The transcriptome of Ixodes scapularis salivary glands at single cell resolution
Applications of transcriptomics have significantly advanced the identification of numerous tick salivary molecules involved in feeding and the transmission of tick-borne pathogens. In this study, we analyze the salivary gland transcriptome of Ixodes scapularis, the main vector of Borrelia burgdorferi in the U.S., at single cell resolution. Overall design: The Ixodes scapularis ticks used in this study were obtained from the tick-rearing facility at Oklahoma State University (Stillwater, OK, USA). Prior to infestation, unfed ticks were maintained at 21°C and 85% relative humidity. One day before feeding, female ticks were paired with males to allow mating. Adult ticks were placed on the outer ear of four naïve female New Zealand rabbits, restricted by orthopedic stockinets that were glued in place. A total of 20 female and 20 male ticks (40 ticks per ear, 80 ticks per rabbit) were allowed to attach. To classify ticks by blood-feeding index, partially fed ticks were collected from the host based on engorgement size, sorted by weight, and divided into biological triplicates: an unfed group (UF) (15 females per replicate, weighing 1.6 mg in average), group G2 (5 females per replicate, weighing 7.2 mg in average), and group G5 (4 females per replicate, weighting 41.5 mg in average). After removal from the host, ticks were rinsed sequentially with 1% bleach, nuclease-free water, 70% ethanol, and a final rinse with nuclease-free water. Ticks were dissected within two hours of removal. Salivary glands (SGs) were isolated and stored in fresh, ice-cold, nuclease-free HBSS buffer (Invitrogen, Carlsbad, CA, USA). To prepare a single-cell suspension, the salivary glands were dissociated following a mechanical and enzymatic protocol. The resulting cell suspension was filtered through a 40 µm Flowmi cell strainer and centrifuged at 600 rcf for five minutes to pellet the cells. The pellet was then resuspended in an appropriate volume of HBSS media to achieve the target cell concentration. Cell counting and viability were assessed using trypan blue staining and a Neubauer chamber for visualization. The single cell suspensions were loaded (7,000 cells) onto a Chromium Controller for GEM (Gel Bead-In-EMulsion) generation in triplicate for three groups and barcoding using the Chromium Single Cell 3' Reagent Kits according to the manufacturer's protocol (10x Genomics). After cDNA synthesis, amplification, and library construction, the libraries were sequenced by the Illumina NovaSeq 6000 System. The raw sequencing data were demultiplexed and processed using the Cell Ranger Software Suite (10x Genomics). Cell Ranger was used to perform sample demultiplexing, barcode processing, and single-cell gene counting. The 'cellranger count' function was applied for alignment, filtering, barcode counting, and unique molecular identifier (UMI) counting. The genome of I. scapularis published in 2021 (University of Maryland) was used as the reference genome for the alignment.  Following generation of gene-cell matrices from Cell Ranger, further analysis was performed using Seurat v3, an R package for analysis of single cell RNAseq data. Quality control was not applied to exclude cells with high mitochondrial gene content, because mitochondrial genes were expected to be upregulated in feeding groups.  The gene expression matrix was then normalized and scaled. Variable features were identified using the 'vst' method in the 'FindVariableFeatures' function. Dimensionality reduction was performed using principal component analysis (PCA). The most significant principal components were selected for downstream analyses. Based on these components, shared nearest neighbor (SNN) graph-based clustering was performed. Clusters were visualized using Uniform Manifold Approximation and Projection (UMAP). For each cluster, differential gene expression was performed to identify cluster-specific marker genes. These markers were used to annotate cell clusters based on known marker genes for cell types.