro Ueti: Writing – review & editing, Conceptualization, Resources, Funding aquisition. Juan Mosqueda: Writing – review & editing, Conceptualization, Resources, Data aquisition. Glen Scoles: Writing – review & editing, Conceptualization, Resources. David M. Wagner: Writing – original draft, Writing – review & editing, Conceptualization, Supervision, Project administration, Funding acquisition. Declarations of competing interest None. Acknowledgments We thank the many US inspectors and veterinarians of the USDA-APHIS and TAHC who scratch cattle to collect cattle fever ticks in the field, and we are grateful for the ticks collected by numerous veterinarians and collaborators in Mexico. References de la Fuente J, Estrada-Pena A, Venzal JM, Kocan KM, Sonenshine DE. Overview: Ticks as vectors of pathogens that cause disease in humans and animals. Front Biosci. 2008;13:6938-46. Jongejan F, Uilenberg G. The global importance of ticks. Parasitology. 2004;129 Suppl:S3-14. Bock R, Jackson L, de Vos A, Jorgensen W. Babesiosis of cattle. Parasitology. 2004;129 Suppl:S247-69. Bram RA, George JE, Reichard RE, Tabachnick WJ. Threat of foreign arthropod-borne pathogens to livestock in the United States. Journal of Medical Entomology. 2002;39(3):405-16. 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Additional Files 5 and 6 Additional file 5: DNA alignments for 14 genes. https://github.com/GrantPem/Busch_etal_2024/AdditionalFile5_DNAalignments Additional file 6: Amino acid alignments for 14 proteins. https://github.com/GrantPem/Busch_etal_2024/AdditionalFile6_AAalignments Additional Declarations No competing interests reported. Supplementary Files AdditionalFile1TickSamples.xlsx Additional files Additional file 1: Table S1.Metadata for all tick samples used in this study. AdditionalFile2SupplementaryMethods.docx Additional file 2: Supplementary methods. AdditionalFile3PrimersForAmpSeq.xlsx Additional file 3: Table S2.Primers used to amplify individual exons of vaccine candidate genes in R. microplus (176 primers to amplify 88 amplicons). AdditionalFile4ExonSuccessRateandGenBankAccessions.xlsx Additional file 4: Table S3.Success rate of each exon across 167 R. microplus ticks and GenBank accession numbers. AdditionalFile7Protein3D.pdf Additional file 7: Supplemental information for amino acid replacements mapped onto predicted 3D structural models of selected proteins. Cite Share Download PDF Status: Published Journal Publication published 15 Apr, 2025 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 01 Sep, 2024 Reviews received at journal 31 Aug, 2024 Reviews received at journal 29 Aug, 2024 Reviewers agreed at journal 06 Aug, 2024 Reviewers agreed at journal 06 Aug, 2024 Reviewers invited by journal 05 Aug, 2024 Editor assigned by journal 02 Aug, 2024 Submission checks completed at journal 02 Aug, 2024 First submitted to journal 01 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4844765","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":344972918,"identity":"89b69004-9a37-4ad3-b488-3a8a40deb6cc","order_by":0,"name":"Joseph D. 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Wagner","email":"","orcid":"","institution":"Northern Arizona University","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"M.","lastName":"Wagner","suffix":""}],"badges":[],"createdAt":"2024-08-02 00:07:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4844765/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4844765/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-025-06683-5","type":"published","date":"2025-04-15T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63492505,"identity":"4ed75577-0d07-4302-a766-e0647fd2a576","added_by":"auto","created_at":"2024-08-28 18:19:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":172307,"visible":true,"origin":"","legend":"\u003cp\u003eProtein conservation in 14 anti-tick targets evaluated in 167 samples of \u003cem\u003eRhipicephalus \u003c/em\u003e(\u003cem\u003eBoophilus\u003c/em\u003e) \u003cem\u003emicroplus\u003c/em\u003e from North America (n=145), South America (n=17), and Pakistan (n=5). The y-axis shows protein similarity; x-axis is set to cross the y-axis at 0.80. Proteins are ranked from most to least conserved; the Bm86 protein (red box) used in all first-generation cattle vaccines is one of the least conserved proteins in our study.\u003c/p\u003e","description":"","filename":"Figure1ProteinSimilarityBarChart20240322JPG1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4844765/v1/028ec8ca127e133c432128f4.jpg"},{"id":63493082,"identity":"7634e768-4952-4562-bdf3-16ab3aa8073d","added_by":"auto","created_at":"2024-08-28 18:27:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":475433,"visible":true,"origin":"","legend":"\u003cp\u003eLocations of amino acid replacements in 10-aa windows of the 14 proteins analyzed in this study.\u003c/p\u003e\n\u003cp\u003eOnly replacements identified in the \u003cem\u003eRhipicephalus \u003c/em\u003e(\u003cem\u003eBoophilus\u003c/em\u003e)\u003cem\u003e microplus\u003c/em\u003e samples from our study (n=167) are used. Amino acid positions are shown on the top scale. Outlined boxes indicate each full-length protein except for VgR; the VgR boxes outline two ligand binding domains (LBDs) that were assayed in this study. Shading key: blue=conserved positions; orange to red=1-5 replacements per window of 10 amino acids; white=missing data.\u003c/p\u003e","description":"","filename":"Figure2SlidingWindowAnalysis20240710JPG1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4844765/v1/363819ae36f8bd03d178ce58.jpg"},{"id":63492506,"identity":"f4c43c49-fd56-49ba-8ea8-48d78147c57e","added_by":"auto","created_at":"2024-08-28 18:19:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1425914,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of amino acid replacements (blue) mapped onto predicted 3D structural models of selected proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e) Voltage-dependent anion channel (RmVDAC); \u003cstrong\u003eB\u003c/strong\u003e) Vitellogenin receptor (VgR); \u003cstrong\u003eC\u003c/strong\u003e) Aquaporin-1 (RmAQP1); \u003cstrong\u003eD\u003c/strong\u003e) Serine protease inhibitor-1 (RmS-1); \u003cstrong\u003eE\u003c/strong\u003e) Subolesin (RmSub); \u003cstrong\u003eF\u003c/strong\u003e) Aquaporin-2 (RmAQP2); \u003cstrong\u003eG\u003c/strong\u003e) Chitinase (Chit); \u003cstrong\u003eH\u003c/strong\u003e) Glycoprotein Bm86 (Bm86). Published short peptide vaccine targets (magenta) are highlighted for RmAQP2, Chit, RmSub, and Bm86; magenta is also used to highlight two lipid-binding domains (LBDs) that were assayed in VgR. Only those replacements identified in our \u003cem\u003eRhipicephalus \u003c/em\u003e(\u003cem\u003eBoophilus\u003c/em\u003e)\u003cem\u003e. microplus \u003c/em\u003edataset from the Americas and Pakistan are highlighted; additional replacements identified from previously published sequences of \u003cem\u003eR. microplus\u003c/em\u003e and other \u003cem\u003eRhipicephalus\u003c/em\u003e species are documented in the amino acid alignments within \u003cstrong\u003eAdditional File 6\u003c/strong\u003e. All 3D protein models were generated using the Alphafold website; specific URL addresses for each protein are provided.\u003c/p\u003e","description":"","filename":"Figure3Protein3D20240720PDF31.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4844765/v1/c2f68a572b28948458e1235b.jpg"},{"id":81050990,"identity":"591297a1-5246-481f-88fa-74af79d0d49e","added_by":"auto","created_at":"2025-04-21 16:09:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3473005,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4844765/v1/6cf61bce-8fcd-4e7b-bd55-110755d108a9.pdf"},{"id":63493081,"identity":"0d2626d8-478a-465a-9c32-29cb22b27375","added_by":"auto","created_at":"2024-08-28 18:27:00","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30609,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional files\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 1: Table S1.\u003c/strong\u003eMetadata for all tick samples used in this study.\u003c/p\u003e","description":"","filename":"AdditionalFile1TickSamples.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4844765/v1/32b966f55d066ec1d6fef74c.xlsx"},{"id":63492509,"identity":"ce15c342-a53a-4a15-8bf4-29746c0c78dc","added_by":"auto","created_at":"2024-08-28 18:19:00","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":60597,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: \u003c/strong\u003eSupplementary methods.\u003c/p\u003e","description":"","filename":"AdditionalFile2SupplementaryMethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-4844765/v1/6136451b0f87cbca8b6d1910.docx"},{"id":63492508,"identity":"69d53bcd-c0a0-46fa-b98b-9277cb129cdc","added_by":"auto","created_at":"2024-08-28 18:19:00","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":44998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 3: Table S2.\u003c/strong\u003ePrimers used to amplify individual exons of vaccine candidate genes in \u003cem\u003eR. microplus\u003c/em\u003e (176 primers to amplify 88 amplicons).\u003c/p\u003e","description":"","filename":"AdditionalFile3PrimersForAmpSeq.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4844765/v1/2071265d4f358ce75fc569f4.xlsx"},{"id":63492511,"identity":"4d0acd3a-5511-4284-b698-1027efc1e0d2","added_by":"auto","created_at":"2024-08-28 18:19:00","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":122938,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 4: Table S3.\u003c/strong\u003eSuccess rate of each exon across 167 \u003cem\u003eR. microplus\u003c/em\u003e ticks and GenBank accession numbers.\u003c/p\u003e","description":"","filename":"AdditionalFile4ExonSuccessRateandGenBankAccessions.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4844765/v1/5515a246057fb86c470ddac7.xlsx"},{"id":63492512,"identity":"38e1ca1d-188b-4199-bba7-d198fe9650ba","added_by":"auto","created_at":"2024-08-28 18:19:00","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1133953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 7: \u003c/strong\u003eSupplemental information for amino acid replacements mapped onto predicted 3D structural models of selected proteins.\u003c/p\u003e","description":"","filename":"AdditionalFile7Protein3D.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4844765/v1/4e93725b29979403e9649e58.