Geographical differences in <i>Pvama1</i> sequences across DI only.

<p>Pairwise F_ST values between Venezuelan sequences presented here and other sites only are highlighted. ^a = 1996 villages, N = 28; ^b = 1997 villages, N = 12; ^c = Koshirowetheri 1997 only, N = 33; ¹ = all Venezuela data irrespective of village, N = 73; ² = <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003366#pone.0003366-Gunasekera1" target="_blank">[15]</a>; ³ = <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003366#pone.0003366-Cheng1" target="_blank">[33]</a>; ⁴ = <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003366#pone.0003366-Figtree1" target="_blank">[34]</a>; ⁵ = <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003366#pone.0003366-Rajesh1" target="_blank">[35]</a>; Sample sizes available are shown in parenthesis after each location; Significant difference (P<0.05) indicated by bold underline. Slightly negative numbers, indicated by italics, are biologically meaningless and are equivalent to F_ST = 0.000.</p

Graphical representation of the distributions of the <i>Pvama1</i> and <i>Pfama1</i> allele frequencies from the Venezuelan Amazon.

<p>A. Distribution of 18 <i>Pvama1</i> haplotypes across surveys in 1996 and 1997. Sequences from the additional village sampled in 1997 (Koshiro) are presented separately from those of the villages sampled in both surveys. B. Haplotype frequency distribution for <i>Pfama1</i> and <i>Pvama1</i> from the 1996 survey only.</p

Linkage disequilibrium indices of <i>Pvama</i>1 and <i>Pfama</i>1.

<p>Linkage disequilibrium plots of D' and R² for <i>Pvama1</i> (Graphs A and B) and <i>Pfama1</i> (Graphs C and D). Sites with significant linkage (P<0.05) are shown as solid circles; non-significant sites are shown as for <i>Pvama1</i> only. No non-significant sites for <i>Pfama1</i> were found.</p

PCR primers and conditions.

*<p>(Polley & Conway, 2001).</p

Within-population analyses of <i>Pvama</i>1 and <i>Pfama</i>1 from the Venezuelan Amazon.

<p>^* = 0.10>P>0.05; ^** = P>0.10; ^*** = P<0.02.</p>†<p> = Effective population size, <i>Ne</i>, = C/4<i>r</i>, where <i>r</i> is the recombination rate and <i>r</i> of <i>P. falciparum</i> is used for all, approximately 6×10⁻⁷, as the recombination rate for <i>P. vivax</i> has not yet been determined (Joy <i>et al.</i>, 2006).</p

Geographical differences in <i>Pvama1</i> and <i>Pfama1</i> sequences across all domains.

<p>Pairwise F_ST values between Venezuelan sequences and other sites are highlighted. Sample sizes are in parenthesis. ¹ = all Venezuela <i>Pvama1</i>data from both 1996 and 1997; ² = all Venezuela <i>Pfama1</i> data from 1996 only; ² = <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003366#pone.0003366-Gunasekera1" target="_blank">[15]</a>; ³ = <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003366#pone.0003366-Rajesh1" target="_blank">[35]</a> one sequence was ignored in the whole gene and DI analysis as it was not full length; ⁴ = <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003366#pone.0003366-Polley2" target="_blank">[19]</a>; ⁵ = <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003366#pone.0003366-Polley1" target="_blank">[18]</a>. Significant difference (P<0.05) indicated by bold text. Slightly negative numbers, indicated by italics, are biologically meaningless and are equivalent to F_ST = 0.000.</p

McDonald-Kreitman analyses of <i>Pvama</i>1 and <i>Pfama</i>1.

1<p> = Neutrality index indicates the extent to which the levels of amino acid polymorphism depart from the expected in the neutral model.</p>2<p> = Fisher's exact test.</p><p>*0.001</p

Sliding window plots of Tajima's D and Fu and Li's F* for <i>Pvama</i>1 and <i>Pfama</i>1.

<p>A and B – <i>Pvama1</i>; C and D – <i>Pfama1</i>. Midpoints where the sequence significantly departs from zero (P<0.05) are shown as solid points. Domains, DI, DII, DIII, are marked.</p

Identification and Characterization of the RouenBd1987 <i>Babesia divergens - Figure 4 </i> Rhopty-Associated Protein 1

<p>A, BdRap-1 binds to a neuraminidase, trypsin and chymotrypsin-resistant RBC receptor. ³⁵S-labelled proteins derived from parasite culture supernatants were mixed with untreated RBCs treated (WT lane), or with RBCs treated with 0.1 U/mL of Neuraminidase (N), 0.1 mg/mL trypsin (T1), 1 mg/mL trypsin (T2), and 1 mg/mL. Separated cells were then lysed and the soluble fraction was immuno-precipitated with anti-rBdRAP-1 antibodies and separated on SDS-PAGE gel. BdRAP-1 appears to be an adhesin that participates in invasion by binding to the RBC surface, as indicated by the presence of a band at the expected size of ∼46 kDa. However, none of the enzyme treatments inhibited or decreased BdRAP-1 binding, as shown the presence of the ∼46 kDa band in all lanes representing treated cells, thus, the binding profile of native BdRAP-1 to the RBC suggests the participation of a novel red cell receptor in merozoite invasion. B, BdRAP-1 binds to a non-proteinacious receptor. Binding profile of BdRAP-1 to untreated RBCs treated (WT lane), or with RBCs treated with 0.5 mg/mL of Proteinase K (PK) or 0.5 mg/mL Papain (P).</p

Antibodies against BdRAP-1 do not significantly inhibit parasite invasion <i>in vitro</i>.

<p><i>B. divergens</i> cultures in human cells were maintained for 36 h in the presence of 2 mg/mL of the purified IgG fraction of anti-rBdRAP-1 antibodies from rabbit serum or in the presence of the equivalent concentration of purified rabbit pre-immune IgG. The level of growth inhibition compared to a no-serum control was determined every 12 h. The presence of BdRAP-1 (indicated by a solid line) was able to inhibit growth of <i>B. divergens</i> by 11% and 16% at 12 h and 24 h, respectively, to a maximum inhibition of 19% at 36 h. however, this is not significantly different from the inhibition due to the presence of pre-immune IgG (indicated by a dashed line), which inhibited growth by 20% at 36 h, and the lack of inhibition specifically due to anti-BdRAP-1 antibodies suggests multiple alternative pathways of invasion are available to the parasite, or the role of this ligand is not restricted to invasion only.</p