Steven W. Adamson

Knockdown of Selenocysteine-Specific Elongation Factor in Amblyomma maculatum Alters the Pathogen Burden of Rickettsia parkeri with Epigenetic Control by the Sin3 Histone Deacetylase Corepressor Complex

Selenocysteine is the 21st naturally-occurring amino acid. Selenoproteins have diverse functions and many remain uncharacterized, but they are typically associated with antioxidant activity. The incorporation of selenocysteine into the nascent polypeptide chain recodes the TGA stop codon and this process depends upon a number of essential factors including the selenocysteine elongation factor (SEF). The transcriptional expression of SEF did not change significantly in tick midguts throughout the blood meal, but decreased in salivary glands to 20% at the end of the fast feeding phase. Since selenoprotein translation requires this specialized elongation factor, we targeted this gene for knockdown by RNAi to gain a global view of the role selenoproteins play in tick physiology. We found no significant differences in tick engorgement and embryogenesis but detected no antioxidant capacity in tick saliva. The transcriptional profile of selenoproteins in R. parkeri-infected Amblyomma maculatum revealed declined activity of selenoprotein M and catalase and increased activity of selenoprotein O, selenoprotein S, and selenoprotein T. Furthermore, the pathogen burden was significantly altered in SEF-knockdowns. We then determined the global impact of SEF-knockdown by RNA-seq, and mapped huge shifts in secretory gene expression that could be the result of downregulation of the Sin3 histone deacetylase corepressor complex.

Choice of a Stable Set of Reference Genes for qRT-PCR Analysis in Amblyomma maculatum (Acari: Ixodidae)

ABSTRACT Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a widely used laboratory tool to quantify mRNA levels of target genes involved in various biological processes. The most commonly used method for analyzing qRT-PCR data are the normalizing technique where a housekeeping gene is used to determine the transcriptional regulation of the target gene. The choice of a reliable internal standard is pivotal for relative gene expression analysis to obtain reproducible results, especially when measuring small differences in transcriptional expression. In this study, we used geNorm, NormFinder, and BestKeeper programs to analyze the gene expression results using qRT-PCR. Five candidate reference genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), &bgr;-actin, &agr;-tubulin, elongation factor 1-&agr;, and glutathione s-transjerase, were used to evaluate the expression stability during prolonged blood-feeding on the vertebrate host. These five genes were evaluated in all life stages of Amblyomma maculatum (Koch) as well as in the salivary gland and midgut tissues of adult females to determine which are the most stably expressed gene for use in qRT-PCR studies. &bgr;-Actin is the most stably expressed gene in salivary glands and midguts of A. maculatum, and throughout all developmental stages both Actin and GAPDH were found to have the most stable expression with the lowest degree of variance. We recommend the use of &bgr;-actin and/or GAPDH as reference genes for qRT-PCR analysis of gene expression in A. maculatum.

Transcriptional activation of antioxidants may compensate for selenoprotein deficiencies in Amblyomma maculatum (Acari: Ixodidae) injected with selK- or selM-dsRNA

The Gulf‐Coast tick, Amblyomma maculatum, possesses an elaborate set of selenoproteins, which prevent the deleterious effects from oxidative stress that would otherwise occur during feeding. In the current work, we examined the role of selenoprotein K (SelK) and selenoprotein M (SelM) in feeding A. maculatum by bioinformatics, transcriptional gene expression, RNA interference and antioxidant assays. The transcriptional expression of SelK did not vary significantly in salivary glands or midguts throughout the bloodmeal. However, there was a 58‐fold increase in transcript levels of SelM in tick midguts. Ticks injected with selK‐dsRNA or selM‐dsRNA did not reveal any observable differences in egg viability but oviposition was reduced. Surprisingly, salivary antioxidant activity was higher in selenoprotein knockouts compared with controls, which is probably the result of compensatory transcriptional expression of genes involved in combating reactive oxygen species. In fact, quantitative real‐time PCR data suggest that the transcriptional expression of catalase increased in ticks injected with selM‐double‐stranded RNA. Additionally, the transcriptional expression of selN decreased ∼90% in both SelK/SelM knockdowns. These data indicate that SelK and SelM are salivary antioxidants but are not essential for tick survival or reproduction and are compensated by other antioxidant systems.

Molecular characterization and functional significance of the Vti family of SNARE proteins in tick salivary glands

Exocytosis involves membrane fusion between secretory vesicles and the plasma membrane. The Soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs) and their receptor proteins (SNAREs) interact to fuse vesicles with the membrane and trigger the release of their sialosecretome out of the tick salivary gland cells. In this study, we examined the functional significance of the Vti family of SNARE proteins of blood-feeding Amblyomma maculatum and Amblyomma americanum. Vti1A and Vti1B have been implicated in multiple functional roles in vesicle transport. QRT-PCR studies demonstrated that the highest transcriptional expression of vti1a and vti1b genes occurs in unfed salivary glands, suggesting that elevated secretory vesicle formation occurs prior to feeding but continues at low rates after blood feeding commences. Vti1A and Vti1B localize to the secretory vesicles in unfed tick salivary glands in immunofluorescence microscopy studies. Knockdown of vti1a and vti1b by RNA interference resulted in a significant decrease in the engorged tick weight compared to the control during prolonged blood-feeding on the host. RNA interference of vti1a or vti1b impaired oviposition and none of the ticks produced eggs masses. Surprisingly, the double knockdown did not produce a strong phenotype and ticks fed normally on the host and produced egg masses, suggesting a compensatory mechanism exists within the secretory system which may have been activated in the double knockdown. These results suggest an important functional role of the Vti family of SNARE proteins in tick blood feeding and ultimately oviposition. Understanding the basic functions of the Vti family of SNARE proteins in salivary glands may lead to better ways to prevent tick attachment and transmission of tick-borne diseases.

