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Targeted silencing of the Aquaporin 2 gene of Rhipicephalus (Boophilus) microplus reduces tick fitness.

ABSTRACT: Ticks are blood-feeding arthropods that can affect human and animal health both directly by blood-feeding and indirectly by transmitting pathogens. The cattle tick Rhipicephalus (Boophilus) microplus is one of the most economically important ectoparasites of bovines worldwide and it is responsible for the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. Aquaporins (AQPs) are water channel proteins implicated in physiological mechanisms of osmoregulation. Members of the AQP family are critical for blood-feeding arthropods considering the extreme osmoregulatory changes that occur during their feeding. We investigated the pattern of expression of a newly identified AQP2 gene of R. microplus (RmAQP2) in different tick tissues and stages. We also examined in vivo the biological implications of silencing expression of RmAQP2 silencing during tick feeding on either uninfected or B. bovis-infected cattle.In silico gene analyses were performed by multiple alignments of amino acid sequences and topology prediction. Levels of RmAQP2 transcripts in different tick tissues and stages were analyzed by reverse transcriptase quantitative PCR. Patterns of expression of RmAQP2 protein were investigated by immunoblots. Gene silencing was performed by RNA interference and in vivo functional analyses carried out by feeding ticks on either uninfected or B. bovis-infected cattle.RmAQP2 transcripts were found in unfed larvae, engorged nymphs, and salivary glands and guts of partially engorged females; however, of all tick tissues and stages examined, RmAQP2 protein was found only in salivary glands of partially engorged females. RmAQP2 silencing significantly reduced tick fitness and completely abrogated protein expression. The effect of RmAQP2 silencing on fitness was more pronounced in females fed on a B. bovis-infected calf than in ticks fed on an uninfected calf and none of their larval progeny survived.Collectively, considering the gene expression and tick fitness data, we conclude that RmAQP2 is critical for tick blood feeding and may be a suitable candidate target for the development of novel strategies to control R. microplus and tick-borne parasites.

SUBMITTER: Hussein HE

PROVIDER: S-EPMC4667534 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Targeted silencing of the Aquaporin 2 gene of Rhipicephalus (Boophilus) microplus reduces tick fitness.

Hussein Hala E HE Scoles Glen A GA Ueti Massaro W MW Suarez Carlos E CE Adham Fatma K FK Guerrero Felix D FD Bastos Reginaldo G RG

Parasites & vectors 20151202

<h4>Background</h4>Ticks are blood-feeding arthropods that can affect human and animal health both directly by blood-feeding and indirectly by transmitting pathogens. The cattle tick Rhipicephalus (Boophilus) microplus is one of the most economically important ectoparasites of bovines worldwide and it is responsible for the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. Aquaporins (AQPs) are water channel proteins implicated in physiological mechanisms o ...[more]

PMID: 26626727

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Project description:BackgroundRhipicephalus microplus is the vector of deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. However, R. microplus populations worldwide have developed resistance to available acaricides, prompting the search for novel acaricide targets. G protein-coupled receptors (GPCRs) are involved in the regulation of many physiological processes and have been suggested as druggable targets for the control of arthropod vectors. Arthropod-specific signaling systems of small neuropeptides are being investigated for this purpose. The pyrokinin receptor (PKR) is a GPCR previously characterized in ticks. Myotropic activity of pyrokinins in feeding-related tissues of Rhipicephalus sanguineus and Ixodes scapularis was recently reported.MethodsThe R. microplus pyrokinin receptor (Rhimi-PKR) was silenced through RNA interference (RNAi) in female ticks. To optimize RNAi, a dual-luciferase assay was applied to determine the silencing efficiency of two Rhimi-PKR double-stranded RNAs (dsRNA) prior to injecting dsRNA in ticks to be placed on cattle. Phenotypic variables of female ticks obtained at the endpoint of the RNAi experiment were compared to those of control female ticks (non-injected and beta-lactamase dsRNA-injected). Rhimi-PKR silencing was verified by quantitative reverse-transcriptase PCR in whole females and dissected tissues.ResultsThe Rhimi-PKR transcript was expressed in all developmental stages. Rhimi-PKR silencing was confirmed in whole ticks 4 days after injection, and in the tick carcass, ovary and synganglion 6 days after injection. Rhimi-PKR silencing was associated with an increased mortality and decreased weight of both surviving females and egg masses (P < 0.05). Delays in repletion, pre-oviposition and incubation periods were observed (P < 0.05).ConclusionsRhimi-PKR silencing negatively affected female reproductive fitness. The PKR appears to be directly or indirectly associated with the regulation of female feeding and/or reproductive output in R. microplus. Antagonists of the pyrokinin signaling system could be explored for tick control.

