Perception of Ticks and Tick-Borne Diseases Worldwide

In this comprehensive review study, we addressed the challenge posed by ticks and tick-borne diseases (TBDs) with growing incidence affecting human and animal health worldwide. Data and perspectives were collected from different countries and regions worldwide, including America, Europe, Africa, Asia, and Oceania. The results updated the current situation with ticks and TBD and how it is perceived by society with information bias and gaps. The study reinforces the importance of multidisciplinary and international collaborations to advance in the surveillance, communication and proposed future directions to address these challenges

Post-Integration Silencing of piggyBac Transposable Elements in Aedes aegypti

The piggyBac transposon, originating in the genome of the Lepidoptera Trichoplusia ni, has a broad host range, making it useful for the development of a number of transposon-based functional genomic technologies including gene vectors, enhancer-, gene- and protein-traps. While capable of being used as a vector for the creation of transgenic insects and insect cell lines, piggyBac has very limited mobility once integrated into the genome of the yellow fever mosquito, Aedes aegypti. A transgenic Aedes aegypti cell line (AagPB8) was created containing three integrated piggyBac elements and the remobilization potential of the elements was tested. The integrated piggyBac elements in AagPB8 were transpositionally silent in the presence of functional transposase, which was shown to be capable of catalyzing the movement of plasmid-borne piggyBac elements in the same cells. The structural integrity of one of the integrated elements along with the quality of element-flanking DNA, which is known to influence transposition rates, were tested in D. melanogaster. The element was found to be structurally intact, capable of transposition and excision in the soma and germ-line of Drosophila melanogaster, and in a DNA sequence context highly conducive to element movement in Drosophila melanogaster. These data show that transpositional silencing of integrated piggyBac elements in the genome of Aedes aegypti appears to be a function of higher scale genome organization or perhaps epigenetic factors, and not due to structural defects or suboptimal integration sites

Plasmids used in this study.

<p>All plasmids are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068454#s2" target="_blank">Material and Methods</a>. Large arrowheads represent the terminal sequences of <i>piggyBac</i>. Act5C, promoter from <i>D. melanogaster</i> gene <i>Actin5C; Hygromycin^R,</i> coding region for bacterial gene <i>hygromycin B phosphotransferase</i>; ie1, promoter from the baculovirus gene <i>immediate early 1</i>; Amp^R, bacterial gene <i>beta-lactamase</i>; hsp70, promoter from <i>D. melanogaster</i> gene <i>hsp70</i>; PB-transposase, coding region for <i>piggyBac</i> transposase; DsRed, coding region for <i>Discosoma sp</i>. gene <i>red fluorescent protein</i>; pUb, promoter from <i>D. melanogaster</i> gene <i>pUbi-p63e</i>; Kan^R, bacterial gene <i>Neomycin phosphotransferase II</i>; gDNA, refers to <i>Aedes aegypti</i> genomic DNA flanking the 5′ and 3 ends of <i>piggyBac</i> elements integrated in the genome of cell line AagPB8 (in pCL1w+) and in transgenic line 40D (in p40Dw+; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068454#pone.0068454-Sethuraman1" target="_blank">[25]</a>); mini-white, the <i>D. melanogaster</i> gene <i>w^+mW.hs;</i> attB, the bacterial attachment site for phage <i>ΦC31.</i></p

Location of <i>piggyBac</i> integration sites in AegPB8.

*<p>Position of underlined nucleotide shown based on <i>Aedes aegypti</i> genome version 66.1 (AegL1)</p

Intrinsic Characteristics of Neighboring DNA Modulate Transposable Element Activity in Drosophila melanogaster

Identifying factors influencing transposable element activity is essential for understanding how these elements impact genomes and their evolution as well as for fully exploiting them as functional genomics tools and gene-therapy vectors. Using a genetics-based approach, the influence of genomic position on piggyBac mobility in Drosophila melanogaster was assessed while controlling for element structure, genetic background, and transposase concentration. The mobility of piggyBac elements varied over more than two orders of magnitude solely as a result of their locations within the genome. The influence of genomic position on element activities was independent of factors resulting in position-dependent transgene expression (“position effects”). Elements could be relocated to new genomic locations without altering their activity if ≥500 bp of genomic DNA originally flanking the element was also relocated. Local intrinsic factors within the neighboring DNA that determined the activity of piggyBac elements were portable not only within the genome but also when elements were moved to plasmids. The predicted bendability of the first 50 bp flanking the 5′ and 3′ termini of piggyBac elements could account for 60% of the variance in position-dependent activity observed among elements. These results are significant because positional influences on transposable element activities will impact patterns of accumulation of elements within genomes. Manipulating and controlling the local sequence context of piggyBac elements could be a powerful, novel way of optimizing gene vector activity

Plasmid-based <i>piggyBac</i> excision assay.

