**1. Introduction**

Small non-coding RNAs (sncRNAs) regulate genes at the post-transcriptional level in animals, plants, and arthropods, including ticks [1–5]. MicroRNAs (miRNAs) are a class of sncRNA, between 18 and 25 nucleotides in length, and they are now known to be important in arthropod immunity and host–pathogen interactions through their involvement in several cellular processes, including development, immunity, and pathogen responses in arthropods [2,6–10]. In animals, miRNAs regulate post-transcriptional gene expression, most often by binding to the 30 -untranslated region (30 -UTR) of target genes. While perfect complementarity of 2–8 nucleotides at the 50 end of the miRNA (seed region) is necessary for miRNA regulation, the remaining sequence can harbor mismatches or bulges [2,11,12]. miRNAs are transcribed as primary miRNA transcripts before processing by Drosha and Pasha proteins into pre-miRNAs. These are then exported to the cytoplasm and processed

**Citation:** Kumar, D.; Downs, L.P.; Embers, M.; Flynt, A.S.; Karim, S. Identification of microRNAs in the Lyme Disease Vector *Ixodes scapularis*. *Int. J. Mol. Sci.* **2022**, *23*, 5565. https://doi.org/10.3390/ijms 23105565

Academic Editors: Denis Sereno and Michail Kotsyfakis

Received: 14 April 2022 Accepted: 10 May 2022 Published: 16 May 2022

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by Dicer into mature miRNAs, which are loaded onto the microRNA-induced silencing complex (miRISC) before targeting complementary mRNA for degradation [13,14].

Several proteomic and transcriptomic studies have identified differentially expressed transcripts and proteins in uninfected and infected ticks [1,15–18], but few have investigated the role of pathogens in the differential modulation of the post-transcriptional tick and host machinery. Although there are over 800 tick species and despite their importance as vectors of human and animal diseases, ticks are underrepresented in available miRNA resources. For example, the miRbase database contains 49 *Ixodes scapularis* miRNAs and 24 *Rhipicephalus microplus* miRNAs, while MirGeneDB 2.1 contains 64 *Ixodes scapularis* miRNAs. However, small RNA sequencing (RNA-seq) and computational approaches are accelerating the discovery of miRNAs from species with incomplete genome sequencing, assembly, and annotation.

*Ixodes scapularis* is a primary vector of human pathogens, including the Lyme disease agent *Borrelia burgdorferi*, which infects vertebrates and ticks through evolved complex mechanisms. There is now a good understanding of tick immune pathways and their interactions with *B. burgdorferi* [19–21], but it is still uncertain how *B. burgdorferi* avoids clearance. *B. burgdorferi* must traverse tick salivary glands during transmission [22]. Since saliva/salivary gland proteins can enhance *B. burgdorferi* transmission into the vertebrate host, characterization of molecular interactions at the tick-bite site and the tick salivary glands is expected to facilitate vaccine development [23,24], since promoting immunity against tick salivary proteins could neutralize tick bites and pathogen transmission. Once *B. burgdorferi* is acquired by ticks from infected hosts, it resides in the tick gut and only migrates to the salivary gland during subsequent blood feeding, which generally lasts for 3–7 days [25–28]. While it is known that other tick-borne pathogens, such as *Anaplasma marginale*, must replicate inside salivary glands for efficient transmission [29], the details of *B. burgdorferi* replication are less well understood. Indeed, borrelial spirochetes invade the tick salivary gland via an unknown mechanism [22] and might be carried to the host dermis via tick saliva. Several salivary gland genes are upregulated in *B. burgdorferi*-infected *Ixodes scapularis* nymphs compared with uninfected ones [30], suggesting a significant role for salivary gland gene regulation in *B. burgdorferi* infection and transmission.

To fill the knowledge gap on miRNA expression in *Ixodes scapularis* salivary glands, here, we performed miRNA profiling of partially fed *B. burgdorferi*-infected and uninfected tick salivary glands to identify miRNAs that might play a role in *B. burgdorferi* survival, colonization, transmission, and host immunomodulation. In doing so, we detected 254 miR-NAs, of which 25 were high confidence miRNAs and 51 low confidence miRNAs. Forty-one of the identified miRNAs were present as *I. scapularis* miRNAs in miRBase (v22.1). Gene ontology and network analysis of target genes of differentially expressed miRNAs have predicted roles in metabolic and cellular development, cellular component biogenesis, and biological regulation processes. Several KEGG pathways, including sphingolipid metabolism; valine, leucine and isoleucine degradation; lipid transport and metabolism; exosome biogenesis and secretion; and phosphate-containing compound metabolic processes, were predicted as targets of differentially expressed miRNAs.