Project description:The success of cattle tick fixation largely depends on the secretion of substances that alter the immune response of the host. The majority of these substances are expressed by the parasite salivary gland and secreted in tick saliva. It is known that hosts can mount immune responses against ticks and bovine European breeds, and bovine industrial crossbreeds are more susceptible to infestations than are Bos indicus cattle. To identify candidates for the development of novel control strategies for the cattle tick Rhipicephalus (Boophilus) microplus, a salivary gland transcriptome analysis of engorged females fed on susceptible or resistant hosts was performed. Using RNA-Seq, transcriptomes were de novo assembled and produced a total of 235,451 contigs with 93.3% transcriptome completeness. Differential expression analysis identified 137 sequences as differentially expressed genes (DEGs) between ticks raised on tick-susceptible or tick-resistant cattle. DEGs predicted to be secreted proteins include innexins, which are transmembrane proteins that form gap junction channels; the transporters Na+/dicarboxylate, Na+/tricarboxylate, and phosphate transporter and a putative monocarboxylate transporter; a phosphoinositol 4-phosphate adaptor protein; a cysteine-rich protein containing a trypsin inhibitor-like (TIL) domain; a putative defense protein 3 containing a reeler domain; and an F-actin-uncapping protein LRRC16A with a CARMIL_C domain; these genes were upregulated in ticks fed on tick-susceptible cattle. DEGs predicted to be non-secreted proteins included a small heat shock protein and the negative elongation factor B-like, both acting in a coordinated manner to increase HSP transcript levels in the salivary glands of the ticks fed on tick-susceptible cattle; the 26S protease regulatory subunit 6B and another chaperone with similarity to calnexin, also upregulated in ticks fed on tick-susceptible cattle; an EF-hand calcium binding protein and a serine carboxypeptidase (SCP), both involved in the blood coagulation cascade and upregulated in ticks fed on tick-susceptible cattle; and two ribosomal proteins, the 60S acidic ribosomal protein P2 and the 60S ribosomal protein L19. These results help to characterize cattle tick salivary gland gene expression in tick-susceptible and tick-resistant hosts and suggest new putative targets for the control of tick infestations, as those genes involved in the mechanism of stress response during blood feeding.

Project description:BACKGROUND: The southern cattle tick, Rhipicephalus (Boophilus) microplus, is an economically important parasite of cattle and can transmit several pathogenic microorganisms to its cattle host during the feeding process. Understanding the biology and genomics of R. microplus is critical to developing novel methods for controlling these ticks. RESULTS: We present a global comparative genomic analysis of a gene index of R. microplus comprised of 13,643 unique transcripts assembled from 42,512 expressed sequence tags (ESTs), a significant fraction of the complement of R. microplus genes. The source material for these ESTs consisted of polyA RNA from various tissues, lifestages, and strains of R. microplus, including larvae exposed to heat, cold, host odor, and acaricide. Functional annotation using RPS-Blast analysis identified conserved protein domains in the conceptually translated gene index and assigned GO terms to those database transcripts which had informative BlastX hits. Blast Score Ratio and SimiTri analysis compared the conceptual transcriptome of the R. microplus database to other eukaryotic proteomes and EST databases, including those from 3 ticks. The most abundant protein domains in BmiGI were also analyzed by SimiTri methodology. CONCLUSION: These results indicate that a large fraction of BmiGI entries have no homologs in other sequenced genomes. Analysis with the PartiGene annotation pipeline showed 64% of the members of BmiGI could not be assigned GO annotation, thus minimal information is available about a significant fraction of the tick genome. This highlights the important insights in tick biology which are likely to result from a tick genome sequencing project. Global comparative analysis identified some tick genes with unexpected phylogenetic relationships which detailed analysis attributed to gene losses in some members of the animal kingdom. Some tick genes were identified which had close orthologues to mammalian genes. Members of this group would likely be poor choices as targets for development of novel tick control technology.