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Leading anti-tick vaccine targets are variably conserved in cattle fever ticks","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003e Ticks are the most important vectors of animal diseases worldwide and an important public health concern (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Species such as the Asian longhorn tick (\u003cem\u003eHaemaphysalis longicornis\u003c/em\u003e) and southern cattle tick (\u003cem\u003eRhipicephalus\u003c/em\u003e [\u003cem\u003eBoophilus\u003c/em\u003e] \u003cem\u003emicroplus\u003c/em\u003e) have become global problems for livestock production due to their invasiveness, use of multiple hosts, and ability to transmit disease-causing pathogens (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). \u003cem\u003eR. microplus\u003c/em\u003e and \u003cem\u003eR. annulatus\u003c/em\u003e both transmit \u003cem\u003eBabesia bovis\u003c/em\u003e and \u003cem\u003eB. bigemina\u003c/em\u003e, which cause severe bovine babesiosis in na\u0026iuml;ve adult cattle, as well as the bacterium \u003cem\u003eAnaplasma marginale\u003c/em\u003e that causes bovine anaplasmosis (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Approximately one billion bovines are at risk of infestation by \u003cem\u003eR. microplus\u003c/em\u003e (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) and the global economic impact on the cattle industry due to this species alone is estimated to be at least USD \u003cspan\u003e$\u003c/span\u003e13.9\u0026ndash;18.7\u0026nbsp;billion per year (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Management of this issue is based primarily on chemical control of ticks on infested hosts, and acaricides have been used on \u003cem\u003eR. microplus\u003c/em\u003e populations for over one century, which has led to human-mediated selection for resistance to multiple chemical classes (\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Frequent treatment of cattle herds means that these one-host ticks experience repeated selection pressure that rapidly selects for high-level resistance; this can lead to resistance for as many as six chemical classes in certain \u003cem\u003eR. microplus\u003c/em\u003e populations (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Alternative control methods, such as anti-tick vaccines and plant-based compounds, are increasingly being evaluated as tools for tick control (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Because cattle fever ticks feed on a single host animal throughout their development from larvae to adult, their life cycle lends itself to vaccination-based control.\u003c/p\u003e \u003cp\u003eThe strategy for anti-tick vaccines involves immunizing a host with one or more tick proteins that stimulate a strong IgG antibody response directed at those proteins within the tick. Once ticks attach and begin to blood feed, the host IgG antibodies bind to these target proteins \u003cem\u003ein situ\u003c/em\u003e and disrupt tick feeding or physiology, leading to mortality or greatly reduced tick fitness. The nature and mechanism of the disruption caused is dependent on the functional role of the target antigen used in the vaccine (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Two main categories of antigens used in anti-tick vaccines are secreted salivary proteins that naturally interact with the host immune system (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), and concealed antigens (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) within the tick that are not normally exposed to the host immune system, but nonetheless can be targeted by host antibodies delivered via the blood meal (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Antibodies targeting secreted salivary proteins will substantially impact the attachment process and feeding interaction, whereas antibodies binding to concealed antigens will not \u0026ndash; instead, they interfere with the function of tick proteins responsible for critical physiological roles within the tick. The first anti-tick vaccination test in the 1930s used homogenates of midgut and salivary gland from American dog tick, \u003cem\u003eDermacentor variabilis\u003c/em\u003e, to raise a polyclonal antibody response in guinea pigs (\u003cem\u003eCavia porcellus\u003c/em\u003e) that clearly impacted ticks upon blood feeding (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In the mid-1970s, Galun (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) proposed the idea of controlling ticks by using IgG antibodies against tick juvenile hormones to interrupt normal tick development. Although not formally tested, her hypothesis is one of the earliest examples of the concealed antigen concept. The seminal work by Willadsen and co-workers (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) in the late 1980s identified a glycoprotein protease (Bm86) as a protective concealed antigen and set the stage for all future work on concealed antigens for vaccine development. Since these early studies, \u0026gt;\u0026thinsp;50 tick proteins have been tested in various host models, especially rabbits (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), and numerous review articles have discussed the successes and challenges of reducing tick burdens using specific tick antigens (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30 CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38 CR39 CR40 CR41\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Several studies have revealed that an important challenge for anti-vector vaccines is to raise a robust, long-lasting, and specific IgG antibody response that is protective against the arthropod pest, which has been difficult to achieve in real world settings (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Another strategy is to use transmission-blocking vaccines to reduce a pathogen\u0026rsquo;s ability to successfully infect a tick vector, rather than killing the ticks themselves (\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll of the vaccines against \u003cem\u003eR. microplus\u003c/em\u003e (and \u003cem\u003eR. annulatus\u003c/em\u003e) that have advanced to commercial development have been based on the glycoprotein protease Bm86, which is expressed in midgut epithelial cells (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). A recombinant rBm86 vaccine (TickGARD-PLUS) was developed from the Yeerongpilly strain of \u003cem\u003eR. australis\u003c/em\u003e from Queensland, Australia (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e); this species was previously considered to be \u003cem\u003eR. microplus\u003c/em\u003e but is now formally recognized as a distinct species (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). However, it has been observed that Bm86 protein sequences of \u003cem\u003eR. microplus\u003c/em\u003e populations in the Americas have diverged significantly from the \u003cem\u003eR. australis\u003c/em\u003e Yeerongpilly strain (91\u0026ndash;99% protein similarity) and sequence divergence correlates with variable vaccine efficacy (0\u0026ndash;91%) (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). To counteract this problem, geographically appropriate protein alleles have been developed for a number of rBm86 vaccines employed in different countries (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), including GAVAC\u0026reg; in Cuba (Concord strain Bm95 allele AF150891.2), Tick Vac\u0026reg; and Go Tick\u0026reg; in Colombia and Brazil (proprietary alleles by Limor de Colombia, Bogot\u0026aacute;, CO), and Bovimune ixovac\u0026reg; in Mexico (proprietary allele by Lapisa S.A., Michoac\u0026aacute;n, MX). An important issue for all full-length rBm86 formulations is that the specific surface-exposed epitopes contributing to a protective IgG response remain largely unknown (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). But directing a more specific antibody response has been made possible using the rSBm7462\u0026reg; formulation, which targets short chimeric peptides developed from three epitopes encoded in exons 1, 4 and 11 of the Bm86 gene (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e); additional Bm86 epitopes are being evaluated in \u003cem\u003eR. microplus\u003c/em\u003e populations in Mexico (Martinez-Alzate et al. 2019) and India (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Protein variation in Bm86 has been characterized in \u003cem\u003eR. microplus\u003c/em\u003e and \u003cem\u003eR. annulatus\u003c/em\u003e ticks from diverse geographic locations in the Americas (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e), India (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e), and Africa and Thailand (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Collectively, this work has uncovered extensive amino acid (aa) diversity in the full-length Bm86 protein, which is hypothesized to be the cause of decreased effectiveness in essentially all Bm86 vaccine formulations.\u003c/p\u003e \u003cp\u003eThe study of protein conservation in anti-tick vaccine targets other than Bm86 remains limited and only two studies have performed large-scale surveys of multiple \u003cem\u003eR. microplus\u003c/em\u003e populations. The first investigated DNA and protein variation within subolesin (Sub) and voltage-dependent anion channel (VDAC) in populations from Mexico (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e), and the second focused on Sub and tropomyosin (TPM) in \u003cem\u003eR. microplus\u003c/em\u003e throughout India (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Other studies have sampled a smaller number of populations to evaluate conservation in subolesin (Sub), vitellogenin receptor (VgR), and the aquaporins of \u003cem\u003eR. microplus\u003c/em\u003e (RmAQP1 and RmAQP2) (\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). To advance the development of next-generation anti-tick vaccines, we evaluated the level of conservation in Bm86 (as a control protein) and 13 other tick proteins, many of which were the focus of the Cattle Vaccine (CatVac) Consortium (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e) and have been considered for vaccine development by a number of research groups (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eTick Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRhipicephalus microplus:\u003c/em\u003e To evaluate global aa conservation within 14 anti-tick vaccine candidates, we examined individual \u003cem\u003eR. microplus\u003c/em\u003e (n=167) collected from geographically diverse locations in North America, South America, and Pakistan (\u003cstrong\u003eAdditional File 1\u003c/strong\u003e). The majority of the samples were collected in the field from \u003cem\u003eBos taurus\u003c/em\u003e cattle in Mexico (n=57 from 14 states) and the US (n=88 from Texas, five counties). The five \u003cem\u003eR. microplus\u003c/em\u003e samples collected in Pakistan for a previous study (67) were included to represent a population of \u003cem\u003eR. microplus\u003c/em\u003e from Asia. All US field samples of \u003cem\u003eR. microplus\u003c/em\u003e were collected from cattle; in certain instances, ticks were sampled on stray cattle from northern Mexico that were apprehended in Texas. The full methods for tick field collection and DNA extraction are described in \u003cstrong\u003eAdditional File 2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eLaboratory colonies of \u003cem\u003eR. microplus\u003c/em\u003e: In addition to field collections, we made use of 21 ticks from seven \u003cem\u003eR. microplus\u003c/em\u003e lab colonies. The first four colonies are maintained by the United States Department of Agriculture Cattle, Agricultural Research Service, Fever Tick Research Laboratory (USDA-ARS-CFTRL) and originated in Texas (Deutsch genome strain), Brazil (Santa Luiza), Colombia (Arauca), and Puerto Rico (Yabucoa). The others are two lab colonies (Porto Alegre [POA], and SLF) maintained by the Instituto de Pesquisas Veterin\u0026aacute;rias Desid\u0026eacute;rio Finamor (IPVDF) in Eldorado do Sul, Brazil and a naturally occurring field population (IPV) at the IPVDF pastures.