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

BMC Genomics Evolutionary conserved microRNAs are ubiquitously expressed compared to tick-specific miRNAs in the cattle tick Rhipicephalus (Boophilus) microplus Roberto A Barrero 0 Gabriel Keeble-Gagnre 0 Bing Zhang Paula Moolhuijzen 0 Kazuho Ikeo Yoshio Tateno Takashi Gojobori Felix D Guerrero Ala Lew-Tabor 0 Matthew Bellgard 0 0 Centre for Comparative Genomics, Murdoch University , WA 6150 , Australia 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. - Background The Arthropods are a diverse group of organisms including Chelicerata (ticks, spiders), Myriapoda (centipedes, millipedes), Crustacea (crabs, shrimps), and Insecta (flies, beetles). Molecular estimates indicate that ticks emerged 300 27 MYA, while the prostriate and metastriate hard tick lineages diverged 241 28 MYA [1]. Rhipicephalus (Boophilus) microplus is considered to be the most economically important tick parasite in the world. R. microplus is a hard tick associated with cattle infestations but can also occasionally be found on other hosts including horses, goats, sheep, pigs and some wild animals living in subtropical and tropical regions worldwide [2,3]. R. microplus is a member of the metastriate lineage of ticks that includes numerous genera and species of medical and veterinary importance. In comparison, I. scapularis is a member of the prostriate lineage that comprises the single genus Ixodes. The prostriate and metastriate lineages differ markedly in many aspects of their biology such as type of developmental cycle (i.e., three-versus one-host ticks), host range and vector competence. Comparative analyses between prostriate and metastriate gene sets including miRNAs may reveal the genetic basis for fundamental differences in the biology of these tick lineages [4]. R. microplus is generally a single host tick spending all parasitic life cycle stages on cattle. The eggs hatch in the environment and the larvae crawl up grass or other plants to find a host. In the summer, R. microplus can survive for as long as 3 to 4 months without feeding. In cooler temperatures, they may live without food for up to six months. Newly attached seed ticks (larvae) are usually found on the softer skin inside the thigh, flanks, and forelegs. After feeding, the larvae molt twice, to become nymphs and male or female adults. Each developmental stage (larval, nymph and adult) feeds only once, but the feeding takes places over several days. Adult male ticks become sexually mature after feeding, and mate with feeding females. An adult female tick that has fed and mated detaches from the host and deposits a single batch of many eggs in the environment. Typically, these eggs are placed in crevices or debris, or under stones. The female tick dies after ovipositing. Ticks in the subgenus Boophilus have a life cycle that can be completed in 3 to 4 weeks; this characteristic can result in a heavy tick burden particularly on tick susceptible cattle in tropical areas [2,3]. MicroRNAs (miRNAs) are small 1925 nucleotide regulatory RNAs that act as post-transcriptional modulators of gene expression in animals and plants [5]. They are estimated to represent 1% of the transcriptome in higher eukaryotes and predicted to control the expression of up to 30% of messenger RNAs [6,7]. Most miRNAs are encoded in intergenic regions and are transcribed by RNA polymerase II as long primary nuclear miRNAs (pri-miRNAs), which range from hundreds to thousands of nucleotides in length [8]. One pri-miRNA typically contains a single or several miRNA precursors (pre-miRNAs) as stem-loop, hairpin structures flanked by unstructured, single stranded RNA sequences [9]. Pre-miRNAs are cleaved near their loops by the cytoplasmic RNase III enzyme Dicer to generate a heteroduplex of two ~23-nt RNAs that are then packed into the RISC complex [10]. Mature miRNA sequences are encoded either in the 5-arm or the 3arm of pre-miRNAs. The decision as to which sequence is incorporated into the silencing complex is influenced by the difference in pairing stabilities between the two ends of the miRNA:miRNA star (miRNA*) duplex, with preferential incorporation of the strand whose 5end is less stably paired [11,12]. In some cases both strands of the miRNA:miRNA* duplex were found expressed at similar levels [13]. About half of the miRNA genes in Drosophila melanogaster are clustered and transcribed from a single polycystronic pri-miRNA [14]. Recently, next generation sequencing technologies have been utilized to profile and discover miRNAs genome-wide. To assist in this process it is normally required to have a reference genome sequence. Currently, there is no reference genome sequence for R. microplus, with the Ixodes scapularis gen (...truncated)