Emergence of the zoonotic tick Dermacentor (Indocentor) auratus Supino, 1897 (Acari: Ixodidae) in Singapore.

Though ticks pose a significant public health risk, until recently, little research had focused on the diversity of ticks and tick-borne diseases in Singapore. To date, only fourteen tick species in five genera have been recorded there. For the first time, Dermacentor auratus is recorded from Singapore from a range of hosts, including humans. DNA sequences are provided at 2 loci, for D. auratus, the cytochrome c oxidase I (COI) for DNA barcoding and the 16S large subunit ribosomal RNA (16S lsu rRNA). The health risk posed by D. auratus in Singapore is discussed.

Molecular evidence for the inhibition of cytochrome p450s and cholinesterases in ticks by the repellent DEET.

For more than 50 years DEET (N,N-Diethyl-m-toluamide) has been considered the gold standard of repellents. It is applied to the skin or clothing to deter mosquitoes and other blood-sucking invertebrate pests from approaching and/or settling, and ultimately it provides temporary protection from bites. Despite rampant global use, surprisingly little is understood about DEET's mode of action and the molecular targets of the active ingredient. Furthermore, the theories into its mechanism for repellency are largely based off fruit fly and mosquito research. Since ticks possess a unique sensory structure, the Haller's organ, the specific genes and pathways associated with DEET avoidance may differ from insects. In these studies, we collected American dog ticks (Dermacentor variabilis) from four natural populations within Manitoba, Canada. We first carried out behavior assays, which showed DEET effectively repelled the ticks. RNA sequencing revealed that DEET caused a rapid and substantial reduction in the abundance of transcripts encoding cytochrome P450 and acetylcholinesterase genes, which gradually recovered over the 24 h time course. Finally, enzymatic kinetics provided functional support for DEET's role as an effective inhibitor of P450 s. While many facets of its mode of action remain to be worked out, our study provides valuable insights into the molecular underpinnings of DEET's repellence in ticks.

A Kunitz protease inhibitor from Dermacentor variabilis, a vector for spotted fever group rickettsiae, limits Rickettsia montanensis invasion.

A defining facet of tick-Rickettsia symbioses is the molecular strategy employed by each partner to ensure its own survival. Ticks must control rickettsial colonization to avoid immediate death. In the current study, we show that rickettsial abundance in the tick midgut increases once the expression of a Kunitz-type serine protease inhibitor from the American dog tick (Dermacentor variabilis) (DvKPI) is suppressed by small interfering RNA (siRNA). A series of in vitro invasion assays suggested that DvKPI limits rickettsial colonization during host cell entry. Interestingly, we observed that DvKPI associates with rickettsiae in vitro as well as in the tick midgut. Collectively, our data demonstrate that DvKPI limits host cell invasion by Rickettsia montanensis, possibly through an association with the bacterium.

Exploring the mialome of ticks: an annotated catalogue of midgut transcripts from the hard tick, Dermacentor variabilis (Acari: Ixodidae).

BACKGROUND: Ticks are obligate blood feeders. The midgut is the first major region of the body where blood and microbes ingested with the blood meal come in contact with the tick's internal tissues. Little is known about protein expression in the digestive tract of ticks. In this study, for analysis of global gene expression during tick attachment and feeding, we generated and sequenced 1,679 random transcripts (ESTs) from cDNA libraries from the midguts of female ticks at varying stages of feeding. RESULTS: Sequence analysis of the 1,679 ESTs resulted in the identification of 835 distinct transcripts, from these, a total of 82 transcripts were identified as proteins putatively directly involved in blood meal digestion, including enzymes involved in oxidative stress reduction/antimicrobial activity/detoxification, peptidase inhibitors, protein digestion (cysteine-, aspartic-, serine-, and metallo-peptidases), cell, protein and lipid binding including mucins and iron/heme metabolism and transport. A lectin-like protein with a high match to lectins in other tick species, allergen-like proteins and surface antigens important in pathogen recognition and/or antimicrobial activity were also found. Furthermore, midguts collected from the 6-day-fed ticks expressed twice as many transcripts involved in bloodmeal processing as midguts from unfed/2-day-fed ticks. CONCLUSION: This tissue-specific transcriptome analysis provides an opportunity to examine the global expression of transcripts in the tick midgut and to compare the gut response to host attachment versus blood feeding and digestion. In contrast to those in salivary glands of other Ixodid ticks, most proteins in the D. variabilis midgut cDNA library were intracellular. Of the total ESTs associated with a function, an unusually large number of transcripts were associated with peptidases, cell, lipid and protein binding, and oxidative stress or detoxification. Presumably, this is consistent with their role in intracellular processing of the blood meal and response to microbial infections. The presence of many proteins with similar functions is consistent with the hypothesis that gene duplication contributed to the successful adaptation of ticks to hematophagy. Furthermore, these transcripts may be useful to scientists investigating the role of the tick midgut in blood-meal digestion, antimicrobial activity or the transmission of tick-borne pathogens.