<p>A) Diagrammatic representation of the <i>piggyBac</i>-containing plasmid, <i>piggyBac</i> 3×P3EGFP used in the excision assay described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068454#s2" target="_blank">Material and Methods</a> (donor plasmid), and the same plasmid following precise excision of the <i>piggyBac</i> element (excision plasmid). The <i>piggyBac</i>-containing donor plasmid, <i>piggyBac</i> 3×P3EGFP, and <i>piggyBac</i> transposase expressing helper plasmid, pHspPBtpase:PubDsRed, were co-transfected into AagPB8 cells. Transfected cells were heat-shocked after 12 hrs and collected after 72 hrs. DNA was extracted and used as a template for PCR. Primers 1 and 2 (shown as labeled short half-arrows) were specific to the donor-plasmid backbone (494donorFWD, 494donorREV) and yield a 751 bp product (grey line) in the presence of the donor and excision plasmids. Primers 3 and 4 (494excisionFWD, 494excisionREV and shown as labeled short half-arrows) are specific to the plasmid DNA flanking the <i>piggyBac</i> element, however under the conditions of this experiment PCR products were only detected if donor plasmids missing the <i>piggyBac</i> element through excision were present, yielding a 540 bp PCR product (grey line). The 5′ and 3′ terminal <i>piggyBac</i> sequences are represented by arrows (5′PB, 3′PB). The duplicated TTAA target sequence into which <i>piggyBac</i> integrated is shown as a black diamond and the 3×P3EGFP transgene within the <i>piggyBac</i> element is shown as a black rectangle. The normally circular plasmids are represented as linear molecules. B) The PCR results from two <i>piggyBac</i> excision assays in AagPB8 cells. Lanes 1 and 2: from cells transfected with donor and <i>pHspPBtpase:PubDsRed</i> (2 independent transfections). Lane 3: from cells transfected with donor and control plasmids (pBluescript SKII+). Lane 4 and 5: positive controls for detecting excision events. The DNA used as a template in these reactions was a purified excision plasmid recovered from a previous excision assay (2 independent transfections). Lane 6: negative control for detecting excision events. DNA used as a template in this reaction came from cells transfected with donor plasmid only, without the transposase helper plasmid. Two PCR reactions were performed on each sample using primer combinations indicated above the lanes numbers. Primers 1+2 (same primers referred to in panel A) detected the presence of donor and excision plasmids and yielded a 751 bp reaction product (white arrow). Primers 3+4 (same primers referred to in panel A) yielded a 540 bp reaction product (white arrow) only when the <i>piggyBac</i> element in the donor plasmid had excised. Only the 540 and 751 bp bands are specific reaction products.</p

<i>piggyBac</i> transposable element display results using DNA isolated from cell line AagPB8.

<p>Lanes 1 are the results using DNA as a template isolated from cell line AagPB8 and shows evidence of the 5′ end of one of the two <i>piggyBac</i> elements that had integrated by canonical cut-and-paste transposition –80 bp band. The 5′ end of the second <i>piggyBac</i> element that integrated by canonical cut-and-paste transposition is not visible. This element can be detected when the 3′ ends of integrated <i>piggyBac</i> elements are visualized using transposable element display (not shown). The band at 250 bp is the <i>piggyBac</i> element associated with a copy of the integrated plasmid pBac:Act5cHyg:ie1EGFP. The sample was loaded into two adjacent lanes. Lanes 2 are the results using DNA as a template isolated from non-transgenic Aag-2 cells and this serves as a negative control for this assay since there are no <i>piggyBac</i> elements in <i>Ae. aegypti</i>. The sample was loaded into two adjacent lanes. Lanes 3 are the results using DNA as a template from AagPB8 cells 72 hours after being transfected with <i>piggyBac</i>-transposase-expressing pHspPBtpase:PubDsRed. The sample was loaded into two adjacent lanes. There was no evidence of <i>piggyBac</i> elements in other positions in the genome in Lane 3 as would be expected if <i>piggyBac</i> transposase mobilized the integrated <i>piggyBac</i> elements in AagPB8 cells. The asterisk indicated the position of a non-specific TE display band present in all samples. The positions of molecular weight markers 80 bp and 250 bp in length are shown.</p

Relative abundance of bacterial genera/species in horn fly eggs after applying the cut-off filter of abundance level ≥0.1% and prevalence of taxa ≥66% within each class respectively or a prevalence ≥55% across all nine samples.

<p>Values are mean percentages (n = 3).</p