\u003c/p\u003e\n\u003cp\u003eOther \u003cem\u003eRhipicephalus\u003c/em\u003e species: To evaluate protein diversity in a wider set of species in the genus \u003cem\u003eRhipicephalus\u003c/em\u003e we analyzed DNA samples from \u003cem\u003eR. annulatus\u003c/em\u003e and \u003cem\u003eR. appendiculatus\u003c/em\u003e. Field collections of \u003cem\u003eR. annulatus\u003c/em\u003e ticks were made by APHIS or TAHC field inspectors from cattle and introduced red deer (\u003cem\u003eCervus elaphus\u003c/em\u003e) and processed as described above. We also sampled the Vega lab colony of \u003cem\u003eR. annulatus\u003c/em\u003e (Texas) maintained at the USDA-ARS-CFTRL and the Muguga lab colony of \u003cem\u003eR. appendiculatus\u003c/em\u003e, originally collected in Kenya and maintained for \u0026gt;20 years at the Roslin institute in Scotland, then at the USDA-ARS-Animal Disease Research Unit in Pullman, WA since 2013.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmplicon Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used amplicon sequencing (AmpSeq) on an Illumina\u0026trade; short read platform (MiSeq\u0026trade;) to obtain exon DNA sequences encoding 14 anti-tick vaccine candidates for this study (\u003cstrong\u003eTable 1\u003c/strong\u003e). Each protein has been used in cattle vaccination trials with published mRNA sequences that served as reference homologs. We chose exon sequencing instead of mRNA sequencing because our archive of field samples (\u0026gt;10,000 \u003cem\u003eR. microplus\u003c/em\u003e) consists entirely of DNA extractions. Gene-specific primers (n=176 for \u003cem\u003eR. microplus\u003c/em\u003e were designed for 88 exon targets from 14 genes, and exon assays were divided into four multiplexed PCRs that maximized primer compatibility (\u003cstrong\u003eAdditional File 3\u003c/strong\u003e). Most priming sites were located at the ends of each exon, and therefore our data comprise partial length sequences for each gene/protein, with the exception of RmAQP2, which is full length. The full details of the PCR and AmpSeq methods are provided in \u003cstrong\u003eAdditional File 2\u003c/strong\u003e, and the success rate of each exon across all 167 \u003cem\u003eR. microplus\u003c/em\u003e is shown in \u003cstrong\u003eAdditional File 4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatic Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur bioinformatic methods follow that of a recent publication (68) and the full details are described in \u003cstrong\u003eAdditional File 2\u003c/strong\u003e. To set up standardized reference sequences for downstream analyses, we downloaded mRNA sequences of all 14 homologs from the first whole genome sequence of \u003cem\u003eR. microplus\u003c/em\u003e based on the USDA Deutsch lab colony from Texas (GenBank WOVZ00000000.1; Bioprojects PRJNA412317 and PRJNA312025) (69, 70). We note that exon sequences could not be concatenated because the relationship of exons in heterozygotes to their source allele (their \u0026ldquo;phase\u0026rdquo;) was unknown (see DNA sequence in \u003cstrong\u003eAdditional File 5\u003c/strong\u003e;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ehttps://github.com/GrantPem/Busch_etal_2024). Each partial exon sequence was then translated \u003cem\u003ein silico\u003c/em\u003e using BioEdit (71) to obtain predicted aa sequences that were aligned against the 14 Deutsch references (aa sequence in \u003cstrong\u003eAdditional File 6\u003c/strong\u003e). Protein similarity to the Deutsch reference was calculated as 1 \u0026ndash; (#aa replacements / total aa positions assayed). The location of aa replacements within each 10-aa window across the full-length proteins was visualized as a heat map of conserved versus variable peptides.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate protein conservation at predicted surface epitopes, we also mapped the specific location of each aa replacement onto predicted 3D protein structures using the AlphaFold website (https://alphafold.ebi.ac.uk/) (72, 73). We chose to visualize eight of the 14 proteins: the five most conserved proteins, two proteins with published short peptide vaccines, and Bm86 because it is used in commercially produced vaccines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e List of 14 published anti-tick vaccine targets used in our evaluation of protein conservation in \u003cem\u003eRhipicephalus\u0026nbsp;\u003c/em\u003e(\u003cem\u003eBoophilus\u003c/em\u003e) \u003cem\u003emicroplus\u003c/em\u003e.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"630\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFull length (aa)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhysiological target\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003emRNA Reference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eRmAQP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eAquaporin-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eWater balance; salivary glands\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eKJ626366.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eRmAQP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eAquaporin-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eWater balance; salivary glands, gut, ovaries\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eKP406519.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eBm86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eGlycoprotein (Bm86/Bm95)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e650\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eIntestinal lining of midgut\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eM29321.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eChit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eChitinase-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e436\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eCell structure and exoskeleton\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eGBBR01000100.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eCOX3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eCytochrome oxidase III\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eMitochondria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eKP143546.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eGST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eGlutathione S-transferase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e216\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eDetoxification\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eKF784792.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eMP4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eMetalloprotease 4 (reprolysin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e559\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eSalivary gland and digestive tract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eDQ118970.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eRmS-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eSerine protease inhibitor 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e380\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eInnate immune response; development\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eKC990100.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eRmS-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eSerine protease inhibitor 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e404\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eInnate immune response; development\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eKC990104.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eRmS-11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eSerine protease inhibitor 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e380\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eInnate immune response; development\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eKC990110.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eSub\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eSubolesin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e161\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eGene expression and regulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eKM115651.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eVDAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eVoltage-dependent anion channel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eMitochondria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eGU994210.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eVgR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eVitellogenin receptor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e1799\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eEgg development\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eKY781176.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.651828298887123%\" valign=\"top\"\u003e\n \u003cp\u003eVora\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.457869634340224%\" valign=\"top\"\u003e\n \u003cp\u003eVoraxin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.128775834658187%\" valign=\"top\"\u003e\n \u003cp\u003e\u003csup\u003ep\u003c/sup\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.842607313195547%\" valign=\"top\"\u003e\n \u003cp\u003eReproduction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91891891891892%\" valign=\"top\"\u003e\n \u003cp\u003eJX502818.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe column \u0026ldquo;Physiological target\u0026rdquo; broadly summarizes the focus of each vaccine as described in the available literature. Each reference sequence (based on mRNA) was chosen from a single published vaccine trial to ensure our data match previously described homologs. We compared each dataset to the Deutsch genome sequence (WOVZ00000000.1) to evaluate protein similarity. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ep\u003c/sup\u003ePartial cds for Voraxin in \u003cem\u003eRhipicephalus\u0026nbsp;\u003c/em\u003e(\u003cem\u003eBoophilus\u003c/em\u003e) \u003cem\u003emicroplus\u003c/em\u003e (see GenBank annotation page).