Project description:The NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to screen the resulting mRNA harvested from ticks treated with the tick Ubiquitin-63E 600bp dsRNA in comparison to controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A stringent bioinformatics analysis was undertaken to predict off target effects (OTE) resulting from the in silico dicing of the 600bp Ubiquitin-63E dsRNA which identified 10 down (including TC6372) and 13 up-regulated ESTs within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196bp and 109bp dsRNAs, and a cocktail of short hairpin RNAs targeting Ubiquitin-63E demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative real time PCR analysis confirmed TC6372 and predicted off target differential expression. Our study demonstrates the effectiveness and specificity for utilising ~100bp dsRNA products for targeted tick gene knockdown with nil identified off targets. The microarray was conducted by NimbleGen Systems Inc following the method reported by Saldivar [Saldivar L et al., Insect Mol Biol 2008, 17(6):597-606]. Briefly, Custom high-density single channel oligonucleotide arrays were constructed by NimbleGen Systems Inc. using 13 601 of the 13 643 members of BmiGI Version 2 with 14 perfect match 50-mer probes per BmiGI target. No mismatched probes were included on the arrays, although probes with randomly generated sequences were included. These random sequence probes were designed to match the melting temperature (Tm) of the other probes on the array and to reflect the distribution of non-specific signal intensities for binding events to probes with approximately the same composition as the perfect match probes but with random sequences. The associated GEO dataset can be found online at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL6373. Each microarray chip includes two in-slide replicates (spot replicates). In this experiment two biological replicates for each sample were hybridized to assess variability between individual samples. Each pooled sample consisted of 30 female adult ticks (N strain) as the source of RNA. Four technical replicates were processed, i.e. repeated measurements of the same pooled R. microplus mRNA, consisting of two chip replicates with two spot replicates. The chip replicates were hybridized on different dates to different chips, while spot replicates indicate the same probe was spotted on different locations on the chip. Four R. microplus samples were hybridized to separate microarrays at NimbleGen Systems Inc. (Madison, WI, USA). Data Processing: Statistical analysis Following array hybridization the raw intensity values were background corrected using convolution and normalized using quantile normalization to adjust for technical sources of variation. Final log2 expression intensities were generated using the Robust Multichip Average (RMA) algorithm [Irizarry et al.,Biostatistics 2003, 4:249-264]; [Groeneveld E, et al.,Proceedings 6th World Cong Genet Appl Livest Prod,: 1998; Armidale, NSW, Australia; 455-458] implemented in R. Differential expression was tested on the RMA normalized intensities using a mixed model of the form yijkr=?+BSrk+Gi+GTij+Eijkr where yijkr are the log2 RMA normalized signal intensities; i =gene, j=treatment, r=block, k=slide/array, the main fixed effect is BSrk (block by slide interaction), Gi is the main random effect of the gene; GTij is the random interaction term of the gene by treatment; E is just the error term; and normal assumptions for the random effects – iid are assumed. The model was fitted using VCE4.0 [Groeneveld E, et al., 1998]. Differentially expressed (DE) probes were considered as those which were three or more standard deviations away from the mean (p-value <0.001). The contrast of interest was the 600pb dsRNA microinjected ticks (R. microplus Ubiquitin-63E homologue as per Kurscheid et al 2009, BMC Molecular Biology 10:26) compared to normal untreated controls in order to examine the knockdown effect associated with phenotype. Kurscheid et al 2009 reported a strong knockdown phenotype following dsRNA knockdown and the subsequent differentially expressed ESTs were examined in this array experiment. The putative identities of the lists of up and down regulated tick sequences (DE probes) were then subsequently analysed using bioinformatic tools to predict function (described below). Bioinfomatics: Nimblegen tick microarray differentially expressed probes with fold changes >1.5 and p-value <0.001 were screened against the following databases on the CCG [Centre for Comparative Genomics: http://ccg.murdoch.edu.au], Grendel HPC system [Hunter A, et al.,Australasian Workshop on Grid Computing and e-Research 2005:2-3]; NCBI protein (nr and patent) [National Centre for Biotechnology Information: http://www.ncbi.nlm.nih.gov], String [Mering von C., et al.,Nucleic Acids Research 2006:00: D01-D05], COG [Tatusov R, et al., BMC Bioinformatics 2003, 4], tigr_bmigi.062608 [Wang et al.,BMC Genomics 8:368], NCBI Conserved Domain database [Goonesekere N, et al., Proteins 2008, 71(2):910-919], and KEGG (Kanehisa & Goto 2000 Nucleic Acids Res. 28, 27-30; Kanehisa et al 2006 Nucleic Acids Research 34, D354-D357; Kanehisa et al 2010 Nucleic Acids Research 38, D355-D360). Further analysis of differentially expressed transcripts identified in the microarray experiments was undertaken using the BLAST program suite (Altschul et al., 1997) for similarity searches in the UniProt database (TheUniProtConsortium, 2010 Nucleic Acids Res 38, D142-D148.) and in the protein sequence database of Ixodes scapularis tick (available from http://www.vectorbase.org/Ixodes_scapularis/Info/Index; last accessed 2010-11-21). The results of the blastx searches against the I. scapularis protein sequences where used to map the R. microplus ESTs to unique identifiers in the DAVID Bioinformatics Resource and perform a subsequent functional annotation to Gene Ontology terms in the categories “Biological Process (BP)”, “Cellular Component (CC)”, and “Molecular Functions (MF)” (Dennis et al 2003 Genome Biology 4: P3; Huang et al 2009 Nature Protoc. 4: 44-57.).

