Global comparative analysis of ESTs from the southern cattle tick, Rhipicephalus (Boophilus) microplus

<p>Abstract</p> <p>Background</p> <p>The southern cattle tick, <it>Rhipicephalus (Boophilus) microplus</it>, 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 <it>R. microplus </it>is critical to developing novel methods for controlling these ticks.</p> <p>Results</p> <p>We present a global comparative genomic analysis of a gene index of <it>R. microplus </it>comprised of 13,643 unique transcripts assembled from 42,512 expressed sequence tags (ESTs), a significant fraction of the complement of <it>R. microplus </it>genes. The source material for these ESTs consisted of polyA RNA from various tissues, lifestages, and strains of <it>R. microplus</it>, 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 <it>R. microplus </it>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.</p> <p>Conclusion</p> <p>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.</p

Distribution patterns of three sodium channel mutations associated with pyrethroid resistance in Rhipicephalus (Boophilus) microplus populations from North and South America, South Africa and Australia

AbstractResistance to synthetic pyrethroids (SP) in the cattle tick Rhipicephalus (Boophilus) microplus is widespread throughout its distribution area. Three single nucleotide substitutions identified in Domains II and III of the sodium channel gene of R. (B.) microplus are known to be associated with target site pyrethroid resistance. We developed a multiplex PCR using allele-specific primers to amplify wild type or mutated genotypes of the three mutations simultaneously. This assay was used to screen tick samples originating from Brazil, Argentina, Mexico, South Africa and Australia whose phenotype to flumethrin and cypermethrin had been determined by the use of the Larval Tarsal test (LTT) or the Larval Packet Test (LPT). These mutations were found to have distinct geographical distributions and result in different resistance phenotypes. The L64I Domain II mutation conferring resistance to several SP compounds was found in all the Brazilian, Argentinean and Australian populations and in one South African population, with frequencies between 38% and 100% in flumethrin and cypermethrin resistant populations. In contrast, this mutation was not found in samples from Mexico, while the Domain III mutation was found exclusively in this country. The G72V Domain II flumethrin-specific mutation was found in a single Australian population, with a very low resistant allele frequency (3%). The homozygous resistant RR genotype of the L64I Domain II mutation correlated significantly with the survival rates at the discriminating doses of flumethrin and cypermethrin. This survey shows the widespread distribution of the L64I Domain II mutation and provides evidence of its geographic separation from the Domain III mutation

Anisotropic Strain Induced Soliton Movement Changes Stacking Order and Bandstructure of Graphene Multilayers

The crystal structure of solid-state matter greatly affects its electronic properties. For example in multilayer graphene, precise knowledge of the lateral layer arrangement is crucial, since the most stable configurations, Bernal and rhombohedral stacking, exhibit very different electronic properties. Nevertheless, both stacking orders can coexist within one flake, separated by a strain soliton that can host topologically protected states. Clearly, accessing the transport properties of the two stackings and the soliton is of high interest. However, the stacking orders can transform into one another and therefore, the seemingly trivial question how reliable electrical contact can be made to either stacking order can a priori not be answered easily. Here, we show that manufacturing metal contacts to multilayer graphene can move solitons by several $\mu$m, unidirectionally enlarging Bernal domains due to arising mechanical strain. Furthermore, we also find that during dry transfer of multilayer graphene onto hexagonal Boron Nitride, such a transformation can happen. Using density functional theory modeling, we corroborate that anisotropic deformations of the multilayer graphene lattice decrease the rhombohedral stacking stability. Finally, we have devised systematics to avoid soliton movement, and how to reliably realize contacts to both stacking configurations

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing

<p>Abstract</p> <p>Background</p> <p>Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, <it>Rhipicephalus </it>(<it>Boophilus</it>) <it>microplus</it>, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the <it>R. microplus </it>microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of <it>R. microplus </it>collected during outbreaks in southern Texas.</p> <p>Results</p> <p>Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include <it>Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum</it>, and <it>Finegoldia magna</it>. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include <it>Wolbachia</it>, <it>Coxiella</it>, and <it>Borrelia</it>. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with <it>R. microplus</it>.</p> <p>Conclusions</p> <p>This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with <it>R. microplus</it>. Additional reports on the identification of new bacterial species maintained in nature by <it>R. microplus </it>that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role <it>R. microplus </it>can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where <it>R. microplus </it>was eradicated.</p

Comparative microarray analysis of Rhipicephalus (Boophilus) microplus expression profiles of larvae pre-attachment and feeding adult female stages on Bos indicus and Bos taurus cattle