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eWe found varying levels of conservation among the 14 proteins examined in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Because we used the Deutsch genome as a standardized reference, we were able to make a direct comparison of conservation across all 14 proteins in this dataset. The highest protein similarity within our sample of 167 \u003cem\u003eR. microplus\u003c/em\u003e ticks was observed in VDAC, which displayed no aa replacements in any of the sampled populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The DNA alignment for VDAC reveals 20 single nucleotide polymorphisms (SNPs) within \u003cem\u003eR. microplus\u003c/em\u003e but all are synonymous, equating to a \u003cem\u003eK\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e ratio of zero (\u003cb\u003eAdditional File 5\u003c/b\u003e). Four other proteins were highly conserved (RmAQP1, VgR, RmS-1, and Sub), with protein similarity values\u0026thinsp;\u0026gt;\u0026thinsp;0.98 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The majority of SNPs in these four genes were synonymous and yielded \u003cem\u003eK\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e estimates of 0.09, 0.07, 0.09, and 0.16, respectively. Intermediate levels of protein conservation were found in seven proteins (COX3, RmAQP2, Chit, GST, RmS-11, RmS-5, and Vora, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and ranged from 0.904\u0026ndash;0.979; the lowest levels were found in Bm86 (0.889) and MP4 (0.802). Although we did not calculate protein similarity values for other \u003cem\u003eRhipicephalus\u003c/em\u003e species due to low sample size, we note that protein conservation appears to decrease when other \u003cem\u003eRhipicephalus\u003c/em\u003e species are included in the comparison (\u003cb\u003eAdditional File 6\u003c/b\u003e). As an extreme example, Bm86 exhibited twice the number of aa replacements across seven \u003cem\u003eRhipicephalus\u003c/em\u003e species as compared to within \u003cem\u003eR. microplus\u003c/em\u003e alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe small number of aa changes in highly conserved proteins were typically spaced far apart. As observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, more variable proteins exhibited evidence of mutational hotspots, with aa changes clustered in multiple short segments of the protein. Despite the high density of changes in some of these proteins, short stretches of highly conserved peptides can also be found in each protein. One caveat for identifying conserved peptide regions is that our sampling design supports the detection of rare aa replacements in North America, but not in other locations due to smaller sample sizes. Another caveat is that the true amount of protein variation is probably underestimated in our dataset, because: 1) not all aa positions were queried due to the location of priming sites inside exons, 2) not all exons amplified equally well, and 3) tick populations from certain regions (Brazil and Pakistan) tended to fail more often than ticks from North America. However, 60 of the 88 exons had success rates\u0026thinsp;\u0026gt;\u0026thinsp;90% across our \u003cem\u003eR. microplus\u003c/em\u003e samples (\u003cb\u003eAdditional File 4\u003c/b\u003e) and these provide high confidence for estimating conservation at these exons, especially in North America.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe predicted 3D structural model for \u003cem\u003eR. microplus\u003c/em\u003e VDAC (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e) is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. In addition to being completely conserved in \u003cem\u003eR. microplus\u003c/em\u003e, the VDAC protein was conserved in all 12 \u003cem\u003eR. annulatus\u003c/em\u003e from Texas, which were identical to the \u003cem\u003eR. microplus\u003c/em\u003e allele (\u003cb\u003eAdditional File 6\u003c/b\u003e). In the DNA sequences, \u003cem\u003eR. annulatus\u003c/em\u003e individuals were variable at the same 20 nucleotide positions as \u003cem\u003eR. microplus\u003c/em\u003e, and all SNPs were synonymous (\u003cb\u003eAdditional File 5\u003c/b\u003e). In contrast, our samples of \u003cem\u003eR. appendiculatus\u003c/em\u003e displayed nine aa replacements in VDAC, and two publicly available \u003cem\u003eR. sanguineus\u003c/em\u003e sequences (XP_037498097.1 and UFQ89927.1) contained eight replacements, five of which were shared with \u003cem\u003eR. appendiculatus\u003c/em\u003e (\u003cb\u003eAdditional File 6\u003c/b\u003e). Interestingly, only one of these replacements (L136V) was located on an external surface loop (\u003cb\u003eAdditional File 7A\u003c/b\u003e) predicted in a 3D structural model for VDAC (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e). The other aa changes from \u003cem\u003eR. appendiculatus\u003c/em\u003e and \u003cem\u003eR. sanguineus\u003c/em\u003e were located within the transmembrane barrel and internal loops that extend into the cytoplasm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAquaporin-1 was the second most conserved protein across \u003cem\u003eR. microplus\u003c/em\u003e from the Americas and Pakistan (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). It is important to note that ticks carry multiple genes in the aquaporin family (18 reported from \u003cem\u003eIxodes scapularis\u003c/em\u003e) (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e) and their annotation has not been standardized across tick species. For instance, RmAQP1 was shown to be a homolog of IsAQP9 in a recent gene tree reconstruction (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). In our dataset, the RmAQP1 protein contained just three changes (I264V, L286I, and T294V) across the full-length protein (316 aa) within \u003cem\u003eR. microplus\u003c/em\u003e (\u003cb\u003eAdditional File 6\u003c/b\u003e). These changes were found in a small number of ticks in lab colonies from Brazil (IPV, POA, and Santa Luiza) and three individual ticks from Texas (Cameron County); no ticks from Mexico, Colombia, or Puerto Rico carried these changes. Ticks from Pakistan all failed to amplify the exon assay (AQP1_E01502) containing these three residues (\u003cb\u003eAdditional File 4\u003c/b\u003e), as did all 12 \u003cem\u003eR. annulatus\u003c/em\u003e samples (data not shown). In the publicly available \u003cem\u003eR. annulatus\u003c/em\u003e genome sequence (WOVY00000000.1; Bioproject PRJNA593711), three replacements occur in AQP1 (T223S, M266V, and T294V). The T223S replacement is the most important because it is located in an external loop of the 3D protein model (\u003cb\u003eAdditional File 7B\u003c/b\u003e). Our \u003cem\u003eR. appendiculatus\u003c/em\u003e samples contained 10 other replacements and two GenBank \u003cem\u003eR. sanguineus\u003c/em\u003e AQP9 sequences (XP_037510823.1 and KAH7963214.1) contained 17 replacements, but only one was at the same aa position as \u003cem\u003eR. microplus\u003c/em\u003e (I264S).\u003c/p\u003e \u003cp\u003eThe third most conserved locus was VgR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), with the caveat that we assayed only 434 positions of 1799 in the full-length protein, and three of the eight exon assays had lower success rates (80\u0026ndash;86%) (\u003cb\u003eAdditional File 4\u003c/b\u003e). Because VgR is a large protein, we focused on two ligand-binding domains (LBDs) encoded by exons 2\u0026ndash;3 (105 aa) and 14\u0026ndash;20 (329 aa). We found three aa replacements across \u003cem\u003eR. microplus\u003c/em\u003e from the Americas, which represented one inter-class change (N19T) and two intra-class changes (R1193H and T1216S). None of the three replacements were found in the five ticks from Pakistan, which carried changes at three other positions (T56I, P1167Q, and A1184G) for a total of six VgR replacements in our overall \u003cem\u003eR. microplus\u003c/em\u003e dataset. R1193H and T1216S are probably linked because they co-occurred in all 60 \u003cem\u003eR. microplus\u003c/em\u003e individuals from the Americas that carried them, as well as the 12 \u003cem\u003eR. annulatus\u003c/em\u003e samples (\u003cb\u003eAdditional File 6\u003c/b\u003e). Furthermore, they were found in all North and South American countries that we sampled (\u003cb\u003eAdditional File 1\u003c/b\u003e). Every \u003cem\u003eR. annulatus\u003c/em\u003e tick from Texas carried four replacements observed in \u003cem\u003eR. microplus\u003c/em\u003e (N19T, R1193H, A1184V, and T1216S), as well as two others (S63N and A921S). The \u003cem\u003eR. appendiculatus\u003c/em\u003e ticks in our study amplified at only two of eight assays (exons 16 and 19), but the data from exon 16 alone identified 11 polymorphic aa residues. A similar level of variation (13 replacements in a 187 aa peptide) is also evident in the partial VgR of \u003cem\u003eR. appendiculatus\u003c/em\u003e Muguga strain from Kenya (ATP60167.1) (\u003cb\u003eAdditional File 6\u003c/b\u003e). Likewise, \u003cem\u003eR. sanguineus\u003c/em\u003e (XP_037521270.1) contained 135 replacements across the entire protein, 45 of which overlap with the two LBDs that we screened in \u003cem\u003eR. microplus\u003c/em\u003e. The T1216S replacement in \u003cem\u003eR. sanguineus\u003c/em\u003e is shared with \u003cem\u003eR. microplus\u003c/em\u003e and \u003cem\u003eR. annulatus\u003c/em\u003e, and \u003cem\u003eR. sanguineus\u003c/em\u003e had an intra-class replacement at position 19 (N19D).\u003c/p\u003e \u003cp\u003eRmS-1 was well conserved across the \u003cem\u003eR. microplus\u003c/em\u003e populations we sampled, with a protein similarity of 0.985 across the 336 aa positions that we assayed of 380 in the full-length protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Five aa replacements (F101L, E140A, E306K, M337I, and I354V) were observed, all of which occurred in ticks from North America; the E140A change was also found in ticks from South America and Pakistan (\u003cb\u003eAdditional File 6\u003c/b\u003e). The first four are inter-class changes and I354V is an intra-class replacement. Two replacements (F101L and E306K) were found in surface loops of the protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). M337I and I354V appear to be linked, because they both were present in every tick that carried them (seven Texas locations). The \u003cem\u003eR. annulatus\u003c/em\u003e ticks sampled in Texas had replacements at four other positions (K52E, E275K, I280M, and L286M), and none carried the E140A replacement that was common in \u003cem\u003eR. microplus\u003c/em\u003e. Seventeen aa replacements were present in \u003cem\u003eR. appendiculatus\u003c/em\u003e ticks in three of the four assays. Assay A0101 failed in our 10 samples of \u003cem\u003eR. appendiculatus\u003c/em\u003e, but a full-length sequence from GenBank (AAK61375.1) shows\u0026thinsp;\u0026gt;\u0026thinsp;20 replacements in this section of the protein alone (positions 1-100) (\u003cb\u003eAdditional File 6\u003c/b\u003e). Likewise, \u003cem\u003eR. sanguineus\u003c/em\u003e (XP_037521270.1) contains 46 replacements across the entire protein. The other two members of the serpin family that we investigated (RmS-5 and RmS-11) showed much more variation in \u003cem\u003eR. microplus\u003c/em\u003e; RmS-5 had 27 replacements in the 360 positions that we assayed (similarity\u0026thinsp;=\u0026thinsp;0.925) and RmS-11 had 19 replacements in 340 assayed positions (similarity\u0026thinsp;=\u0026thinsp;0.944).\u003c/p\u003e \u003cp\u003eSubolesin was highly conserved in \u003cem\u003eR. microplus\u003c/em\u003e from the Americas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) with only a single aa change (I41V) in the 52 positions that we assayed (of 162 in the full-length protein). Our \u003cem\u003eR. microplus\u003c/em\u003e samples from Colombia and Pakistan are missing data at the assay that covers position 41, due to failed amplification. The I41V replacement is an intra-class change (isoleucine and valine are both aliphatic acids) and it is possible that the valine replacement would not significantly impact IgG reactivity, but this remains unknown. We found this replacement to be rare but widespread in Texas and Mexico (states of Tamaulipas, Zacatecas, and Campeche) yet absent in our samples from southern Brazil. It was also present in 10 of 12 \u003cem\u003eR. annulatus\u003c/em\u003e from Texas. We were unable to obtain data for positions 53\u0026ndash;161 in our \u003cem\u003eR. microplus\u003c/em\u003e samples because two AmpSeq assays failed to amplify (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), despite multiple attempts at designing new forward and reverse primer pairs. Our primer sets for Sub did not amplify any of the \u003cem\u003eR. appendiculatus\u003c/em\u003e individuals in our sample set. However, \u003cem\u003eR. appendiculatus\u003c/em\u003e GenBank accession QKY58555.1 has one inter-class replacement (N62S), and a second sample (ABA62331.1) has one intra-class (H95R) and two inter-class (A90T and P82A) replacements (\u003cb\u003eAdditional File 6\u003c/b\u003e). An \u003cem\u003eR. sanguineus\u003c/em\u003e sequence from GenBank (XP_037520396.1) carries H95R, plus three different replacements (S84C, A80T and H86P).