Project description:BackgroundCattle babesiosis is a tick-borne disease of cattle that has severe economic impact on cattle producers throughout the world's tropical and subtropical countries. The most severe form of the disease is caused by the apicomplexan, Babesia bovis, and transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus, with the most prevalent species being Rhipicephalus (Boophilus) microplus. We studied the reaction of the R. microplus larval transcriptome in response to infection by B. bovis.MethodsTotal RNA was isolated for both uninfected and Babesia bovis-infected larval samples. Subtracted libraries were prepared by subtracting the B. bovis-infected material with the uninfected material, thus enriching for expressed genes in the B. bovis-infected sample. Expressed sequence tags from the subtracted library were generated, assembled, and sequenced. To complement the subtracted library method, differential transcript expression between samples was also measured using custom high-density microarrays. The microarray probes were fabricated using oligonucleotides derived from the Bmi Gene Index database (Version 2). Array results were verified for three target genes by real-time PCR.ResultsTicks were allowed to feed on a B. bovis-infected splenectomized calf and on an uninfected control calf. RNA was purified in duplicate from whole larvae and subtracted cDNA libraries were synthesized from Babesia-infected larval RNA, subtracting with the corresponding uninfected larval RNA. One thousand ESTs were sequenced from the larval library and the transcripts were annotated. We used a R. microplus microarray designed from a R. microplus gene index, BmiGI Version 2, to look for changes in gene expression that were associated with infection of R. microplus larvae. We found 24 transcripts were expressed at a statistically significant higher level in ticks feeding upon a B. bovis-infected calf contrasted to ticks feeding on an uninfected calf. Six transcripts were expressed at a statistically significant lower level in ticks feeding upon a B. bovis-infected calf contrasted to ticks feeding on an uninfected calf.ConclusionOur experimental approaches yielded specific differential gene expression associated with the infection of R. microplus by B. bovis. Overall, an unexpectedly low number of transcripts were found to be differentially expressed in response to B. bovis infection. Although the BmiGI Version 2 gene index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=b_microplus) was a useful database to help assign putative function to some transcripts, a majority of the differentially expressed transcripts did not have annotation that was useful for assignment of function and specialized bioinformatic approaches were necessary to increase the information from these transcriptome experiments.

Dataset Information

Targeted silencing of the Aquaporin 2 gene of Rhipicephalus (Boophilus) microplus reduces tick fitness.

Publications

Targeted silencing of the Aquaporin 2 gene of Rhipicephalus (Boophilus) microplus reduces tick fitness.

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