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.
Highlights
Amblyomma americanum, the Lone Star Tick, is widespread across the entire eastern seaboard of the USA and westward as far as central Texas, and it has begun to invade the central plains [1,2,3,4,5,6]
The salivary glands and midgut were analyzed separately to identify any differences in the time-dependent profiles of housekeeping gene expression during blood feeding
Reference genes were primarily selected by comparing their expression in multiple tissues and selecting the housekeeping gene most stably expressed in all tissues
Summary
The Lone Star Tick, is widespread across the entire eastern seaboard of the USA and westward as far as central Texas, and it has begun to invade the central plains [1,2,3,4,5,6]. The nonspecific and aggressive nature of A. americanum is very important to the veterinary and medical communities, as it is a vector of diseases such as spotted fever group rickettsiosis, human monocytic ehrlichiosis, southern-tick-associated rash illness, theileriosis, tularemia, Heartland virus infection, and infection with Tacaribe virus, newly discovered in this tick (for a review, see [1,7,8,9,10]). Tick feeding requires the insertion of the hypostome into the host’s skin and the formation of a blood pool beneath the dermis at the bite site. The molecules and proteins secreted into the host through the saliva are responsible for: 1) preventing clot formation, to maintain the blood pool; 2) blocking the host immune signaling molecules, to prevent immune cascades; 3) preventing the host inflammatory response, to reduce swelling, erythema, and localized pyrexia; and 4) the transmission of pathogens from the tick salivary glands to the host [14]. Even with increasing access to “big data”, these biomolecules require further functional characterization
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