Anthropogenic electromagnetic radiation alters the transcription levels of the genes encoding the SIFamide and myoinhibitory peptide and their receptors in Ixodes ricinus synganglion.

The research of the influences of man-made electromagnetic fields on tick physiology has been very sparse and long neglected since the pioneer studies published in 1996 and 2000. Once multiple behavioral tests confirmed an attraction and possible perception of electromagnetic fields in ticks, a new interest in this topic erupted in recent years. In this study, qRT-PCR is utilized to determine the changes in the mRNA transcript levels of neuropeptides SIFamide and myoinhibitory peptide (mip and sifa) and their representative receptors (mip-r1 and sifa-r1) in the synganglia of the tick Ixodes ricinus irradiated by 900 MHz radiofrequency electromagnetic field. It was determined that 40 V/m intensity has a significant suppressory effect on the transcript levels of all genes after at least 60 minutes of constant exposure in both sexes. Commonly occurring intensity of radiation in urban areas (2 V/m) produced an elevation in mRNA levels after various timespans in every gene. A significant decrease of transcript abundances was detected in females after one hour of exposure to 2 V/m. Results of this study widen the knowledge of EMF-induced alterations in the neurophysiology of I. ricinus, the most commonly distributed hard tick in Europe.

Genome resequencing reveals population divergence and local adaptation of blacklegged ticks in the United States.

Tick vectors and tick-borne disease are increasingly impacting human populations globally. An important challenge is to understand tick movement patterns, as this information can be used to improve management and predictive modelling of tick population dynamics. Evolutionary analysis of genetic divergence, gene flow and local adaptation provides insight on movement patterns at large spatiotemporal scales. We develop low coverage, whole genome resequencing data for 92 blacklegged ticks, Ixodes scapularis, representing range-wide variation across the United States. Through analysis of population genomic data, we find that tick populations are structured geographically, with gradual isolation by distance separating three population clusters in the northern United States, southeastern United States and a unique cluster represented by a sample from Tennessee. Populations in the northern United States underwent population contractions during the last glacial period and diverged from southern populations at least 50 thousand years ago. Genome scans of selection provide strong evidence of local adaptation at genes responding to host defences, blood-feeding and environmental variation. In addition, we explore the potential of low coverage genome sequencing of whole-tick samples for documenting the diversity of microbial pathogens and recover important tick-borne pathogens such as Borrelia burgdorferi. The combination of isolation by distance and local adaptation in blacklegged ticks demonstrates that gene flow, including recent expansion, is limited to geographical scales of a few hundred kilometres.

Tick-Borne pathogens and defensin genes expression: A closer look at Ixodes ricinus and Dermacentor reticulatus.

The immune system of ticks, along with that of other invertebrates, is comparatively simpler than that of vertebrates, relying solely on innate immune responses. Direct antimicrobial defence is provided by the synthesis of antimicrobial peptides (AMPs), including defensins. The aim of this study was to investigate the differences in defensin genes expression between questing and engorged Ixodes ricinus (def1 and def2) and Dermacentor reticulatus (defDr) ticks, in the presence of selected pathogens: Borrelia spp., Rickettsia spp., Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis in the natural environment. After pathogen screening by PCR/qPCR, the expression of defensin genes in pathogen positive ticks and ticks without any of the tested pathogens, was analysed by reverse transcription qPCR. The results showed an increased expression of defensin genes in I. ricinus ticks after blood feeding and I. ricinus and D. reticulatus ticks during in cases of co-infection. In particular, the expression of defensins genes was higher in questing D. reticulatus than in questing and engorged I. ricinus ticks, when borreliae were detected. This study contributes to uncovering the expression patterns of defensin genes in the presence of several known tick pathogens, the occurrence of these pathogens and possible regulatory mechanisms of defensins in tick vector competence.

First detection and a new avian host of the tick Ixodes ventalloi Gil Collado, 1936, in Slovakia.

This study describes the first detection of Ixodes ventalloi in Slovakia. Two engorged females of I. ventalloi were collected from Dunnocks (Prunella modularis) captured in eastern Slovakia. The identification of females was based on morphological and molecular 16S rRNA gene features. Phylogenetic analysis revealed a classification of the females into distinct genogroups. Moreover, comparative morphological analysis highlighted variations between the two females, particularly in the curvature of the auriculae, the shape of coxa I, and the internal spur. These findings suggest the potential for varied phenotypes of I. ventalloi correlated with their genogroups. Nonetheless, I. ventalloi population establishment within Slovakia necessitates further investigation through flagging or drag sampling.