Functional characterization and novel rickettsiostatic effects of a Kunitz-type serine protease inhibitor from the tick Dermacentor variabilis.

Here we report the novel bacteriostatic function of a five-domain Kunitz-type serine protease inhibitor (KPI) from the tick Dermacentor variabilis. As ticks feed, they release anticoagulants, anti-inflammatory and immunosuppressive molecules that mediate the formation of the feeding lesion on the mammalian host. A number of KPIs have been isolated and characterized from tick salivary gland extracts. Interestingly, we observe little D. variabilis KPI gene expression in the salivary gland and abundant expression in the midgut. However, our demonstration of D. variabilis KPI's anticoagulant properties indicates that D. variabilis KPI may be important for blood meal digestion in the midgut. In addition to facilitating long-term attachment and blood meal acquisition, gene expression studies of Drosophila, legumes, and ticks suggest that KPIs play some role in the response to microbial infection. Similarly, in this study, we show that challenge of D. variabilis with the spotted fever group rickettsia, Rickettsia montanensis, results in sustained D. variabilis KPI gene expression in the midgut. Furthermore, our in vitro studies show that D. variabilis KPI limits rickettsial colonization of L929 cells (mouse fibroblasts), implicating D. variabilis KPI as a bacteriostatic protein, a property that may be related to D. variabilis KPI's trypsin inhibitory capability. This work suggests that anticoagulants play some role in the midgut during feeding and that D. variabilis KPI may be involved as part of the tick's defense response to rickettsiae.

Differential expression of two glutathione S-transferases identified from the American dog tick, Dermacentor variabilis.

Reciprocal signalling and gene expression play a cardinal role during pathogen-host molecular interactions and are prerequisite to the maintenance of balanced homeostasis. Gene expression repertoire changes during rickettsial infection and glutathione-S-transferases (GSTs) were among the genes found up-regulated in Rickettsia-infected Dermacentor variabilis. GSTs are well known to play an important part in cellular stress responses in the host. We have cloned two full-length GSTs from D. variabilis (DvGST1 and DvGST2). Comparison of these two DvGST molecules with those of other species indicate that DvGST1 is related to the mammalian class theta and insect class delta GSTs, while DvGST2 does not seem to fall in the same family. Northern blotting analyses revealed differential expression patterns, where DvGST1 and DvGST2 transcripts are found in the tick gut, with DvGST2 transcripts also present in the ovaries. Both DvGST transcripts are up-regulated upon tick feeding. Challenge of fed adult ticks with Escherichia coli injection showed decreased transcript amounts compared with ticks injected with phosphate-buffered saline (sham) and naïve ticks.

Immune-responsive lysozymes from hemocytes of the American dog tick, Dermacentor variabilis and an embryonic cell line of the Rocky Mountain wood tick, D. andersoni.

Immune-responsive lysozyme encoding cDNAs were identified from two medically important tick species by an expressed sequence tag approach of D. variabilis hemocytes (Dv Lys) and a D. andersoni embryonic derived cell line, DAE100. Comparative sequence analyses indicated the Dermacentor molecules to be products of orthologous genes and to be most similar to arthropod c-type lysozymes. Northern blotting analyses demonstrated that Dv Lys expression levels were most abundant in tick hemocytes and to a much lesser degree in the midgut while barely detectable in ovary, salivary gland, and Malpighian tubule tissues. Involvement of the Dermacentor c-type lysozymes in innate immunity was demonstrated by Escherichia coli challenges of D. variabilis ticks by injection resulting in a temporal profile of significantly elevated transcript abundances above those of naive controls that was similarly observed of the D. andersoni cells co-cultured with E. coli. In contrast to that reported of the digestive gut lysozyme of the soft tick Ornithodoros moubata, Dv Lys levels were not statistically differentially regulated by blood meal digestion. Additionally, given the differences in tissue distribution, sequence characteristics and phylogenetic placements between the Dermacentor and Ornithodoros lysozymes demonstrates that ticks possess differently adapted c-type lysozymes that are spatially and temporally differentially expressed.

Cloning and sequencing of putative acetylcholinesterase cDNAs from the American dog tick, Dermacentor variabilis, and the brown dog tick, Rhipicephalus sanguineus (Acari: Ixodidae).