\u003c/p\u003e \u003cp\u003eIt is worth noting that RmAQP2 (homolog of IsAQP1 and RsAQP7) stands out as being well conserved in \u003cem\u003eR. microplus\u003c/em\u003e from the Americas but not Pakistan (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). A total of seven aa replacements were found in the full-length protein (294 aa) across all of our \u003cem\u003eR. microplus\u003c/em\u003e samples, but three of these (R8H, A136T, G175V) were only found in \u003cem\u003eR. microplus\u003c/em\u003e from the Americas (\u003cb\u003eAdditional File 6\u003c/b\u003e). These aa changes were rare; R8H and G175V were found in just one tick each from Mexico and Colombia. The A136T change was also rare, found only in Brazil and Pakistan. Therefore, AQP2 is more conserved in \u003cem\u003eR. microplus\u003c/em\u003e from the Americas than the S-1 protein. The other four replacements (V249L, L254I, D275H, and E276G) occurred only in ticks from Pakistan (\u003cb\u003eAdditional File 6\u003c/b\u003e). This disproportionate number of changes compared to ticks from the Americas is consistent with the long-term spatial and temporal separation of populations from Asia and the Americas. One change, A136T, sits on an extracellular loop in the middle of published vaccine peptide 2 (residues 125\u0026ndash;156) (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). Other \u003cem\u003eRhipicephalus\u003c/em\u003e species contained greater variation within RmAQP2, including \u003cem\u003eR. annulatus\u003c/em\u003e (eight changes) and \u003cem\u003eR. appendiculatus\u003c/em\u003e (12 changes). The RmAQP2 homolog in \u003cem\u003eR. sanguineus\u003c/em\u003e is RsAQP7 (XP_037518224.1), which had 21 replacements and one indel.\u003c/p\u003e \u003cp\u003eIn all other proteins, we found decreasing levels of conservation within \u003cem\u003eR. microplus\u003c/em\u003e, with MP4 being the least conserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The Bm86 protein was the second least conserved protein in our samples from the Americas and Pakistan, with 53 replacements (\u003cb\u003eAdditional File 7H\u003c/b\u003e) in the 476 positions (of 650 total) that we assayed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Many segments of the protein show evidence of mutational hotspots with clusters of aa changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e); 70% of the 10-aa sliding windows contain 1\u0026ndash;5 replacements. The only highly conserved region occurs at aa positions 400\u0026ndash;480. This region is encoded by Bm86 exons 11 and 12, both of which had a high success rate (96%) in \u003cem\u003eR. microplus\u003c/em\u003e and yielded data for all ticks from Brazil and Colombia, as well as three of the five ticks from Pakistan. Therefore, this conserved region was assayed with high confidence.\u003c/p\u003e \u003cp\u003eUnderstanding the specific location of aa replacements is important for evaluating the risk of vaccine escape from short peptide vaccines developed from proteins such as Sub (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e), RmAQP2 (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e), Chit (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e), and Bm86 (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). In our 167 \u003cem\u003eR. microplus\u003c/em\u003e samples, four of 12 (33%) published short peptides contained at least one aa replacement (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). When all GenBank entries are included, the number rises to eight of 12 (67%), and each of these four proteins carries at least one aa replacement in at least one short peptide target (\u003cb\u003eAdditional File 6\u003c/b\u003e). The SBm7462\u0026reg; construct for Bm86 (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) has multiple replacements within each short peptide, although some may be restricted to certain regions of the world.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study we provide insights into the conservation of 14 protein candidates for anti-tick vaccines and compare them to Bm86 protein used in commercially available vaccines for cattle. We found that conservation varies across these proteins, with the greatest levels observed in VDAC, AQP1, VgR, RmS-1, and Sub across \u003cem\u003eR. microplus\u003c/em\u003e samples from the Americas and Pakistan. When considering protein conservation alone, these five proteins each rank as high priority vaccine candidates. In DNA sequences, the \u003cem\u003ed\u003c/em\u003e\u003csub\u003eN\u003c/sub\u003e/\u003cem\u003ed\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e ratios (estimated by \u003cem\u003eK\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e) of these five genes were close to (or at) zero and consistent with a signature of past purifying selection (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e). We propose that screening \u003cem\u003ed\u003c/em\u003e\u003csub\u003eN\u003c/sub\u003e/\u003cem\u003ed\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e ratios of gene sequences will be a useful filtering step for identifying conserved vaccine targets. The top five proteins do not have mutational hotspots that are found in other proteins, and the small number of aa replacements could readily be incorporated into, or avoided in, future vaccine formulations. These proteins display a fraction of the number of Bm86 replacements observed within \u003cem\u003eR. microplus\u003c/em\u003e alone. Despite the density of changes we observed in the less conserved proteins, we also note that short, conserved stretches exist in each protein and could potentially serve as targets for future vaccines based on specific epitopes, even if there is a large amount of variation across the rest of the protein sequence. For the top five conserved proteins and three others (RmAQP2, Chit, and Bm86), we provide coordinates of replacements observed in our dataset and illustrate their locations using 3D predictive models of each protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The ideal vaccine target would be a highly conserved functional epitope on a protein with a critical biological activity that is exposed on the surface where it is available for antibody binding. Ideally, antibody binding to this epitope would abrogate a critical biological function that will result in tick mortality or reproductive failure.\u003c/p\u003e \u003cp\u003eScreening tick populations for genetic variation at potential vaccine targets and other population genetic markers (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e) has become an important goal for vaccine development, and the use of AmpSeq has great utility for rapidly screening large numbers of individuals, especially when RNA is not available. We chose to employ exon sequencing because it allowed us to survey diverse \u003cem\u003eR. microplus\u003c/em\u003e samples from a large DNA collection representing\u0026thinsp;\u0026gt;\u0026thinsp;10,000 field-collected ticks. Exon sequences provide information on coding regions that are important for vaccine development without the need for whole genome sequences in multiple tick populations. Once the DNA sequence is obtained, it is straightforward to find non-synonymous mutations that lead to aa changes. The AmpSeq method will be especially efficient for investigating conservation within short epitopes that are known to be highly protective against \u003cem\u003eR. microplus\u003c/em\u003e (and other tick species) in experimental trials. In our sample set, we found amino acid changes in one-third of the existing published short peptides for RmAQP2, Chit, and Bm86. That said, one potential limitation of the AmpSeq approach is that priming sites will typically need to be located within exons due to the high density of intronic SNPs, resulting in a small amount of missing data from each exon. Fortunately, this is not a problem if the research goal is to examine linear IgG epitopes of 8\u0026ndash;12 aa or vaccines based on short peptides (\u0026lt;\u0026thinsp;30 aa).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eConserved Proteins\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eVoltage-dependent anion channel\u003c/h2\u003e \u003cp\u003eVDAC stands out in our study as being the highest priority vaccine target for global populations of \u003cem\u003eR. microplus\u003c/em\u003e, based on the complete absence of aa replacements in the ticks we surveyed in this study. The small number of publicly available VDAC sequences are also fully conserved (\u003cb\u003eAdditional File 6\u003c/b\u003e), one of which is from a lab strain from China (Rmic-2018) used for genome sequencing (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e). We did not find the three VDAC replacements that have been reported previously in \u003cem\u003eR. microplus\u003c/em\u003e from Mexico (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e) (K27G in Jalisco, P133L in Tabasco, and N238P in Sinaloa) in our North American \u003cem\u003eR. microplus\u003c/em\u003e sample set (n\u0026thinsp;=\u0026thinsp;139), which might suggest they are either rare or possibly artifacts from PCR and cloning prior to Sanger sequencing. Thus, our findings suggest that VDAC is likely to have a very low risk of vaccine failure due to protein variation. This anion channel is the most abundant protein in the outer mitochondrial membrane of eukaryotic cells (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e) and has a central role in apoptotic machinery (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e). In vaccination stall trials, VDAC showed an 82% efficacy for reducing \u003cem\u003eR. microplus\u003c/em\u003e on vaccinated cattle (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e). Surprisingly, VDAC appears to be targeted by \u003cem\u003eBabesia\u003c/em\u003e during the infection of tick midgut cells, and infected ticks experience increased expression of this mRNA and redistribution of VDAC protein compared to uninfected ticks (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e); therefore, it also is being investigated for its potential as a transmission-blocking vaccine (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eR. annulatus\u003c/em\u003e in our sample set were also fully conserved at VDAC, suggesting that this target could also be effective against the \u003cem\u003eR. annulatus\u003c/em\u003e population from northern Mexico. The shared protein sequence in both \u003cem\u003eR. microplus\u003c/em\u003e and \u003cem\u003eR. annulatus\u003c/em\u003e is ideal for the development of a future vaccine that could be used by tick control programs against both species. Conservation in the predicted external loops of VDAC was also very high in \u003cem\u003eR. appendiculatus\u003c/em\u003e and \u003cem\u003eR. sanguineus\u003c/em\u003e (\u003cb\u003eAdditional File 7A\u003c/b\u003e), which potentially means that a VDAC vaccine targeted at these peptides will be useful against multiple \u003cem\u003eRhipicephalus\u003c/em\u003e species. However, other aa replacements in the transmembrane barrel and internal cytoplasmic loops could potentially reduce IgG antibody reactivity for a vaccine based on full-length protein, and these species-specific changes would need to be incorporated before vaccinating against other tick species.