Dataset Information

Functional characterisation of a nicotinic acetylcholine receptor ? subunit from the brown dog tick, Rhipicephalus sanguineus.

ABSTRACT: Ticks and tick-borne diseases have a major impact on human and animal health worldwide. Current control strategies rely heavily on the use of chemical acaricides, most of which target the CNS and with increasing resistance, new drugs are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are targets of highly successful insecticides. We isolated a full-length nAChR ? subunit from a normalised cDNA library from the synganglion (brain) of the brown dog tick, Rhipicephalus sanguineus. Phylogenetic analysis has shown this R. sanguineus nAChR to be most similar to the insect ?1 nAChR group and has been named Rsan?1. Rsan?1 is distributed in multiple tick tissues and is present across all life-stages. When expressed in Xenopus laevis oocytes Rsan?1 failed to function as a homomer, with and without the addition of either Caenorhabditis elegans resistance-to-cholinesterase (RIC)-3 or X. laevis RIC-3. When co-expressed with chicken ?2 nAChR, Rsan?1 evoked concentration-dependent, inward currents in response to acetylcholine (ACh) and showed sensitivity to nicotine (100 ?M) and choline (100 ?M). Rsan?1/?2 was insensitive to both imidacloprid (100 ?M) and spinosad (100 ?M). The unreliable expression of Rsan?1 in vitro suggests that additional subunits or chaperone proteins may be required for more robust expression. This study enhances our understanding of nAChRs in arachnids and may provide a basis for further studies on the interaction of compounds with the tick nAChR as part of a discovery process for novel acaricides.

SUBMITTER: Lees K

PROVIDER: S-EPMC4029082 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus.

Lees Kristin K Jones Andrew K AK Matsuda Kazuhiko K Akamatsu Miki M Sattelle David B DB Woods Debra J DJ Bowman Alan S AS

International journal for parasitology 20131201 1

Ticks and tick-borne diseases have a major impact on human and animal health worldwide. Current control strategies rely heavily on the use of chemical acaricides, most of which target the CNS and with increasing resistance, new drugs are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are targets of highly successful insecticides. We isolated a full-length nAChR α subunit from a normalised cDNA library from the synganglion (brain) of the brown dog tick, Rhipicephalus sanguineus. Phyl ...[more]

PMID: 24291321

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Project description:Rhipicephalus sanguineus, known as the brown dog tick, is a common ectoparasite of domestic dogs and can be found worldwide. R.sanguineus is recognized as the primary vector of the etiological agent of canine monocytic ehrlichiosis and canine babesiosis. Here we present the first description of a R. sanguineus salivary gland transcriptome by the production and analysis of 2,034 expressed sequence tags (EST) from two cDNA libraries, one consctructed using mRNA from dissected salivary glands from female ticks fed for 3-5 days (early to mid library, RsSGL1) and the another from ticks fed for 5 days (mid library, RsSGL2), identifying 1,024 clusters of related sequences.Based on sequence similarities to nine different databases, we identified transcripts of genes that were further categorized according to function. The category of putative housekeeping genes contained approximately 56% of the sequences and had on average 2.49 ESTs per cluster, the secreted protein category contained 26.6% of the ESTs and had 2.47 EST's/clusters, while 15.3% of the ESTs, mostly singletons, were not classifiable, and were annotated as "unknown function". The secreted category included genes that coded for lipocalins, proteases inhibitors, disintegrins, metalloproteases, immunomodulatory and antiinflammatory proteins, as Evasins and Da-p36, as well as basic-tail and 18.3 kDa proteins, cement proteins, mucins, defensins and antimicrobial peptides. Comparison of the abundance of ESTs from similar contigs of the two salivary gland cDNA libraries allowed the identification of differentially expressed genes, such as genes coding for Evasins and a thrombin inhibitor, which were over expressed in the RsSGL1 (early to mid library) versus RsSGL2 (mid library), indicating their role in inhibition of inflammation at the tick feeding site from the very beginning of the blood meal. Conversely, sequences related to cement (64P), which function has been correlated with tick attachment, was largely expressed in the mid library.Our survey provided an insight into the R. sanguineus sialotranscriptome, which can assist the discovery of new targets for anti-tick vaccines, as well as help to identify pharmacologically active proteins.

Project description:AIM:Rickettsioses have an epidemiological importance that includes pathogens, vectors, and hosts. The dog tick Rhipicephalus sanguineus and the camel tick Hyalomma dromedarii play important roles as vectors and reservoirs of Rickettsiae. The aim of this study was to determine the prevalence of Rickettsiae in ixodid ticks species infesting dogs and camels in Egypt, in addition to, the morphological and molecular identification of R. sanguineus and H. dromedarii. MATERIALS AND METHODS:A total of 601 and 104 of ticks' specimens were collected from dogs and camels, respectively, in Cairo, Giza and Sinai provinces. Hemolymph staining technique and OmpA and gltA genes amplification were performed to estimate the prevalence rate of Rickettsiae in ticks. For morphological identification of tick species, light microscope (LM) and scanning electron microscope (SEM) were used. In addition to the phylogenetic analyses of 18S rDNA, Second internal transcript spacer, 12S rDNA, cytochrome c oxidase subunit-1, and 16S rDNA were performed for molecular identification of two tick species. RESULTS:The prevalence rate of Rickettsiae in ticks was 11.6% using hemolymph staining technique and 6.17% by OmpA and gltA genes amplification. Morphological identification revealed that 100% of dogs were infested by R. sanguineus while 91.9% of camels had been infested by H. dromedarii. The phylogenetic analyses of five DNA markers confirmed morphological identification by LM and SEM. The two tick species sequences analyses proved 96-100% sequences identities when compared with the reference data in Genbank records. CONCLUSION:The present studies confirm the suitability of mitochondrial DNA markers for reliable identification of ticks at both intra- and inter-species level over the nuclear ones. In addition to, the detection of Rickettsiae in both ticks' species and establishment of the phylogenetic status of R. sanguineus and H. dromedarii would be useful in understanding the epidemiology of ticks and tick borne rickettsioses in Egypt.