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

Insects have about 50 neuropeptide genes and about 70 genes, coding for neuropeptide G protein-coupled receptors (GPCRs). An important, but small family of evolutionarily related insect neuropeptides consists of adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP). Normally, insects have one specific GPCR for each of these neuropeptides. The tick Ixodes scapularis is not an insect, but belongs to the subphylum Chelicerata, which comprises ticks, scorpions, mites, spiders, and horseshoe crabs. Many of the neuropeptides and neuropeptide GPCRs occurring in insects, also occur in chelicerates, illustrating that insects and chelicerates are evolutionarily closely related. The tick I. scapularis is an ectoparasite and health risk for humans, because it infects its human host with dangerous pathogens during a blood meal. Understanding the biology of ticks will help researchers to prevent tick-borne diseases. By annotating the I. scapularis genome sequence, we previously found that ticks contain as many as five genes, coding for presumed ACP receptors. In the current paper, we cloned these receptors and expressed each of them in Chinese Hamster Ovary (CHO) cells. Each expressed receptor was activated by nanomolar concentrations of ACP, demonstrating that all five receptors were functional ACP receptors. Phylogenetic tree analyses showed that the cloned tick ACP receptors were mostly related to insect ACP receptors and, next, to insect AKH receptors, suggesting that ACP receptor genes and AKH receptor genes originated by gene duplications from a common ancestor. Similar duplications have probably occurred for the ligand genes, during a process of ligand/receptor co-evolution. Interestingly, chelicerates, in contrast to all other arthropods, do not have AKH or AKH receptor genes. Therefore, the ancestor of chelicerates might have lost AKH and AKH receptor genes and functionally replaced them by ACP and ACP receptor genes. For the small family of AKH, ACP, and corazonin receptors and their ligands, gene losses and gene gains occur frequently between the various ecdysozoan clades. Tardigrades, for example, which are well known for their survival in extreme environments, have as many as ten corazonin receptor genes and six corazonin peptide genes, while insects only have one of each, or none.

Genetic structure and Rickettsia infection rates in Ixodes ovatus and Haemaphysalis flava ticks across different altitudes.

Ixodid ticks, such as Ixodes ovatus and Haemaphysalis flava, are important vectors of tick-borne diseases in Japan, such as Japanese spotted fever caused by Rickettsia japonica. This study describes the Rickettsia infection rates influenced by the population genetic structure of I.ovatus and H. flava along an altitudinal gradient. A total of 346 adult I. ovatus and 243 H. flava were analyzed for the presence of Rickettsia by nested PCR targeting the 17kDA, gltA, rOmpA, and rOmpB genes. The population genetic structure was analyzed utilizing the mitochondrial cytochrome oxidase 1 (cox1) marker. The Rickettsia infection rates were 13.26% in I. ovatus and 6.17% in H. flava. For I. ovatus, the global FST value revealed significant genetic differentiation among the different populations, whereas H. flava showed non-significant genetic differentiation. The cox1 I. ovatus cluster dendrogram showed two cluster groups, while the haplotype network and phylogenetic tree showed three genetic groups. A significant difference was observed in Rickettsia infection rates and mean altitude per group between the two cluster groups and the three genetic groups identified within I. ovatus. No significant differences were found in the mean altitude or Rickettsia infection rates of H. flava. Our results suggest a potential correlation between the low gene flow in I. ovatus populations and the spatially heterogeneous Rickettsia infection rates observed along the altitudinal gradient. This information can be used in understanding the relationship between the tick vector, its pathogen, and environmental factors, such as altitude, and for the control of tick-borne diseases in Japan.

Age- and Sex-Associated Pathogenesis of Cell Culture-Passaged Kemerovo Virus in IFNAR(-/-) Mice.