Background: Rhipicephalus (Boophilus) microplus is an obligate blood feeder which is host specific to cattle. Existing knowledge pertaining to the host or host breed effects on tick transcript expression profiles during the tick - host interaction is poor. Results: Global analysis of gene expression changes in whole R. microplus ticks during larval, pre-attachment and early adult stages feeding on Bos indicus and Bos taurus cattle were compared using gene expression microarray analysis. Among the 13,601 R. microplus transcripts from BmiGI Version 2 we identified 297 high and 17 low expressed transcripts that were significantly differentially expressed between R. microplus feeding on tick resistant cattle [Bos indicus (Brahman)] compared to R. microplus feeding on tick susceptible cattle [Bos taurus (Holstein-Friesian)] (p <= 0.001). These include genes encoding enzymes involved in primary metabolism, and genes related to stress, defence, cell wall modification, cellular signaling, receptor, and cuticle formation. Microarrays were validated by qRT-PCR analysis of selected transcripts using three housekeeping genes as normalization controls. Conclusion: The analysis of all tick stages under survey suggested a coordinated regulation of defence proteins, proteases and protease inhibitors to achieve successful attachment and survival of R. microplus on different host breeds, particularly Bos indicus cattle. R. microplus ticks demonstrate different transcript expression patterns when they encounter tick resistant and susceptible breeds of cattle. In this study we provide the first transcriptome evidence demonstrating the influence of tick resistant and susceptible cattle breeds on transcript expression patterns and the molecular physiology of ticks during host attachment and feeding

Integrated Strategy for Sustainable Cattle Fever Tick Eradication in USA is Required to Mitigate the Impact of Global Change

The ticks Rhipicephalus (Boophilus) annulatus and R. (B.) microplus, commonly known as cattle and southern cattle tick, respectively, impede the development and sustainability of livestock industries throughout tropical and other world regions. They affect animal productivity and wellbeing directly through their obligate blood-feeding habit and indirectly by serving as vectors of the infectious agents causing bovine babesiosis and anaplasmosis. The monumental scientific discovery of certain arthropod species as vectors of infectious agents is associated with the history of research on bovine babesiosis and R. annulatus. Together, R. microplus and R. annulatus are referred to as cattle fever ticks (CFT). Bovine babesiosis became a regulated foreign animal disease in the United States of America (U.S.) through efforts of the Cattle Fever Tick Eradication Program (CFTEP) established in 1906. The U.S. was declared free of CFT in 1943, with the exception of a permanent quarantine zone in south Texas along the border with Mexico. This achievement contributed greatly to the development and productivity of animal agriculture in the U.S. The permanent quarantine zone buffers CFT incursions from Mexico where both ticks and babesiosis are endemic. Until recently, the elimination of CFT outbreaks relied solely on the use of coumaphos, an organophosphate acaricide, in dipping vats or as a spray to treat livestock, or the vacation of pastures. However, ecological, societal, and economical changes are shifting the paradigm of systematically treating livestock to eradicate CFT. Keeping the U.S. CFT-free is a critical animal health issue affecting the economic stability of livestock and wildlife enterprises. Here, we describe vulnerabilities associated with global change forces challenging the CFTEP. The concept of integrated CFT eradication is discussed in reference to global change

Reassociation kinetics-based approach for partial genome sequencing of the cattle tick, Rhipicephalus (Boophilus) microplus

Background: The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence fiscally and technically problematic. To selectively obtain gene-enriched regions of this tick's genome, Cot filtration was performed, and Cot-filtered DNA was sequenced via 454 FLX pyrosequencing.Results: The sequenced Cot-filtered genomic DNA was assembled with an EST-based gene index of 14,586 unique entries where each EST served as a potential "seed" for scaffold formation. The new sequence assembly extended the lengths of 3,913 of the 14,586 gene index entries. Over half of the extensions corresponded to extensions of over 30 amino acids. To survey the repetitive elements in the tick genome, the complete sequences of five BAC clones were determined. Both Class I and II transposable elements were found. Comparison of the BAC and Cot filtration data indicates that Cot filtration was highly successful in filtering repetitive DNA out of the genomic DNA used in 454 sequencing.Conclusion: Cot filtration is a very useful strategy to incorporate into genome sequencing projects on organisms with large genome sizes and which contain high percentages of repetitive, difficult to assemble, genomic DNA. Combining the Cot selection approach with 454 sequencing and assembly with a pre-existing EST database as seeds resulted in extensions of 27% of the members of the EST database

Evolutionary conserved microRNAs are ubiquitously expressed compared to tick-specific miRNAs in the cattle tick Rhipicephalus (Boophilus) microplus