#### **2. Materials and Methods**

#### *2.1. Ethics Statement*

All animal experiments were performed in strict accordance with the recommendations in the NIH Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of the University of Southern Mississippi approved the protocol for blood feeding of field-collected ticks (protocol # 15101501.1).

#### *2.2. Ticks and Tissue Dissections*

Ticks were purchased from the Oklahoma State University Tick-Rearing Facility. Adult male and female *I. scapularis* were kept according to standard practices [31] and maintained in the laboratory as described in our previously published work [32,33]. Unfed female adult *I. scapularis* were infected with laboratory-grown *B. burgdorferi* strain B31.5A19 using the capillary feeding method at Tulane National Primate Research Center [34]. *Borrelia*-infected and uninfected ticks (*n* = 45 in each group) were placed on each ear of a rabbit host for tick blood feeding. Blood-fed adult female *I. scapularis* were dissected within 60 min of removal from the rabbit. Tick tissues were dissected and washed in M-199 buffer as described previously [35]. Salivary glands and midguts from individual *I. scapularis* were stored in RNAlater (Life Technologies, Carlsbad, CA, USA) at −80 ◦C until use.

#### *2.3. RNA Isolation, cDNA Synthesis, and PCR-Based B. burgdorferi Detection in Tick Tissues*

The TRIzol method was used for RNA extraction from individually dissected midgut tissues, and cDNA was synthesized as described previously [36,37]. *B. burgdorferi* was detected in tick midguts using the *flaB* gene in a PCR assay [36,38]. After testing for *B. burgdorferi* infection in tick midguts, the corresponding salivary gland tissues from the same uninfected/infected ticks (*n* = 10 salivary glands from each group) were pooled in separate tubes and RNA isolated using the TRIzol method [37].

#### *2.4. Small RNA Sequencing (RNA-Seq)*

Small RNA libraries were made using the Illumina TruSeq Kit following the manufacturer's protocol (Illumina, San Diego, CA, USA). Briefly, short adapter oligonucleotides were ligated to each end of the small RNAs in the sample, cDNA made with reverse transcriptase, and PCR used to add sample-specific barcodes and Illumina sequencing adapters. The final concentration of all sequencing libraries was determined using a Qubit fluorometric assay (Thermo Fisher Scientific, Waltham, MA, USA), and the DNA fragment size of each library was assessed using a DNA 1000 high-sensitivity chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). After purification by polyacrylamide gel electrophoresis, sample libraries were pooled and sequenced on an Illumina NextSeq 500 (single end 36 base) using the TruSeq SBS kit v3 (Illumina, San Diego, CA, USA) and protocols defined by the manufacturer. Four small RNA libraries of clean and *B. burgdorferi*-infected, partially fed, and pooled salivary glands were sequenced. RNA library preparation and indexing were performed at the University of Mississippi Medical Center (UMMC) sequencing facility. The small RNA sequencing data used in this study was deposited in the National Center for Biotechnology Information (NCBI) under the Sequence Read Archive (SRA) accession number PRJNA837369.

#### *2.5. Data Analysis*

A schematic of the experimental plan and data analysis is shown in Supplementary Figure S1. miRDeep2 v.2.0.0.8 [39,40] was used to process RNA-seq data. To predict novel miRNAs, the reads from all samples were combined. The mapper function in miRDeep2 first trims the adapter sequences from the reads and converts the read files from FASTQ to FASTA format. Reads shorter than 18 bases were discarded and the remaining reads mapped to the *I. scapularis* reference genome using default miRDeep2 mapper function parameters. Reads mapping to the genome were used to predict novel miRNAs. The *Drosophila melanogaster* genome was also used as another reference genome, and mapped reads were aligned to available miRNAs of *D. melanogaster* in miRBase (v22) and quantified. Reads were mapped to the reference genomes of *D. melanogaster* and *I. scapularis* and locations of potential miRNA read accumulations identified. The regions immediately surrounding the mapped reads were examined for miRNA biogenesis features including mature miRNAs, star and precursor reads, and stem-loop folding properties. miRDeep2 models the miRNA biogenesis pathway and uses a probabilistic algorithm to score compatibility of the position and frequency of sequencing reads with the secondary structure of the miRNA precursor.

For miRNA expression, a count table was generated using bedtools multicov, which counts alignments from indexed BAM files that overlap intervals in BED files provided from the miRDeep2 analysis.