Kemerovo virus (KEMV) is a tick-borne orbivirus transmitted by ticks of the genus Ixodes. Previous animal experimentation studies with orbiviruses, in particular the interferon receptor double knock-out (IFNAR(-/-)) mouse model, did not indicate bias that is related to age or sex. We endeavoured to assess the effect of serial and alternated passages of KEMV in mammalian or Ixodes cells on virus replication and potential virulence in male or female IFNAR(-/-) mice, with important age differences: younger males (4-5 months old), older males (14-15 months old), and old females (14-15 months old). After 30 serial passages in mammalian or tick cells, or alternated passages in the two cell types, older female mice which were inoculated with the resulting virus strains were the first to show clinical signs and die. Younger males behaved differently from older males whether they were inoculated with the parental strain of KEMV or with any of the cell culture-passaged strains. The groups of male and female mice inoculated with the mammalian cell culture-adapted KEMV showed the lowest viraemia. While older female and younger male mice died by day 6 post-inoculation, surprisingly, the older males survived until the end of the experiment, which lasted 10 days. RNA extracted from blood and organs of the various mice was tested by probe-based KEMV real-time RT-PCR. Ct values of the RNA extracts were comparable between older females and younger males, while the values for older males were >5 Ct units higher for the various organs, indicating lower levels of replication. It is noteworthy that the hearts of the old males were the only organs that were negative for KEMV RNA. These results suggest, for the first time, an intriguing age- and sex-related bias for an orbivirus in this animal model. Changes in the amino acid sequence of the RNA-dependent RNA polymerase of Kemerovo virus, derived from the first serial passage in Ixodes cells (KEMV Ps.IRE1), were identified in the vicinity of the active polymerase site. This finding suggests that selection of a subpopulation of KEMV with better replication fitness in tick cells occurred.

From the fat body to the hemolymph: Profiling tick immune and storage proteins through transcriptomics and proteomics.

Ticks are blood-feeding arachnids that are known to transmit various pathogenic microorganisms to their hosts. During blood feeding, ticks activate their metabolism and immune system to efficiently utilise nutrients from the host's blood and complete the feeding process. In contrast to insects, in which the fat body is known to be a central organ that controls essential metabolic processes and immune defense mechanisms, the function of the fat body in tick physiology is still relatively unexplored. To fill this gap, we sought to uncover the repertoire of genes expressed in the fat body associated with trachea (FB/Tr) by analyzing the transcriptome of individual, partially fed (previtellogenic) Ixodes ricinus females. The resulting catalog of individual mRNA sequences reveals a broad repertoire of transcripts encoding proteins involved in nutrient storage and distribution, as well as components of the tick immune system. To gain a detailed insight into the secretory products of FB/Tr specifically involved in inter-tissue transport and humoral immunity, the transcriptomic data were complemented with the proteome of soluble proteins in the hemolymph of partially fed female ticks. Among these proteins, the hemolipoglyco-carrier proteins were predominant. When comparing immune peptides and proteins from the fat body with those produced by hemocytes, we found that the fat body serves as a unique producer of certain immune components. Finally, time-resolved transcriptional regulation of selected immune transcripts from the FB/Tr was examined in response to experimental challenges with model microbes and analyzed by RT-qPCR. Overall, our data show that the fat body of ticks, similar to insects, is an important metabolic tissue that also plays a remarkable role in immune defense against invading microbes. These findings improve our understanding of tick biology and its impact on the transmission of tick-borne pathogens.

mRNA vaccination of rabbits alters the fecundity, but not the attachment, of adult Ixodes scapularis.

19ISP is a nucleoside-modified mRNA-lipid nanoparticle vaccine that targets 19 Ixodes scapularis proteins. We demonstrate that adult I. scapularis have impaired fecundity when allowed to engorge on 19ISP-immunized rabbits. 19ISP, therefore, has the potential to interrupt the tick reproductive cycle, without triggering some of the other effects associated with acquired tick resistance. This may lead to the development of new strategies to reduce I. scapularis populations in endemic areas.

Comparative analysis of Presence-Absence gene Variations in five hard tick species: impact and functional considerations.

Tick species are vectors of harmful human and animal diseases, and their expansion is raising concerns under the global environmental changes' scenario. Ticks host and transmit bacteria, protozoa and viruses, making the understanding of host-pathogen molecular pathways critical to development of effective disease control strategies. Despite the considerable sizes and repeat contents of tick genomes, individual tick genomics is perhaps the most effective approach to reveal genotypic traits of interest. Presence-Absence gene Variations (PAVs) can contribute to individual differences within species, with dispensable genes carried by subsets of individuals possibly underpinning functional significance at individual or population-levels. We exploited 350 resequencing datasets of Dermacentor silvarum, Haemaphysalis longicornis, Ixodes persulcatus, Rhipicephalus microplus and Rhipicephalus sanguineus hard tick specimens to reveal the extension of PAV and the conservation of dispensable genes among individuals and, comparatively, between species. Overall, we traced 550-3,346 dispensable genes per species and were able to reconstruct 5.3-7 Mb of genomic regions not included in the respective reference genomes, as part of the tick pangenomes. Both dispensable genes and de novo predicted genes indicated that PAVs preferentially impacted mobile genetic elements in these tick species.