Two putative cDNAs of acetylcholinesterase (AChE), one from Dermacentor variabilis, and the other from Rhipicephalus sanguineus, were amplified and sequenced. The deduced amino acid sequences have high amino acid identities (between 70 and 94%) to known tick AChE sequences deposited in GenBank. Furthermore, these two AChEs also possess common features in their primary AChE structure such as catalytic active sites. A 2,220-bp contiguous sequence, containing a 1,791-bp open reading frame encoding an AChE precursor with 596 amino acid residues, was obtained from D. variabilis. The deduced proteins of R. sanguineus are different in size by 6 amino acids because of alternative splicing at the 5' end. A gene tree deduced from phylogenetic analysis indicates that there are at least three lineages of AChE in arthropods.

Nitric oxide synthase and cGMP activity in the salivary glands of the American dog tick Dermacentor variabilis.

We colocalized nitric oxide synthase (NOS) activity in epithelial cells that surround the salivary gland duct in female Dermacentor variabilis with NADPH diaphorase histochemistry and immunohistochemistry using a polyclonal anti-endothelial NOS. Using size-exclusion chromatography, a fraction with a molecular mass of about 185 kDa that had diaphorase activity was eluted from tick salivary gland homogenate. This fraction converted arginine to citrulline with the production of nitric oxide (NO), which was detected by using electron spin resonance spectroscopy. The complete activity of the diaphorase fraction was dependent on NADPH, FAD, tetrahydrobiopterin, calmodulin, (CaM), and Ca(2+), but was not dependent on dithiothreitol. The arginine analog N(G)-monomethyl-L-arginine inhibited the activity of this fraction. NO and arginine activated soluble guanylate cyclase to produce cGMP in dopamine-stimulated isolated salivary glands. Dopamine-stimulated isolated salivary glands treated with tick saline containing either EDTA, the NOS inhibitor N(G)-nitro-L-arginine methyl ester, or the calcium/CaM binding inhibitor W-7 showed no increase in cGMP. The NO donor sodium nitroprusside significantly increased cGMP levels in unstimulated isolated salivary glands. A possible function for NO in salivation by this ixodid tick is discussed.

Assay for phenoloxidase activity in Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis.

Phenoloxidase activity in Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis was assayed. No phenoloxidase activity was detected compared to high activity through time in larvae of the greater wax moth (Galleria mellonella). We conclude that activated prophenoloxidase does not act as an opsonin in ixodid ticks tested to date.

Ultrastructural localization of acetylcholinesterase in the synganglion of the tick, Dermacentor variabilis (Say).

Light- and electron-microscopic enzyme cytochemistry was used to localize acetylcholinesterase (AChE) activity in the synganglion (brain) of the tick Dermacentor variabilis. High AChE activity was observed throughout the neuropil as well as adjacent to most neuronal perikarya. Intracellular activity was not observed by light microscopy. By electron microscopy, reaction product was localized at the plasma membrane of glia and neurons. Enzyme activity was not associated with the olfactory globuli neurons. In other types of neurons, small amounts of reaction product were observed in the Golgi apparatus and nuclear envelope. Large neurosecretory neurons contained activity that appeared to be associated with deep invaginations of the plasma membrane as well as intracellular membranes. AChE activity was also associated with processes of both neurons and glia. In most peripheral nerves AChE activity was associated with virtually all axons. Clearly then, AChE is associated with glia and non-cholinergic neurons as well as with presumed cholinergic neurons. The widespread localization and large amounts of AChE in the tick brain exceeds that reported for other invertebrates and vertebrates. As has been suggested for other animals, AChE in the tick brain may have functions in addition to its known role in cholinergic neurotransmission.

Expression, inheritance, and linkage relationships among eight enzyme genes in Dermacentor albipictus (Packard) (Acarina: Ixodidae).

Starch gel electrophoresis was used to examine the inheritance, expression, and linkage relationships among eight enzyme genes in the winter tick, Dermacentor albipictus. A fructose-specific hexokinase (FHK), adenylate kinase (ADK), and two forms of aconitase (ACON-A, ACON-C) appeared to have monomeric quaternary structures. A glycylleucine peptidase (PEP), isocitrate dehydrogenase (IDH), and anodally migrating malate dehydrogenase (MDH-A) were apparently dimers. The quaternary structure of glucose phosphate isomerase (GPI) could not be determined because of the similarity in relative mobility of the two available electromorphs. The genes for GPI, FHK, and ADK are located on the X chromosome in the following order: Adk - 37.4 - Gpi - 24.6 - Fhk, with Adk - Fhk being 46.5 map units apart. The remaining five genes were autosomally inherited. Of the 10 possible paired combinations of these genes, only the data for two pairs, Idh-Mdh (44.5% recombinants) and Acon-A--Acon-C (46.4% recombinants), suggested statistically significant linkage.