RNA Interference in Ticks: A Functional Genomics Tool for the Study of Physiology

Abstract Ticks are an example of an efficient ectoparasite that feeds on a variety of hosts including humans, domestic and wild animals. Tick salivary glands are critical to the biological success of ticks both during extended periods off the host and during the feeding period on the host. The salivary glands are also the sites of pathogen development and saliva is the route of transmission. The importance of multifunctional salivary glands to tick survival and vector competency makes the glands potential targets for intervention. RNA interference (RNAi) has the potential to revolutionize genetic manipulation and enhance the development of therapeutic and control agents in many arthropod-borne diseases and vectors. This reverse genetics tool has already been successfully used to study tick–host and tick–pathogen interactions. The translation of RNAi from an effective functional genomics tool into field application has been hindered by the challenge of delivering RNAi molecules to their target tissues by systemic administration. This review highlights the current status of RNAi in defining the physiological role of tick molecules in vector competence and vector–host interactions.

A study of ticks and tick-borne livestock pathogens in Pakistan

Background As obligate blood-feeding arthropods, ticks transmit pathogens to humans and domestic animals more often than other arthropod vectors. Livestock farming plays a vital role in the rural economy of Pakistan, and tick infestation causes serious problems with it. However, research on tick species diversity and tick-borne pathogens has rarely been conducted in Pakistan. In this study, a systematic investigation of the tick species infesting livestock in different ecological regions of Pakistan was conducted to determine the microbiome and pathobiome diversity in the indigenous ticks. Methodology/Principal findings A total of 3,866 tick specimens were morphologically identified as 19 different tick species representing three important hard ticks, Rhipicephalus, Haemaphysalis and Hyalomma, and two soft ticks, Ornithodorus and Argas. The bacterial diversity across these tick species was assessed by bacterial 16S rRNA gene sequencing using a 454-sequencing platform on 10 of the different tick species infesting livestock. The notable genera detected include Ralstonia, Clostridium, Staphylococcus, Rickettsia, Lactococcus, Lactobacillus, Corynebacterium, Enterobacter, and Enterococcus. A survey of Spotted fever group rickettsia from 514 samples from the 13 different tick species generated rickettsial-specific amplicons in 10% (54) of total ticks tested. Only three tick species Rhipicephalus microplus, Hyalomma anatolicum, and H. dromedarii had evidence of infection with “Candidatus Rickettsia amblyommii” a result further verified using a rompB gene-specific quantitative PCR (qPCR) assay. The Hyalomma ticks also tested positive for the piroplasm, Theileria annulata, using a qPCR assay. Conclusions/Significance This study provides information about tick diversity in Pakistan, and pathogenic bacteria in different tick species. Our results showed evidence for Candidatus R. amblyommii infection in Rhipicephalus microplus, H. anatolicum, and H. dromedarii ticks, which also carried T. annulata.

RNA Interference in Ticks

Abstract Ticks are an example of an efficient ectoparasite that feeds on a variety of hosts including humans, domestic and wild animals. Tick salivary glands are critical to the biological success of ticks both during extended periods off the host and during the feeding period on the host. The salivary glands are also the sites of pathogen development and saliva is the route of transmission. The importance of multifunctional salivary glands to tick survival and vector competency makes the glands potential targets for intervention. RNA interference (RNAi) has the potential to revolutionize genetic manipulation and enhance the development of therapeutic and control agents in many arthropod-borne diseases and vectors. This reverse genetics tool has already been successfully used to study tick–host and tick–pathogen interactions. The translation of RNAi from an effective functional genomics tool into field application has been hindered by the challenge of delivering RNAi molecules to their target tissues by systemic administration. This review highlights the current status of RNAi in defining the physiological role of tick molecules in vector competence and vector–host interactions.

Chapter 4 - RNA Interference in Ticks: A Functional Genomics Tool for the Study of Physiology

Abstract Ticks are an example of an efficient ectoparasite that feeds on a variety of hosts including humans, domestic and wild animals. Tick salivary glands are critical to the biological success of ticks both during extended periods off the host and during the feeding period on the host. The salivary glands are also the sites of pathogen development and saliva is the route of transmission. The importance of multifunctional salivary glands to tick survival and vector competency makes the glands potential targets for intervention. RNA interference (RNAi) has the potential to revolutionize genetic manipulation and enhance the development of therapeutic and control agents in many arthropod-borne diseases and vectors. This reverse genetics tool has already been successfully used to study tick–host and tick–pathogen interactions. The translation of RNAi from an effective functional genomics tool into field application has been hindered by the challenge of delivering RNAi molecules to their target tissues by systemic administration. This review highlights the current status of RNAi in defining the physiological role of tick molecules in vector competence and vector–host interactions.