Diversity and host specificity of Borrelia burgdorferi's outer surface protein C (ospC) alleles in synanthropic mammals, with a notable ospC allele U absence from mixed infections.

Interactions among pathogen genotypes that vary in host specificity may affect overall transmission dynamics in multi-host systems. Borrelia burgdorferi, a bacterium that causes Lyme disease, is typically transmitted among wildlife by Ixodes ticks. Despite the existence of many alleles of B. burgdorferi's sensu stricto outer surface protein C (ospC) gene, most human infections are caused by a small number of ospC alleles ["human infectious alleles" (HIAs)], suggesting variation in host specificity associated with ospC. To characterize the wildlife host association of B. burgdorferi's ospC alleles, we used metagenomics to sequence ospC alleles from 68 infected individuals belonging to eight mammalian species trapped at three sites in suburban New Brunswick, New Jersey (USA). We found that multiple allele ("mixed") infections were common. HIAs were most common in mice (Peromyscus spp.) and only one HIA was detected at a site where mice were rarely captured. ospC allele U was exclusively found in chipmunks (Tamias striatus), and although a significant number of different alleles were observed in chipmunks, including HIAs, allele U never co-occurred with other alleles in mixed infections. Our results suggest that allele U may be excluding other alleles, thereby reducing the capacity of chipmunks to act as reservoirs for HIAs.

Cross-alteration of murine skin and tick microbiome concomitant with pathogen transmission after Ixodes ricinus bite.

BACKGROUND: Ticks are major vectors of diseases affecting humans such as Lyme disease or domestic animals such as anaplasmosis. Cross-alteration of the vertebrate host skin microbiome and the tick microbiome may be essential during the process of tick feeding and for the mechanism of pathogen transmission. However, it has been poorly investigated. METHODS: We used mice bitten by field-collected ticks (nymphs and adult ticks) in different experimental conditions to investigate, by 16S rRNA gene metabarcoding, the impact of blood feeding on both the mouse skin microbiome and the tick microbiome. We also investigated by PCR and 16S rRNA gene metabarcoding, the diversity of microorganisms transmitted to the host during the process of tick bite at the skin interface and the dissemination of the pathogen in host tissues (blood, heart, and spleen). RESULTS: Most of the commensal bacteria present in the skin of control mice were replaced during the blood-feeding process by bacteria originating from the ticks. The microbiome of the ticks was also impacted by the blood feeding. Several pathogens including tick-borne pathogens (Borrelia/Borreliella, Anaplasma, Neoehrlichia, Rickettsia) and opportunistic bacteria (Williamsia) were transmitted to the skin microbiome and some of them disseminated to the blood or spleen of the mice. In the different experiments of this study, skin microbiome alteration and Borrelia/Borreliella transmission were different depending on the tick stages (nymphs or adult female ticks). CONCLUSIONS: Host skin microbiome at the bite site was deeply impacted by the tick bite, to an extent which suggests a role in the tick feeding, in the pathogen transmission, and a potentially important impact on the skin physiopathology. The diversified taxonomic profiles of the tick microbiome were also modified by the blood feeding. Video Abstract.

Molecular-phylogenetic analyses of Ixodes species from South Africa suggest an African origin of bird-associated exophilic ticks (subgenus Trichotoixodes).