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAquaporins\u003c/h2\u003e \u003cp\u003eThe aquaporins are an important family of osmoregulatory proteins for diverse organisms, including animals, plants, and bacteria (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e). To maintain water balance, ticks secrete excess water and ions from bloodmeals back into the host (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e). For example, IsAQP1 in \u003cem\u003eI. scapularis\u003c/em\u003e (a homolog of RmAQP2) is expressed at high levels in salivary glands during blood feeding but decreases once ticks are engorged (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). Due to their metabolic importance, aquaporins are being considered as a target for anti-tick vaccines (\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e) and have been the focus of \u003cem\u003ein-silico\u003c/em\u003e analyses to identify potential epitopes (\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e). We found the RmAQP1 protein to be highly conserved in \u003cem\u003eR. microplus\u003c/em\u003e, with only three aa changes in our samples from North and South America. None of these replacements sit in the extracellular loops of the predicted 3D protein structure model (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Therefore, RmAQP1 ranks as another high priority vaccine target for global populations of \u003cem\u003eR. microplus\u003c/em\u003e. However, wider survey of protein conservation is needed to determine if RmAQP1 could be protective against other closely related species, such as \u003cem\u003eR. annulatus\u003c/em\u003e. A potentially significant aa replacement (T223S) found in the \u003cem\u003eR. annulatus\u003c/em\u003e genome sequence is located in one of the external loops of the 3D model, emphasizing the need to characterize additional populations of \u003cem\u003eR. annulatus\u003c/em\u003e and other tick species of interest for a future vaccine.\u003c/p\u003e \u003cp\u003eThe RmAQP2 protein is also well conserved in our tick samples from the Americas, and the three aa replacements we detected were rare. The A136T change is probably the most important of these because it sits on an extracellular loop in the middle of vaccine peptide 2 (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e) and could potentially reduce IgG reactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Other than the presence of A136T in two Brazilian lab colonies (IPV and POA), all aa positions within the three published peptides were fully conserved in \u003cem\u003eR. microplus\u003c/em\u003e from North America. We also note that A136T sits at the end of a short, predicted epitope (M8; positions 124\u0026ndash;136), which a modeling study (\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e) predicts will be highly immunogenic in IsAQP1, a homolog of RmAQP2. Other \u003cem\u003eRhipicephalus\u003c/em\u003e species display greater variation within AQP2, including \u003cem\u003eR. annulatus\u003c/em\u003e, which carries an A126T replacement in peptide 2. Fortunately, conservation was much higher in peptide 1 (only an A60G in \u003cem\u003eR. sanguineus\u003c/em\u003e) and peptide 3 (S241A/D in \u003cem\u003eR. sanguineus\u003c/em\u003e and \u003cem\u003eR. appendiculatus\u003c/em\u003e, respectively). Our RmAQP2 findings further demonstrate the utility of screening exons with AmpSeq to detect any aa changes in short peptide vaccines. This information can then be used to tailor vaccine formulations to insure effectiveness against targeted tick populations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eVitellogenin receptor\u003c/h2\u003e \u003cp\u003eVitellogenin receptor regulates the absorption of yolk proteins such as vitellin, the most abundant lipoglycoprotein in tick eggs (\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e). It stands out as a valuable vaccine candidate because vitellogenin (the precursor molecule to vitellin) is manufactured in the fat bodies and midgut of females and transported to oocytes via hemolymph (\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e); therefore, disruption of this receptor is expected to reduce the acquisition of vitellogenin essential to building egg mass and decrease tick fitness (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). We report the first in-depth survey of variation in the two ligand-binding domains (LBDs) of this protein, which had just three aa changes in \u003cem\u003eR. microplus\u003c/em\u003e from the Americas. Of these, the most important is probably R1193H because it is located inside a predicted low-density lipid (LDL) binding region of the protein (\u003cb\u003eAdditional File 6\u003c/b\u003e) (\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e). This widespread replacement was found in all countries that we sampled in North and South America and should be taken into consideration for any future vaccines that include this aa position. Due to the high level of conservation in \u003cem\u003eR. microplus\u003c/em\u003e (and perhaps \u003cem\u003eR. annulatus\u003c/em\u003e), VgR ranks as a high priority anti-tick vaccine candidate. The level of conservation in VgR quickly decreases in alignments that include \u003cem\u003eR. appendiculatus\u003c/em\u003e and \u003cem\u003eR. sanguineus\u003c/em\u003e (\u003cb\u003eAdditional File 6\u003c/b\u003e), and future vaccine formulations targeting multiple tick species would need to account for this extensive cross-species variation. Another potential solution might be to focus on shorter, highly conserved peptides within the two LBD domains that occur in all \u003cem\u003eRhipicephalus\u003c/em\u003e species that we evaluated. A surprising feature of VgR is that \u003cem\u003eB. bovis\u003c/em\u003e parasites likely access developing oocytes by hitchhiking on vitellogenin molecules as they pass through the VgR (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e). Thus, blocking the VgR could potentially serve a dual role that decreases egg quality and blocks the entry of \u003cem\u003eB. bovis\u003c/em\u003e to any eggs and hatched larvae, effectively disrupting the \u003cem\u003eBabesia\u003c/em\u003e life cycle by blocking transmission. Because male ticks do not transmit \u003cem\u003eB. bovis\u003c/em\u003e in cattle, only females need to be impacted by a VgR vaccine.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSerine Protease Inhibitor-1\u003c/h2\u003e \u003cp\u003eProteins in the serpin family are involved with diverse physiological functions in eukaryotes (\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e). In ticks, serpins modulate the host interaction during bloodfeeding, but also play a role in development and reproduction (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e). Twenty-four serpins have been described in \u003cem\u003eR. microplus\u003c/em\u003e and are hypothesized to be functional in the extracellular environment (\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e). Due to their importance in gene regulation, serpins have been investigated as anti-tick vaccine candidates against multiple tick species (\u003cspan additionalcitationids=\"CR96\" citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e), and vaccination with recombinant \u003cem\u003eH. longicornis\u003c/em\u003e serpin-2 (rHLS2) provided rabbits partial protection against \u003cem\u003eH. longicornis\u003c/em\u003e ticks (\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e). We found RmS-1 to be well conserved in \u003cem\u003eR. microplus\u003c/em\u003e from the Americas and Pakistan. The two most significant replacements are likely F101L and E306K because of their position in surface loops of the protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), which may have the potential to impact IgG reactivity if epitopes exist on these loops. E306K was common in Texas, but F101L was very rare and we only detected it in Mexico (n\u0026thinsp;=\u0026thinsp;2) and Colombia (n\u0026thinsp;=\u0026thinsp;2). These replacements should be taken into account to reduce the risk of vaccine escape in this candidate. Because RmS-1 is expressed in salivary gland, midgut, and ovary (\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e), it might have the potential to simultaneously affect multiple physiological functions in ticks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSubolesin\u003c/h2\u003e \u003cp\u003eSubolesin has been frequently investigated as a vaccine candidate and is one of the leading targets for a universal vaccine against ticks and other arthropod disease vectors (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan additionalcitationids=\"CR99\" citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e). It plays a broad role in gene regulation and affects the expression of tick reproduction and aspects of the innate immune system (\u003cspan additionalcitationids=\"CR102\" citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e). In an experimental field trial in Mexico, a Sub vaccine provided 67% efficacy against \u003cem\u003eR. microplus\u003c/em\u003e in calves grazing on infested pastures (\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e). A field trial in Uganda is also being planned to evaluate the efficacy of Sub to protect cattle against \u003cem\u003eR. appendiculatus\u003c/em\u003e and \u003cem\u003eR. decoloratus\u003c/em\u003e (\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e). Sub is one of the few vaccine candidates that has been surveyed for genetic variation in \u003cem\u003eR. microplus\u003c/em\u003e populations from Mexico, and the study by P\u0026eacute;rez Soria et al. (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e) reported just one aa replacement (S19T) from a single \u003cem\u003eR. microplus\u003c/em\u003e in Nayarit, Mexico. We did not find this change in our 139 \u003cem\u003eR. microplus\u003c/em\u003e samples from North America (including three \u003cem\u003eR. microplus\u003c/em\u003e from Nayarit), which may indicate it is rare. In our \u003cem\u003eR. microplus\u003c/em\u003e samples, we found just one intra-class replacement (I41V) in the N-terminal half of Sub. It was shared by \u003cem\u003eR. microplus\u003c/em\u003e and \u003cem\u003eR. annulatus\u003c/em\u003e in Mexico and Texas but not observed outside of North America. This aa change maintains aliphatic residues (isoleucine and valine) that may be less likely to impact IgG reactivity than inter-class changes. Position 41 does not occur within the linear epitopes designed previously from tick Sub and insect akirin sequences in the Q38 chimera vaccine (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e) and, thus, is not expected to impact the efficacy of this engineered vaccine. Sub is relatively less conserved in \u003cem\u003eR. microplus\u003c/em\u003e sequences from India (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e), where most ticks carry 1\u0026ndash;2 aa replacements compared to the Deutsch reference (\u003cb\u003eAdditional File 6\u003c/b\u003e). Other GenBank sequences of \u003cem\u003eR. microplus\u003c/em\u003e from Mexico reveal Sub replacements between aa positions 98\u0026ndash;122 (L100P, K115R, and I121M), which lie within