Detection of parasites after experimental transovum transmission of <i>Leptomonas wallacei</i> parasites by <i>Oncopeltus fasciatus</i>.

<p>(A) Detection of parasite infection by PCR. Lane 1- The DNA extracted from an axenic culture of <i>L. wallacei</i> was amplified with primers specific for parasite detection. Lane 2- A pool of DNA samples extracted from gut of parasite-free insects was concomitantly amplified with primers specific for parasite and insect DNA detection. Lanes 3–7- Representative DNA samples extracted from guts of insects that fed on sunflower seeds contaminated with eggshels collected from infected colony were concomitantly amplified with primers specific for parasite and insect DNA detection. On the left, the positions of molecular size markers are shown in base pairs. The figure represents a negative image of the gel. (B) Detection of parasite infection by optical microscopy. The representative micrograph shows Giemsa-stained parasites (arrows) in the gut contents of newly infected insects. The arrowheads indicate the nucleus (N) and kinetoplast (K) of the parasite. Magnification = 400 x.</p

Scanning electron micrograph of feces and eggs of <i>Oncopeltus fasciatus</i> infected with <i>Leptomonas wallacei.</i>

<p>(A) Promastigote forms of <i>L. wallacei</i> in fresh feces showing typical characteristics of live cells and cystic forms (arrow). Bar = 5 µm. (B) High magnification of a promastigote form showing one cystic form (arrow) near to its flagella. Bar = 1 µm. (C) Promastigote form present in the naturally dried feces showing extensive membrane damage and three cystic forms. Note that the cystic forms (arrows) do not show any surface damage. Bar = 1 µm. (D) Low magnification of the surface of the eggs. Bar = 500 µm. (E) High magnification of egg surface showing typical cystic forms of <i>L. wallacei</i> (arrows). Bar = 1 µm.</p

Scanning electron microscopy of <i>Oncopeltus fasciatus</i> guts infected with <i>Leptomonas wallacei</i>.

<p>(A) Midgut of <i>L. wallacei</i>-infected <i>O. fasciatus</i>. The arrows indicate large numbers of parasites near the midgut wall. (B) Hindgut of <i>L. wallacei</i>-infected <i>O. fasciatus</i>. The image shows a massive presence of flagellates. Most of those are attached to the intestinal wall of the hindgut by their flagella, so only their slender bodies can be seen. One of the short-sized flagellates can be seen in the lumen (arrow). Bars = 10 µm.</p

<i>Oncopeltus fasciatus</i> hatched from eggs submitted to surface asepsis were <i>Leptomonas wallacei</i>-free.

<p>(A) Representative gel electrophoresis of PCR-amplified DNA samples extracted from eggs and whole insect guts. Lane 1- The DNA extracted from an axenic culture of <i>L. wallacei</i> was amplified with primers specific for parasite detection. Lane 2- Sample of pooled DNA extracted from eggs collected at the infected colony and submitted to surface asepsis was concomitantly amplified with primers specific for parasite or insect DNA detection. Lanes 3 and 4- Sample of pooled DNA, extracted from 3 pools of five insect guts of insects hatched from eggs submitted to asepsis, was PCR-amplified with primers specific for parasite (lane 3) or insect DNA (lane 4) detection, respectively. On the left, the positions of molecular size markers are shown in base pairs. The figure represents a negative image of the gel. (B and C) Scanning electron microscopy of <i>O. fasciatus</i> midgut and hindgut, respectively. The micrographs show the presence of bacteria (arrows) but absence of parasites. Bars = 10 µm.</p

Horizontal and vertical transmission of <i>Leptomonas wallacei</i> by <i>Oncopeltus fasciatus</i>.

<p>The insects get infected by feeding on feces contaminated with parasites. <i>L. wallacei</i> produces cystic forms, which are present in the insect guts; the feces contaminate the eggshells. Newly hatched nymphs feed on egg remnants and acquire infection. The cartoon represents a model of transmission based upon previous data and the results shown here.</p

Infection rates of insects hatched from eggs laid by <i>Leptomonas wallacei</i>-infected <i>Oncopeltus fasciatus</i> females.

<p>*Groups 1–3 are biological replicates.</p><p>Infection rates of insects hatched from eggs laid by <i>Leptomonas wallacei</i>-infected <i>Oncopeltus fasciatus</i> females.</p

Immunoblotting of the 130 kDa salivary gland protein probed with anti-human laminin-5 β3 chain antibodies.

<p>Total salivary gland proteins were extracted and separated by 10% SDS-PAGE (<b>a</b>). The 130 kDa band was cut and purified from the gel and the purity of the product was evaluated by 10% SDS-PAGE stained with silver nitrate (<b>b</b>). The purified 130 kDa protein was probed with anti-human laminin-5 β3 chain antibodies (<b>c</b>). The arrow shows the position of the 130 kDa protein on SDS-PAGE and the stained band by immunoblotting.</p

Scanning electron microscopy of <i>Oncopeltus fasciatus</i> salivary glands infected with <i>Phytomonas serpens</i>.

<p>The parasites were injected laterally into the thorax of the insects and the salivary glands were explanted at different time points after injection. (<b>A</b>) Outer surface of the salivary gland showing a high number of parasites between two salivary gland lobes and a few parasites attached to the gland, 48 h post-infection. Scale bar: 10 µm. (<b>B–C</b>) Large numbers of parasites bound to the salivary gland, 72 h post-infection. Scale bars: 100 µm (<b>B</b>) and 50 µm (<b>C</b>). SG: salivary gland; P: parasite; SGL1, SGL2 and SGL3: salivary gland lobes. The asterisk (*) indicates the salivary duct. For further details see the Methods.</p

Comparison of laminin subunit γ through pairwise alignment of γ1 of <i>Homo sapiens</i> (LamG1Hs, gi|145309326), γ1-like of the hemipteran <i>Acyrtosiphon pisum</i> (LamG1Ap, gi|328717115) and γ1-like of <i>O. fasciatus</i> (LamG1Of).

<p>(<b>A</b>) The alignment shows two regions with highly conserved amino acids, consistent with the patterns found in EGF-like domains of γ1 subunits (highlighted in the red rectangles). (<b>B</b>) The alignment of the three γ1 subunits also reveals a sequence similar to the domain VI of γ1 of <i>O. fasciatus</i> transcriptome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048170#pone.0048170-EwenCampen1" target="_blank">[28]</a>. The region similar to domain VI is highlighted in the red rectangles in the alignment. Both in (<b>A</b>) and (<b>B</b>) black shaded residues are identical or similar amino acids present in all three sequences. Grey shaded residues are identical or similar amino acids present in two of the three sequences. The consensus sequence is represented under alignment lines. The alignments were performed using GENEDOC software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048170#pone.0048170-Nicholas1" target="_blank">[90]</a>.</p

Matching of the amino acid sequences of the 130 kDa protein of <i>Oncopeltus fasciatus</i> salivary glands with the human laminin-5 β3 chain amino acid sequence.

<p>(<b>A</b>). Protein spots cut from the gel were treated with porcine trypsin and the peptides were spotted onto a MALDI-TOF sample plate (Voyager- DE, Applied Biosystem, CA, USA). Peptide mass fingerprints were analyzed using Protein Prospector MS-Fit interface (<a href="http://prospector.ucsf.edu" target="_blank">http://prospector.ucsf.edu</a>). (<b>B</b>). Underlined letters represent the amino acid sequences of the salivary gland protein that matched the mass spectrometry data to protein sequences in the NCBI database. The underlined sequences indicate the peptides of the salivary gland protein that were matched with the amino acid sequence of the precursor of the human laminin-5 β3 chain.</p