Temporal Gene Expression Analysis and RNA Silencing of Single and Multiple Members of Gene Family in the Lone Star Tick Amblyomma americanum

Abstract

Saliva is an integral factor in the feeding success of veterinary and medically important ticks. Therefore, the characterization of the proteins present in tick saliva is an important area of tick research. Here, we confirmed previously generated sialotranscriptome data using quantitative real-time PCR. The information obtained in this in-depth study of gene expression was used to measure the effects of metalloprotease gene silencing on tick feeding. We analyzed the temporal expression of seven housekeeping genes and 44 differentially expressed salivary molecules selected from a previously published Amblyomma americanum sialotranscriptome. Separate reference genes were selected for the salivary glands and midgut from among the seven housekeeping genes, to normalize the transcriptional expression of differentially expressed genes. The salivary gland reference gene, ubiquitin, was used to normalize the expression of 44 salivary genes. Unsurprisingly, each gene family was expressed throughout the blood meal, but the expression of specific genes differed at each time point. To further clarify the complex nature of the many proteins found in the saliva, we disrupted the translation of several members of the metalloprotease family. Intriguingly, the nucleotide sequence similarity of the reprolysin metalloprotease gene family is so homologous that a single synthesized dsRNA sequence knocked down multiple members of the family. The use of multigene knockdown yielded a more significant picture of the role of metalloproteases in tick feeding success, and changes were observed in the female engorgement weight and larval hatching success. Interestingly, the depletion of metalloprotease transcripts also reduced the total number of bacteria present in the salivary glands. These data provide insight into the expression and functions of tick salivary proteins expressed while feeding on its host.

Sialotranscriptomics of Rhipicephalus zambeziensis reveals intricate expression profiles of secretory proteins and suggests tight temporal transcriptional regulation during blood-feeding

“…Further assessment of the expression regulation in the most differentially expressed secretory protein families (Lipocalin, Bovine pancreatic trypsin inhibitor, Reprolysin and Glycine rich), revealed similar patterns of intricate transcript up- and downregulation and a trend towards preferential expression of transcripts in a single time point in female salivary glands (data not shown). Correspondingly, members of multi-gene secretory protein families showed remarkable differential expression in the salivary glands of A. americanum [ 33 , 94 ] and I. ricinus [ 23 ], where certain transcripts were uniquely expressed during different developmental stages or feeding time points. Similar profiles were described in I. scapularis ticks based on proteomic analyses of saliva collected in 24-h intervals during feeding [ 92 ], indicating that transcriptomic profiles of salivary glands are transferable to the proteome being secreted into the host.…”

Tick attachment cement – reviewing the mysteries of a biological skin plug system

“…Nevertheless it became clear that the sheer quantity of proteins and other products of the salivary glands (Alarcon‐Chaidez, 2014) make assignment to particular functions such as cement formation difficult. To address this problem, temporary expression patterns of the salivary glands were analysed (Radulovic et al ., 2014; Kim et al ., 2016; Bullard, Williams & Karim, 2016), showing which salivary products were produced at certain points during feeding. These subsets of proteins were then tested for their functions by specific inactivation in living ticks and verification in feeding trials (Kim, Curran & Mulenga, 2014).…”

“…These subsets of proteins were then tested for their functions by specific inactivation in living ticks and verification in feeding trials (Kim, Curran & Mulenga, 2014). However, some proteins of salivary glands have very similar nucleotide sequences, hampering efforts at specific silencing (Bullard et al ., 2016). …”

“…It was suggested that the latter might be the result of degenerating processes of the salivary glands at that point (Kim et al ., 2016). Further analysis of tick cement composition used Fourier transform infrared spectroscopy attenuated total reflectance (FTIR‐ATR) to obtain information about the secondary structures of proteins at the surface of the cement cone (Bullard et al ., 2016). They observed significant differences between in vivo and in vitro collected cement cones.…”

Amblyomma maculatum SECIS binding protein 2 and putative selenoprotein P are indispensable for pathogen replication and tick fecundity

“…The methods used for RNA extraction from tick tissues (time points, RNAi tissues and eggs), cDNA synthesis and qRT-PCR have been published previously (Bullard et al, 2016). Transcriptional gene expression was normalized against the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.…”