Background: MicroRNAs (miRNAs) are small non-coding RNAs that act as regulators of gene expression in eukaryotes modulating a large diversity of biological processes. The discovery of miRNAs has provided new opportunities to understand the biology of a number of species. The cattle tick, Rhipicephalus (Boophilus) microplus, causes significant economic losses in cattle production worldwide and this drives us to further understand their biology so that effective control measures can be developed. To be able to provide new insights into the biology of cattle ticks and to expand the repertoire of tick miRNAs we utilized Illumina technology to sequence the small RNA transcriptomes derived from various life stages and selected organs of R. microplus. Results: To discover and profile cattle tick miRNAs we employed two complementary approaches, one aiming to find evolutionary conserved miRNAs and another focused on the discovery of novel cattle-tick specific miRNAs. We found 51 evolutionary conserved R. microplus miRNA loci, with 36 of these previously found in the tick Ixodes scapularis. The majority of the R. microplus miRNAs are perfectly conserved throughout evolution with 11, 5 and 15 of these conserved since the Nephrozoan (640 MYA), Protostomian (620MYA) and Arthropoda (540 MYA) ancestor, respectively. We then employed a de novo computational screening for novel tick miRNAs using the draft genome of I. scapularis and genomic contigs of R. microplus as templates. This identified 36 novel R. microplus miRNA loci of which 12 were conserved in I. scapularis. Overall we found 87 R. microplus miRNA loci, of these 15 showed the expression of both miRNA and miRNA* sequences. R. microplus miRNAs showed a variety of expression profiles, with the evolutionary-conserved miRNAs mainly expressed in all life stages at various levels, while the expression of novel tick-specific miRNAs was mostly limited to particular life stages and/or tick organs. Conclusions: Anciently acquired miRNAs in the R. microplus lineage not only tend to accumulate the least amount of nucleotide substitutions as compared to those recently acquired miRNAs, but also show ubiquitous expression profiles through out tick life stages and organs contrasting with the restricted expression profiles of novel tick-specific miRNAs

Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

Background The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype. Results We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying. Conclusion We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects

The complexity of Rhipicephalus (Boophilus) microplus genome characterised through detailed analysis of two BAC clones

<p>Abstract</p> <p>Background</p> <p><it>Rhipicephalus (Boophilus) microplus (Rmi) </it>a major cattle ectoparasite and tick borne disease vector, impacts on animal welfare and industry productivity. In arthropod research there is an absence of a complete Chelicerate genome, which includes ticks, mites, spiders, scorpions and crustaceans. Model arthropod genomes such as <it>Drosophila </it>and <it>Anopheles </it>are too taxonomically distant for a reference in tick genomic sequence analysis. This study focuses on the <it>de-novo </it>assembly of two <it>R. microplus </it>BAC sequences from the understudied <it>R microplus </it>genome. Based on available <it>R. microplus </it>sequenced resources and comparative analysis, tick genomic structure and functional predictions identify complex gene structures and genomic targets expressed during tick-cattle interaction.</p> <p>Results</p> <p>In our BAC analyses we have assembled, using the correct positioning of BAC end sequences and transcript sequences, two challenging genomic regions. Cot DNA fractions compared to the BAC sequences confirmed a highly repetitive BAC sequence BM-012-E08 and a low repetitive BAC sequence BM-005-G14 which was gene rich and contained short interspersed elements (SINEs). Based directly on the BAC and Cot data comparisons, the genome wide frequency of the SINE Ruka element was estimated. Using a conservative approach to the assembly of the highly repetitive BM-012-E08, the sequence was de-convoluted into three repeat units, each unit containing an 18S, 5.8S and 28S ribosomal RNA (rRNA) encoding gene sequence (rDNA), related internal transcribed spacer and complex intergenic region.</p> <p>In the low repetitive BM-005-G14, a novel gene complex was found between to 2 genes on the same strand. Nested in the second intron of a large 9 Kb <it>papilin </it>gene was a <it>helicase </it>gene. This <it>helicase </it>overlapped in two exonic regions with the <it>papilin</it>. Both these genes were shown expressed in different tick life stage important in ectoparasite interaction with the host. Tick specific sequence differences were also determined for the <it>papilin </it>gene and the protein binding sites of the 18S subunit in a comparison to <it>Bos taurus</it>.</p> <p>Conclusion</p> <p>In the absence of a sequenced reference genome we have assembled two complex BAC sequences, characterised novel gene structure that was confirmed by gene expression and sequencing analyses. This is the first report to provide evidence for 2 eukaryotic genes with exon regions that overlap on the same strand, the first to describe <it>Rhipicephalinae papilin</it>, and the first to report the complete ribosomal DNA repeated unit sequence structure for ticks. The Cot data estimation of genome wide sequence frequency means this research will underpin future efforts for genome sequencing and assembly of the <it>R. microplus </it>genome.</p