published linear epitope #1 of the Q38 Sub/akirin chimera sequence (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). The geographically widespread diversity of Sub has implications for epitope #1 that could reduce its global effectiveness. In contrast, linear epitope #2 of the Q38 chimera is completely conserved in all publicly available sequences for \u003cem\u003eR. microplus\u003c/em\u003e and seven other \u003cem\u003eRhipicephalus\u003c/em\u003e species; this epitope is based on Sub positions 130\u0026ndash;139 (STKLAEQYDT). However, the published Q38 chimera sequence reports an alanine in position 131, rather than the threonine found in all other sequences. Other than this synthetic change in Q38, linear epitope #2 is one of the most highly conserved vaccine peptides yet reported within the genus \u003cem\u003eRhipicephalus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLess conserved proteins\u003c/h2\u003e \u003cp\u003eThe proteins that exhibited intermediate levels of conservation (0.904\u0026ndash;0.979) have each shown promise as anti-tick vaccine candidates in published cattle trials; however, their effectiveness in field settings will need to factor in any existing aa variation within the tick populations being targeted for control. Additional diversity is likely present in other globally distributed populations of \u003cem\u003eR. microplus\u003c/em\u003e, and future surveys of genetic variation would be recommended before using any of the less conserved vaccine candidates. One potential solution is to focus on epitopes that are both highly antigenic and highly conserved. For instance, a recent vaccine trial used epitope prediction in Chit to develop four small peptide candidates (Soria-Perez et al. 2024), one of which (chitinase 3) had 71% efficacy against \u003cem\u003eR. microplus\u003c/em\u003e in an experimental cattle trial. This peptide is fully conserved in \u003cem\u003eR. microplus\u003c/em\u003e from the Americas (\u003cb\u003eAdditional File 8\u003c/b\u003e) and would likely be appropriate for use against populations in Mexico and Texas. However, aa replacements do occur in \u003cem\u003eR. microplus\u003c/em\u003e sequences from Brazil and China, and the use of chitinase 3 in these regions would need to account for these changes (and perhaps others). Likewise, GST has shown promise in past studies of tick control (\u003cspan additionalcitationids=\"CR107\" citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e) but is only moderately conserved in \u003cem\u003eR. microplus\u003c/em\u003e (0.95). Future investigation will need to account for GST variation in \u003cem\u003eR. microplus\u003c/em\u003e (eight positions) and \u003cem\u003eR. annulatus\u003c/em\u003e (five positions). In the RmS-11 protein, we found that many \u003cem\u003eR. microplus\u003c/em\u003e individuals have a premature stop codon at residue 141; this is a significant aa change because the full length RmS-11 is 380 aa. It remains unknown whether this severely truncated protein would be functional, but if so, any epitopes in the downstream half of the protein would be missing and could significantly decrease the efficacy of an RmS-11 vaccine based on a full-length protein.\u003c/p\u003e \u003cp\u003eThe Bm86 protein has been the basis of all commercially available vaccine formulations against \u003cem\u003eR. microplus\u003c/em\u003e and \u003cem\u003eR. annulatus\u003c/em\u003e. As such, it is the most highly studied vaccine target and the current model for comparison for all vaccine candidates that have followed it (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). This protein makes up a very minor fraction of the midgut proteome (\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e) but was immunodominant in early trials and induced a strong enough IgG response to be highly protective against \u003cem\u003eR. australis\u003c/em\u003e. Unfortunately, this protein is not well conserved globally (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan additionalcitationids=\"CR58 CR59\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e) and studies of sequence variation were not performed until after the vaccine had been developed. Our results confirm the overall lack of conservation as we found 53 replacements (of 476 assayed positions) across examined \u003cem\u003eR. microplus\u003c/em\u003e from the Americas and Pakistan. The use of AmpSeq proved to be a straightforward method for evaluating conservation in short Bm86 peptides such as SBm7462\u0026reg; (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e), which appears to be appropriate for most Brazilian \u003cem\u003eR. microplus\u003c/em\u003e populations in peptide 1, but peptides 2 and 3 each have single aa replacements in the four Brazilian lab colonies of \u003cem\u003eR. microplus\u003c/em\u003e that we analyzed. None of the three peptides were fully conserved in ticks from North America or other countries. Likewise, four highly ranked Bm86 epitopes from a recent modeling study (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e) each have multiple aa replacements, including aa positions 18\u0026ndash;45 (2 changes), 97\u0026ndash;129 (2 changes), 280\u0026ndash;311 (5 changes), and 563\u0026ndash;606 (5 changes).\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIt is important to note that protein conservation is only one of the factors that affect vaccine efficacy. Gene expression at specific life stages could also prevent effective protection (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), even when a well-designed vaccine with a high binding affinity to its target protein. The expression of redundant proteins coded by multi-gene families could also reduce the protectiveness of a vaccine, such as in the sialome (\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e). One way to address these limitations may be to use two or more antigens that are expressed at different parasitic life stages or in different tissue compartments of the tick, allowing host antibodies more than one chance at causing damage to ticks (\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e). Raising a strong antibody response to more than one antigen can be difficult to accomplish (\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e), but co-immunization at different body sites shows promise as a way to ramp up the IgG response against multiple antigens (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e), as do vaccines delivered as DNA (\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e) and mRNA (\u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e). Adjuvants differ in their ability to stimulate the bovine immune response (\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e), and can even favor specific IgG subtypes (\u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e). A delivery platform that continuously presents antigens to the host immune system in an optimum way (\u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e) could lead to improved protection against ticks. Information on specific aa changes in target proteins will complement the current advances in vaccine development and lead to more appropriate vaccine formulations with minimal risk of vaccine escape.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAn increasing focus of anti-tick vaccine studies is the use of protein modeling to predict B-cell epitopes that are highly immunogenic (\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e), and a number of \u003cem\u003eR. microplus\u003c/em\u003e proteins (AQP1, Bm86, Chit, Sub) have been evaluated with Bepipred, IMGT\u0026reg; IEDB, Pepitope, VaxiJen and machine learning methods (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e). Epitope prediction is valuable for prioritizing important peptides in a protein, but epitope choice will also need to be informed by a study of peptide sequence conservation. To provide an improved understanding of polyclonal antibody responses to a full-length protein vaccine, future studies of highly specific bovine immunological responses will benefit from the use of 10x Genomics sequencing of individual B-cells to identify IgG binding motifs and specific IgG subtypes (IgG1, IgG2, etc.) (\u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e121\u003c/span\u003e), and new tools enabled by liquid synthetic peptide arrays (PepSeq\u003csup\u003e\u0026trade;\u003c/sup\u003e platform) to identify specific linear epitopes (\u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e122\u003c/span\u003e). In combination, these different sources of information will provide a strong foundation for the development of the next generation of anti-tick vaccines. The importance of genetic surveys for specific vaccine targets is now recognized and an increasing number of large-scale surveys of variation are being published. However, finding globally useful targets will require diverse sampling sets from all continents where \u003cem\u003eR. microplus\u003c/em\u003e has invaded. The same will be true for other highly invasive ticks that have dispersed globally, such as \u003cem\u003eR. sanguineus\u003c/em\u003e sensu lato, \u003cem\u003eH. longicornis\u003c/em\u003e, and \u003cem\u003eAmblyomma variegatum\u003c/em\u003e (tropical bont tick).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eaa, amino acid; RmAQP1, aquaporin 1; RmAQP2, aquaporin 2; Chit, chitinase, S-1, serpin-1; Sub, subolesin; VDAC, voltage-dependent anion channel; VgR, vitellogenin receptor.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the following funding: USDA-NIFA-AFRI 2018-67015-28301 award to JDB, USDA-NIFA-AFRI 2015-67015-23047 award to DMW, USDA-APHIS-VS AP20VSSPRS00C117 award to JDB, USDA-ARS (NACA 2021 58-2090-1-037) award to JDB, a Northern Arizona University Hooper Undergraduate Research Award (HURA) to RET, and a\u0026nbsp;Pakistan-US Science and Technology Cooperation program award (US Department of State #PGA-P21049) to SK.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe findings and conclusions in this report are those of the authorsand do not necessarily represent the views of the United States Department of Agriculture. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity provider and employer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the conclusions of this article are included within the article and its additional files. All GenBank accessions are listed in \u003cstrong\u003eAdditional File 4\u003c/strong\u003e. Aligned DNA and protein sequences are available at https://github.com/GrantPem/Busch_etal_2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u0026nbsp;\u003cstrong\u003e\u0026nbsp;Joseph D. Busch:\u0026nbsp;\u003c/strong\u003eWriting\u0026nbsp;–\u0026nbsp;original draft, Writing\u0026nbsp;–\u0026nbsp;review\u0026nbsp;\u0026amp;\u0026nbsp;editing, Conceptualization, Funding acquisition, Methodology, Investigation, Supervision, Project administration, Visualization.\u0026nbsp;\u003cstrong\u003eNathan E. Stone:\u0026nbsp;\u003c/strong\u003eWriting\u0026nbsp;–\u0026nbsp;original draft, Writing\u0026nbsp;–\u0026nbsp;review\u0026nbsp;\u0026amp;\u0026nbsp;editing, Methodology, Investigation, Supervision, Formal analysis, Software, Data curation.\u0026nbsp;\u003cstrong\u003eGrant L. Pemberton:\u0026nbsp;\u003c/strong\u003eWriting\u0026nbsp;–\u0026nbsp;review\u0026nbsp;\u0026amp;\u0026nbsp;editing, Software, Investigation, Formal analysis, Data curation.