Project description:Tick salivary glands secrete a complex saliva into their hosts which modulates vertebrate hemostasis, immunity and tissue repair mechanisms. Transcriptomic studies revealed a large number of transcripts coding for structural and secreted protein products in a single tick species. These transcripts are organized in several large families according to their products. Not all transcripts are expressed at the same time, transcription profile switches at intervals, characterizing the phenomenon of "sialome switching". In this work, using transcriptomic and proteomic analysis we explored the sialome of Rhipicephalus sanguineus (s.l.) adult female ticks feeding on a rabbit. The correlations between transcriptional and translational results in the different groups were evaluated, confirming the "sialome switching" and validating the idea that the expression switch may serve as a mechanism of escape from the host immunity. Recombination breakpoints were identified in lipocalin and metalloprotease families, indicating this mechanism could be a possible source of diversity in the tick sialome. Another remarkable observation was the identification of host-derived proteins as a component of tick salivary gland content. These results and disclosed sequences contribute to our understanding of tick feeding biology, to the development of novel anti-tick methods, and to the discovery of novel pharmacologically active products. SIGNIFICANCE: Ticks are a burden by themselves to humans and animals, and vectors of viral, bacterial, protozoal and helminthic diseases. Their saliva has anti-clotting, anti-platelet, vasodilatory and immunomodulatory activities that allows successful feeding and pathogen transmission. Previous transcriptomic studies indicate ticks to have over one thousand transcripts coding for secreted salivary proteins. These transcripts code for proteins of diverse families, but not all are transcribed simultaneously, but rather transiently, in a succession. Here we explored the salivary transcriptome and proteome of the brown dog tick, Rhipicephalus sanguineus. A protein database of over 20 thousand sequences was "de novo" assembled from over 600 million nucleotide reads, from where over two thousand polypeptides were identified by mass spectrometry. The proteomic data was shown to vary in time with the transcription profiles, validating the idea that the expression switch may serve as a mechanism of escape from the host immunity. Analysis of the transcripts coding for lipocalin and metalloproteases indicate their genes to contain signals of breakpoint recombination suggesting a new mechanism responsible for the large diversity in tick salivary proteins. These results and the disclosed sequences contribute to our understanding of the success ticks enjoy as ectoparasites, to the development of novel anti-tick methods, and to the discovery of novel pharmacologically active products.

Project description:BackgroundThe brown dog tick Rhipicephalus sanguineus (sensu stricto) is reputed to be the most widespread tick of domestic dogs worldwide and has also been implicated in the transmission of many pathogens to dogs and humans. For more than two centuries, Rh. sanguineus (s.s.) was regarded as a single taxon, even considering its poor original description and the inexistence of a type specimen. However, genetic and crossbreeding experiments have indicated the existence of at least two distinct taxa within this name: the so-called "temperate" and "tropical" lineages of Rh. sanguineus (sensu lato). Recent genetic studies have also demonstrated the existence of additional lineages of Rh. sanguineus (s.l.) in Europe and Asia. Herein, we assessed the biological compatibility between two lineages of Rh. sanguineus (s.l.) found in southern Europe, namely Rhipicephalus sp. I (from Italy) and Rhipicephalus sp. II (from Portugal).MethodsTicks morphologically identified as Rh. sanguineus (s.l.) were collected in southern Portugal and southern Italy. Tick colonies were established and crossbreeding experiments conducted. Morphological, biological and genetic analyses were conducted.ResultsCrossbreeding experiments confirmed that ticks from the two studied lineages were able to mate and generate fertile hybrids. Hybrid adult ticks always presented the same genotype of the mother, confirming maternal inheritance of mtDNA. However, larvae and nymphs originated from Rhipicephalus sp. I females presented mtDNA genotype of either Rhipicephalus sp. I or Rhipicephalus sp. II, suggesting the occurrence of paternal inheritance or mitochondrial heteroplasmy. While biologically compatible, these lineages are distinct genetically and phenotypically.ConclusionsThe temperate lineages of Rh. sanguineus (s.l.) studied herein are biologically compatible and genetic data obtained from both pure and hybrid lines indicate the occurrence of paternal inheritance or mitochondrial heteroplasmy. This study opens new research avenues and raises question regarding the usefulness of genetic data and crossbreeding experiments as criteria for the definition of cryptic species in ticks.

Dataset Information

Functional characterisation of a nicotinic acetylcholine receptor ? subunit from the brown dog tick, Rhipicephalus sanguineus.

Publications

Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus.

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