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

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

Pyrosequencing-Based Analysis of the Microbiome Associated with the Horn Fly, <em>Haematobia irritans</em>

<div><p>The horn fly, <em>Haematobia irritans</em>, is one of the most economically important pests of cattle. Insecticides have been a major element of horn fly management programs. Growing concerns with insecticide resistance, insecticide residues on farm products, and non-availability of new generation insecticides, are serious issues for the livestock industry. Alternative horn fly control methods offer the promise to decrease the use of insecticides and reduce the amount of insecticide residues on livestock products and give an impetus to the organic livestock farming segment. The horn fly, an obligatory blood feeder, requires the help of microflora to supply additional nutrients and metabolize the blood meal. Recent advancements in DNA sequencing methodologies enable researchers to examine the microflora diversity independent of culture methods. We used the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) method to carry out the classification analysis of bacterial flora in adult female and male horn flies and horn fly eggs. The bTEFAP method identified 16S rDNA sequences in our samples which allowed the identification of various prokaryotic taxa associated with the life stage examined. This is the first comprehensive report of bacterial flora associated with the horn fly using a culture-independent method. Several rumen, environmental, symbiotic and pathogenic bacteria associated with the horn fly were identified and quantified. This is the first report of the presence of <em>Wolbachia</em> in horn flies of USA origin and is the first report of the presence of <em>Rikenella</em> in an obligatory blood feeding insect.</p> </div

List of predominant bacteria in adult male horn fly, adult female horn fly and horn fly egg after filtering with a cut-off filter based on abundance level ≥0.1% and prevalence of taxa ≥66% within each class (or a prevalence ≥55% across all nine samples).

<p>List of predominant bacteria in adult male horn fly, adult female horn fly and horn fly egg after filtering with a cut-off filter based on abundance level ≥0.1% and prevalence of taxa ≥66% within each class (or a prevalence ≥55% across all nine samples).</p

Heat map depicting bacterial diversity and relative abundance in life stages from the horn fly.

<p>Letters (A–C) used to identify individual life stage replicates. Double hierarchical dendogram shows different bacteria distribution between genera based on complete linkage clustering, and Manhattan distance methods with no scaling. Dendrogram linkages and distance of the bacterial genus or trace back groups are not phylogenetic, but based upon relative abundance of the genus within the samples. Traceback means bacterial classifications were based upon the percent identity of the sample sequence to known sequences, the percent divergence was then used to adjust identifications to the taxonomic level with the highest degree of confidence (e.g. a percent divergence <3% can be expected to provide confidence at the species level, >3% but <5% at the genera level, etc). Classifications were compiled after traceback. Legend and scale shown in upper left corner of the figure represent colors in heat map associated with the relative percentage of each traceback grouping of bacteria (cluster of variables in Y-axis) within each fly sample (X-axis clustering). Fly samples along the X-axis with Manhattan distances are indicated by branch length and associated with the scale located at the upper right corner of the figure. Bacterial genera along the Y-axis are also clustered according to Manhattan distances; the respective scale is indicated in the figure's lower left corner.</p

Relative abundance of bacterial genera/species in adult male horn fly 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

Selected 16S rDNA sequences from the male horn fly bacteriome.

<p><b>a</b>. Phylogenetic tree of selected 16S rDNA sequences from the male horn fly bacteriome based on 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. The scale bar represents 0.1 changes per nucleotide position. <i>Halobacterium noricense</i> was used as the outgroup. b. The percent relative abundance of the major phyla found within the 16S rDNA sequences from the male horn fly bacteriome. The relative abundance of <i>Wolbachia</i> is included for comparison.</p