\u0026nbsp;\u003cstrong\u003eMackenzie L. Roberts:\u0026nbsp;\u003c/strong\u003eInvestigation.\u0026nbsp;\u003cstrong\u003eRebekah E. Turner:\u0026nbsp;\u003c/strong\u003eInvestigation.\u0026nbsp;\u003cstrong\u003eNatalie Thornton:\u0026nbsp;\u003c/strong\u003eInvestigation.\u0026nbsp;\u003cstrong\u003eJason W. Sahl:\u0026nbsp;\u003c/strong\u003eWriting\u0026nbsp;–\u0026nbsp;review\u0026nbsp;\u0026amp;\u0026nbsp;editing, Software, Formal analysis, Data curation.\u0026nbsp;\u003cstrong\u003eDarrin Lemmer:\u003c/strong\u003e Sofware, Formal analysis.\u0026nbsp;\u003cstrong\u003eGreta Buckmeier:\u0026nbsp;\u003c/strong\u003eResources.\u0026nbsp;\u003cstrong\u003eSara K. Davis:\u0026nbsp;\u003c/strong\u003eResources.\u0026nbsp;\u003cstrong\u003eRoberto I. Guerrero Solorio:\u0026nbsp;\u003c/strong\u003eResources.\u0026nbsp;\u003cstrong\u003eShahid Karim:\u0026nbsp;\u003c/strong\u003eWriting\u0026nbsp;–\u0026nbsp;review\u0026nbsp;\u0026amp;\u0026nbsp;editing, Resources.\u0026nbsp;\u003cstrong\u003eGuilherme Klafke:\u0026nbsp;\u003c/strong\u003eWriting\u0026nbsp;–\u0026nbsp;review\u0026nbsp;\u0026amp;\u0026nbsp;editing, Resources.\u0026nbsp;\u003cstrong\u003eDonald B. Thomas:\u0026nbsp;\u003c/strong\u003eWriting – review \u0026amp; editing, Conceptualization, Resources. \u003cstrong\u003ePia U. Olafson:\u0026nbsp;\u003c/strong\u003eWriting – review \u0026amp; editing, Conceptualization, Resources.\u0026nbsp;\u003cstrong\u003eMassaro Ueti:\u0026nbsp;\u003c/strong\u003eWriting\u0026nbsp;–\u0026nbsp;review\u0026nbsp;\u0026amp;\u0026nbsp;editing, Conceptualization, Resources, Funding aquisition. \u003cstrong\u003eJuan Mosqueda:\u0026nbsp;\u003c/strong\u003eWriting – review \u0026amp; editing, Conceptualization, Resources, Data aquisition. \u003cstrong\u003eGlen Scoles:\u0026nbsp;\u003c/strong\u003eWriting – review \u0026amp; editing, Conceptualization, Resources. \u003cstrong\u003eDavid M. Wagner:\u003c/strong\u003e Writing – original draft, Writing – review \u0026amp; editing, Conceptualization, Supervision, Project administration, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the many US inspectors and veterinarians of the USDA-APHIS and TAHC who scratch cattle to collect cattle fever ticks in the field, and we are grateful for the ticks collected by numerous veterinarians and collaborators in Mexico.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ede la Fuente J, Estrada-Pena A, Venzal JM, Kocan KM, Sonenshine DE. Overview: Ticks as vectors of pathogens that cause disease in humans and animals. Front Biosci. 2008;13:6938-46.\u003c/li\u003e\n\u003cli\u003eJongejan F, Uilenberg G. The global importance of ticks. 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Insect Biochem Mol Biol. 2016;71:12-28.\u003c/li\u003e\n\u003cli\u003eImamura S, da Silva Vaz Junior I, Sugino M, Ohashi K, Onuma M. A serine protease inhibitor (serpin) from Haemaphysalis longicornis as an anti-tick vaccine. Vaccine. 2005;23(10):1301-11.\u003c/li\u003e\n\u003cli\u003eMulenga A, Sugino M, Nakajima M, Sugimoto C, Onuma M. Tick-encoded serine proteinase inhibitors (Serpins); Potential target antigens for tick vaccine development. Journal of Veterinary Medical Science. 2001;63(10):1063-9.\u003c/li\u003e\n\u003cli\u003eZhao X, Zhao J, Wang J, Liao C, Guan Q, Han Q. Immune protection of three serine protease inhibitors vaccine in mice against \u003cem\u003eRhipicephalus sanguineus\u003c/em\u003e. Sci Rep. 2024;14(1):7703.\u003c/li\u003e\n\u003cli\u003eParizi LF, Githaka NW, Logullo C, Zhou J, Onuma M, Termignoni C, et al. Universal tick vaccines: Candidates and remaining challenges. Animals (Basel). 2023;13(12).\u003c/li\u003e\n\u003cli\u003ede la Fuente J, Moreno-Cid JA, Galindo RC, Almazan C, Kocan KM, Merino O, et al. Subolesin/Akirin vaccines for the control of arthropod vectors and vectorborne pathogens. Transbound Emerg Dis. 2013;60 Suppl 2:172-8.\u003c/li\u003e\n\u003cli\u003eLee SH, Li J, Moumouni PFA, Okado K, Zheng W, Liu M, et al. Subolesin vaccination inhibits blood feeding and reproduction of \u003cem\u003eHaemaphysalis longicornis\u003c/em\u003e in rabbits. Parasit Vectors. 2020;13(1):478.\u003c/li\u003e\n\u003cli\u003ede la Fuente J, Moreno-Cid JA, Canales M, Villar M, de la Lastra JM, Kocan KM, et al. Targeting arthropod subolesin/akirin for the development of a universal vaccine for control of vector infestations and pathogen transmission. Vet Parasitol. 2011;181(1):17-22.\u003c/li\u003e\n\u003cli\u003ede la Fuente J, Almazan C, Blas-Machado U, Naranjo V, Mangold AJ, Blouin EF, et al. The tick protective antigen, 4D8, is a conserved protein involved in modulation of tick blood ingestion and reproduction. Vaccine. 2006;24(19):4082-95.\u003c/li\u003e\n\u003cli\u003eArtigas-Jeronimo S, Villar M, Cabezas-Cruz A, Valdes JJ, Estrada-Pena A, Alberdi P, et al. Functional Evolution of Subolesin/Akirin. Front Physiol. 2018;9:1612.\u003c/li\u003e\n\u003cli\u003eMendoza-Martinez N, Alonso-Diaz MA, Merino O, Fernandez-Salas A, Lagunes-Quintanilla R. Protective efficacy of the peptide Subolesin antigen against the cattle tick \u003cem\u003eRhipicephalus microplus\u003c/em\u003e under natural infestation. Vet Parasitol. 2021;299:109577.\u003c/li\u003e\n\u003cli\u003eKabi F, Dhikusooka M, Matovu M, Mugerwa S, Kasaija P, Emudong P, et al. Monitoring the subolesin vaccine field trial for safer control of cattle ticks amidst increasing acaricide resistance in Uganda. Vaccines (Basel). 2022;10(10).\u003c/li\u003e\n\u003cli\u003eNdawula C, Jr., Sabadin GA, Parizi LF, da Silva Vaz I, Jr. Constituting a glutathione S-transferase-cocktail vaccine against tick infestation. Vaccine. 2019;37(14):1918-27.\u003c/li\u003e\n\u003cli\u003eParizi LF, Reck J, Jr., Oldiges DP, Guizzo MG, Seixas A, Logullo C, et al. Multi-antigenic vaccine against the cattle tick Rhipicephalus (Boophilus) microplus: a field evaluation. Vaccine. 2012;30(48):6912-7.\u003c/li\u003e\n\u003cli\u003eSabadin GA, Parizi LF, Kiio I, Xavier MA, da Silva Matos R, Camargo-Mathias MI, et al. Effect of recombinant glutathione S-transferase as vaccine antigen against \u003cem\u003eRhipicephalus appendiculatus\u003c/em\u003e and \u003cem\u003eRhipicephalus sanguineus \u003c/em\u003einfestation. Vaccine. 2017;35(48 Pt B):6649-56.\u003c/li\u003e\n\u003cli\u003eWilladsen P, Kemp DH. Vaccination with \u0026apos;concealed\u0026apos; antigens for tick control. 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Cell Rep Med. 2021;2(1):100189.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Additional Files 5 and 6","content":"\u003cp\u003e\u003cstrong\u003eAdditional file 5:\u0026nbsp;\u003c/strong\u003eDNA alignments for 14 genes. https://github.com/GrantPem/Busch_etal_2024/AdditionalFile5_DNAalignments\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 6:\u0026nbsp;\u003c/strong\u003eAmino acid alignments for 14 proteins. https://github.com/GrantPem/Busch_etal_2024/AdditionalFile6_AAalignments\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rhipicephalus microplus, R. annulatus, anti-tick vaccine, conserved targets, surface-exposed epitopes","lastPublishedDoi":"10.21203/rs.3.rs-4844765/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4844765/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eRhipicephalus\u003c/em\u003e (\u003cem\u003eBoophilus\u003c/em\u003e) \u003cem\u003emicroplus\u003c/em\u003e causes significant cattle production losses worldwide because it transmits \u003cem\u003eBabesia bovis\u003c/em\u003e and \u003cem\u003eB. bigemina\u003c/em\u003e causative agents of bovine babesiosis. Control of these ticks primarily has relied on treatment of cattle with chemical acaricides, but frequent use, exacerbated by the one-host life cycle of these ticks, has led to high-level resistance to multiple classes of acaricides. Consequently, new approaches for control, such as anti-tick vaccines, are critically important. Key to this approach is targeting highly conserved antigenic epitopes to reduce the risk of vaccine escape in heterologous tick populations.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe evaluated amino acid conservation within 14 tick proteins across 167 \u003cem\u003eR. microplus\u003c/em\u003e collected from geographically diverse locations in the Americas and Pakistan using PCR amplicon sequencing and \u003cem\u003ein silico\u003c/em\u003e translation of exons.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that amino acid conservation varied considerably across these proteins. Only one target, the voltage-dependent anion channel, was fully conserved in all 167 \u003cem\u003eR. microplus\u003c/em\u003e samples (protein similarity 1.0). Five other proteins were highly conserved: the aquaporin RmAQP1 (0.989), vitellogenin receptor (0.985), serpin-1 (0.985), and subolesin (0.981). In contrast, the glycoprotein protease Bm86 was one of the least conserved (0.889). The Bm86 sequence used in the original Australian TickGARD vaccine carried many amino acid replacements compared to the \u003cem\u003eR. microplus\u003c/em\u003e populations examined here, supporting the hypothesis that this vaccine target is not optimal for use in the Americas. By mapping amino acid replacements onto predicted 3D protein models, we also identified amino acid changes within several small peptide vaccines targeting portions of the aquaporin RmAQP2, chitinase, and Bm86.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings emphasize the importance of thoroughly analyzing protein variation within anti-tick vaccine targets across diverse tick populations before selecting candidate vaccine antigens. When considering protein conservation alone, RmAQP1, vitellogenin receptor, serpin-1, subolesin, and especially the voltage-dependent anion channel rank as high priority anti-tick vaccine candidates for use in the Americas and perhaps globally.\u003c/p\u003e","manuscriptTitle":"Leading anti-tick vaccine targets are variably conserved in cattle fever ticks","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-28 18:18:55","doi":"10.21203/rs.3.rs-4844765/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-01T14:26:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-31T13:50:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-29T19:36:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96601517178422959524688960297439038929","date":"2024-08-07T03:55:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229779227598859218247285013825570788958","date":"2024-08-06T18:16:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-05T15:25:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-02T05:45:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-02T05:36:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2024-08-02T00:06:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e10d078c-97d1-4cf9-b1e0-c8ee206b24b8","owner":[],"postedDate":"August 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-21T16:04:18+00:00","versionOfRecord":{"articleIdentity":"rs-4844765","link":"https://doi.org/10.1186/s13071-025-06683-5","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-04-15 15:57:46","publishedOnDateReadable":"April 15th, 2025"},"versionCreatedAt":"2024-08-28 18:18:55","video":"","vorDoi":"10.1186/s13071-025-06683-5","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06683-5","workflowStages":[]},"version":"v1","identity":"rs-4844765","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4844765","identity":"rs-4844765","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}