BACKGROUND: Among hard ticks (Acari: Ixodidae), the genus Ixodes comprises the highest number of species, which in turn are most numerous in the Afrotropical zoogeographic region. In South Africa extensive morphological studies have been performed on Ixodes species but only few reports included molecular analyses. METHODS: In this study, 58 Ixodes spp. ticks, collected from ten mammalian and eight avian host species in South Africa, were molecularly and phylogenetically analyzed. In addition, a newly collected sample of the Palearctic Ixodes trianguliceps was included in the analyses. RESULTS: Among the ticks from South Africa, 11 species were identified morphologically. The majority of ticks from mammals represented the Ixodes pilosus group with two species (n = 20), followed by ticks resembling Ixodes rubicundus (n = 18) and Ixodes alluaudi (n = 3). In addition, single specimens of Ixodes rhabdomysae, Ixodes ugandanus, Ixodes nairobiensis and Ixodes simplex were also found. Considering bird-infesting ticks, Ixodes theilerae (n = 7), Ixodes uriae (n = 4) and ticks most similar to Ixodes daveyi (provisionally named I. cf. daveyi, n = 2) were identified. Molecular analyses confirmed two species in the I. pilosus group and a new species (I. cf. rubicundus) closely related to I. rubicundus sensu stricto. Phylogenetic trees based on concatenated mitochondrial or mitochondrial and nuclear gene sequences indicated that the subgenus Afrixodes forms a monophyletic clade with bird-associated exophilic ticks (subgenus Trichotoixodes). Ixodes trianguliceps clustered separately whereas I. alluaudi with their morphologically assigned subgenus, Exopalpiger. CONCLUSIONS: Phylogenetic analyses shed new lights on the relationships of Ixodes subgenera when including multiple sequences from subgenus Afrixodes and African as well as Palearctic species of subgenera Trichotoixodes and Exopalpiger. Subgenera Afrixodes and bird-associated Trichotoixodes share common ancestry, suggesting that the latter might have also originated in Africa. Regarding the subgenus Exopalpiger, I. alluaudi is properly assigned as it clusters among different Australian Ixodes, whereas I. trianguliceps should be excluded.

The highly improved genome of Ixodes scapularis with X and Y pseudochromosomes.

Ixodes scapularis, the black-legged tick, is the principal vector of the Lyme disease spirochete, Borrelia burgdorferi, and is responsible for most of the ∼470,000 estimated Lyme disease cases annually in the USA. Ixodes scapularis can transmit six additional pathogens of human health significance. Because of its medical importance, I. scapularis was the first tick genome to be sequenced and annotated. However, the first assembly, I. scapularis Wikel (IscaW), was highly fragmented because of the technical challenges posed by the long, repetitive genome sequences characteristic of arthropod genomes and the lack of long-read sequencing techniques. Although I. scapularis has emerged as a model for tick research because of the availability of new tools such as embryo injection and CRISPR-Cas9-mediated gene editing yet the lack of chromosome-scale scaffolds has slowed progress in tick biology and the development of tools for their control. Here we combine diverse technologies to produce the I. scapularis Gulia-Nuss (IscGN) genome assembly and gene set. We used DNA from eggs and male and female adult ticks and took advantage of Hi-C, PacBio HiFi sequencing, and Illumina short-read sequencing technologies to produce a chromosome-level assembly. In this work, we present the predicted pseudochromosomes consisting of 13 autosomes and the sex pseudochromosomes: X and Y, and a markedly improved genome annotation compared with the existing assemblies and annotations.

Bulk and single-nucleus RNA sequencing highlight immune pathways induced in individuals during an Ixodes scapularis tick bite.

Ticks are hematophagous arthropods that use a complex mixture of salivary proteins to evade host defenses while taking a blood meal. Little is known about the immunological and physiological consequences of tick feeding on humans. Here, we performed the first bulk and single-nucleus RNA sequencing (snRNA-seq) of skin and blood of four persons presenting with naturally acquired, attached Ixodes scapularis ticks. Pathways and individual genes associated with innate and adaptive immunity were identified based on bulk RNA sequencing, including interleukin-17 signaling and platelet activation pathways at the site of tick attachment or in peripheral blood. snRNA-seq further revealed that the Hippo signaling, cell adhesion, and axon guidance pathways were involved in the response to an I. scapularis bite in humans. Features of the host response in these individuals also overlapped with that of laboratory guinea pigs exposed to I. scapularis and which acquired resistance to ticks. These findings offer novel insights for the development of new biomarkers for I. scapularis exposure and anti-tick vaccines for human use.

Genetic modification, characterization, and co-infection of Franken Sphingomonas and Anaplasma phagocytophilum in tick cells.

Paratransgenesis through genetic manipulation of symbiotic or commensal microorganisms has been proposed as an effective and environmentally sound approach for the control of vector-borne diseases, including tick bite-related pathologies, and reducing pathogen transmission. Here, we present a protocol for Sphingomonas transformation with Anaplasma phagocytophilum major surface protein 4 and heat shock protein 70. We describe a step-by-step protocol for in vitro study of interactions between transformed Franken Sphingomonas and Ixodes scapularis ISE6 tick cells during A. phagocytophilum infection. For complete details on the use and execution of this protocol, please refer to Mazuecos et al. (2023).1.

