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. 2016 Feb 12;11(2):e0147966.
doi: 10.1371/journal.pone.0147966. eCollection 2016.

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

Rebekah L Bullard  1 , Jaclyn Williams  1 , Shahid Karim  1
Affiliations

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

Rebekah L Bullard et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Temporal expression of housekeeping genes.
Raw Ct values for the mRNA of seven A. americanum housekeeping genes from tick salivary glands (A) and midgut (B) throughout a blood meal (unfed–168 h). Each gene was amplified from each sample and the point at which the amplification curve crosses the threshold is considered the Ct.
Fig 2
Fig 2. Temporal expression of genes encoding protease inhibitor domains.
Six genes from two protein families are shown.
Fig 3
Fig 3. Temporal expression of lipocalin genes.
AamerSigP-18604 was not expressed at any time point, according to qRT–PCR.
Fig 4
Fig 4. Temporal expression of glycine-rich protein genes.
Fig 5
Fig 5. Temporal expression of seven tick-specific genes with unknown functions.
AamerSigp-22563 was not expressed, according to qRT–PCR.
Fig 6
Fig 6. Temporal expression of immunity-related genes.
Notably, gene AamerSigP-41992 was not expressed, according to qRT–PCR.
Fig 7
Fig 7. Temporal gene expression of a miscellaneous pool of protein families.
Aam-5252 was not expressed, according to qRT–PCR.
Fig 8
Fig 8. Temporal expression of seven metalloprotease genes encoding two protein families, measured with qRT–PCR.
Fig 9
Fig 9. Multiple sequence alignment of reprolysin MP nucleotide sequences identified for multiple-gene RNAi.
Knockdown was observed in four MPs. Nucleotides with more than 60% homology are highlighted in blue. Red underlining shows the locations of the primers. Actual primer sequences are listed in S3 Table.
Fig 10
Fig 10. RNA interference (RNAi)-mediated silencing of preprolysin metalloproteases.
(A) With RNAi, all members of a family of MP genes (Aam-41580, Aam-41579, AamerSigP-41680, and AamerSigP-35996) were successfully knocked down. Compensatory mechanisms were detected in response, with a three-fold up regulation of AamerSigP-20700 and a three-fold upregulation of the neprolysin AamerSigP-41953 gene. (B) Single RNAi Aam-41580 also resulted in a statistically significant reduction in Aam-41579 expression. No statistically significant compensatory mechanisms are shown. All knockdowns were 95% or more and * indicates P < 0.05 (statistically significant).
Fig 11
Fig 11. Engorgement weights of female ticks.
Tick engorgement weights (mg) from both Rep-MP RNAi and Aa-41580 RNAi ticks fed on sheep. Am-41580 RNAi knockdown caused a significant reduction in tick weights (P < 0.05) compared with the (No Treatment) control; weights were measured on day 10. The size of the ds-Lac-Z (irrelevant RNA)-treated sample was significantly reduced during feeding and was not included in the analysis. RNAi knockdown of the reprolysin family significantly reduced tick weight (P < 0.05) compared with both controls.
Fig 12
Fig 12. Larval hatching of tick eggs after ds-Reprolysin multiple gene knockdown.
Depletion of MP transcripts negatively affected the hatching of larvae, whereas no changes in the control samples were observed.
Fig 13
Fig 13. Analysis of microbial load in female salivary glands.
The salivary glands of ticks treated with both Rep-Family RNAi and Aam-41580 RNAi were assessed for their total bacterial loads on day 8 of feeding. MP depletion in the salivary glands caused a significant reduction in the total microbial load in ticks in which both the MP family and single genes were knocked down. Bacterial loads after both types of knockdown were 5–12-fold lower than those of the Lac-Z-RNAi-treated and no-treatment control salivary glands. *P < 0.05.
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