Anaplasma phagocytophilum in European bison (Bison bonasus) and their ticks from Lithuania and Poland.

The increasing population of European bison (Bison bonasus) can contribute to the prevalence of zoonotic pathogens. The aim of the present study was to assess the presence of A. phagocytophilum infection in European bison tissues as well as ticks removed from European bison in Lithuania and Poland. A further objective of this work was to compare the detected A. phagocytophilum strains. A total of 85 tissue samples (spleen) of European bison and 560 ticks belonging to two species, Ixodes ricinus (n = 408) and Dermacentor reticulatus (n = 152) were tested. DNA of A. phagocytophilum was detected based on RT-PCR in 40% of the European bison samples, 8.8% of the I. ricinus and 5.9% of the D. reticulatus ticks. Analysis of the obtained partial 16S rRNA gene sequences of A. phagocytophilum revealed the presence of three variants with two polymorphic sites. Furthermore, phylogenetic analysis with partial msp4 gene sequences grouped A. phagocytophilum variants into three clusters. This study revealed that the groEL gene sequences of A. phagocytophilum from European bison and their ticks grouped into ecotype I and only one sequence from Lithuanian European bison belonged to ecotype II. The results of the present study indicated that European bison may play a role as a natural reservoir of A. phagocytophilum.

Detection of Bartonella DNA in Yellow Flies, Lone Star Ticks, and a Human Patient with Concurrent Evidence of Borrelia burgdorferi Infection in Northeast Florida, USA.

Background: Bartonella species and Borrelia burgdorferi sensu lato (Bbsl) are emerging zoonotic pathogens. The vectors and frequency of infections with both pathogen groups in the southern United States is understudied. This study describes an investigation of Bartonella and Bbsl in yellow flies collected at a residence in northeast Florida, USA, that led to subsequent discoveries of both organisms in lone star ticks (Amblyomma americanum) and a human patient. Materials and Methods: DNA samples from flies, ticks, and human patient blood specimens were tested via polymerase chain reaction assays for Bartonella or Bbsl species. DNA sequences were compared to reference strains for identification and characterization. Results: An exploratory investigation of arthropod-borne pathogens in yellow flies collected at a residence in northeast Florida revealed the presence of uncharacterized Bartonella species DNA sequences similar to ones previously detected in two lone star ticks from Virginia. Subsequent testing of several lone star ticks from the area detected similar sequences of Bartonella in three ticks. Testing of stored blood samples from a resident of the site, who had experienced chronic relapsing and remitting symptoms for over a decade, identified nearly identical Bartonella DNA sequences in multiple samples collected over a 10-year period. Two lone star ticks and several samples from the same patient and time period also tested positive for Bo. burgdorferi DNA, suggesting possible long-term coinfection of the patient with both organisms. Conclusion: This investigation identified highly similar Bartonella DNA sequences in yellow flies, lone star ticks, and a human patient in northeast Florida. Similarly, Bo. burgdorferi DNA was detected in two lone star ticks and multiple specimens from the patient. Positive PCR results from archived patient blood samples documented the presence of both organisms at multiple time points over more than a decade. More studies on human patients with chronic undefined illness and on the presence of Bartonella and Bbsl in hematophagous arthropods and animal hosts in the southeastern United States are needed.

Specific mRNA lipid nanoparticles and acquired resistance to ticks.

Acquired resistance to ticks can develop when animals are repeatedly exposed to ticks. Recently, acquired resistance to Ixodes scapularis was induced in guinea pigs immunized with an mRNA-lipid nanoparticle vaccine (19ISP) encoding 19 I. scapularis proteins. Here, we evaluated specific mRNAs present in 19ISP to identify critical components associated with resistance to ticks. A lipid nanoparticle containing 12 mRNAs which included all the targets within 19ISP that elicited strong humoral responses in guinea pigs, was sufficient to induce robust resistance to ticks. Lipid nanoparticles containing fewer mRNAs or a single mRNA were not able to generate strong resistance to ticks. All lipid nanoparticles containing salp14 mRNA, however, were associated with increased redness at the tick bite site - which is the first manifestation of acquired resistance to ticks. This study demonstrates that more than one I. scapularis target within 19ISP is required for resistance to ticks, and that additional targets may also play a role in this process.