Relative expression levels of <i>Tetrahymena thermophila</i><i>MTT1</i>, <i>MTT3</i> and <i>MTT5</i> genes obtained by quantitative RT-PCR.

<p>Panels A, B and C: fold-induction for each gene (<i>MTT1</i>, <i>MTT3</i>, and <i>MTT5</i>, respectively) after treatment by different heavy metals. Gene expression levels are shown relative to an untreated control (which is set at 1±0.0 for every gene). Normalization of expression was achieved against the amplification of an endogenous gene (α-tubulin). Each bar of the histogram corresponds to an average value ±SD of two or three independent experiments. Heavy metal concentrations are as reported in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000291#s4" target="_blank">Material & Methods</a>; Cd+ refers to 27 µM Cd plus 80 µM Cu; treatments times were: 1 h (light bars) and 24 h (dark bars) for all heavy metals. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000291#pone.0000291.s002" target="_blank">Table S2</a> for numerical values. Panel D. Relative expression levels for each gene, in the order <i>MTT1</i> (blue bars), <i>MTT3</i> (green bars) and <i>MTT5</i> (red bars), after induction by stress treatments other than heavy metals. Temperature treatments were for 2h, pH and Paraquat treatments were for 24 h, while starvation treatments were for 24 hrs and 4 days.</p

Members of <i>Tetrahymena</i> subfamily 7a metallothioneins are composed of modules.

<p>Panel A. Alignment of all 15 modules. The MTs were divided up into modules as described in the text; multiple alignments were made using the T-coffee program, followed by visual inspection and manual adjustment. Linkers and submodules (see text) have been separated from one another by a blank column. The bottom four lines represent the highly conserved C-terminal modules of four MTs, missing in MTT5. Panel B. Amino acid conservation at every module position. The height of each letter represents the extent of conservation. At any given position in the module alignment, diversity is minimal (and conservation and logo height maximal) when every module has the same amino acid and there is no gap in any module. Conversely, a blank (0-height) logo column represents minimal conservation, i.e., maximal diversity, at that module position.</p

Diagnostic characteristics of MT subfamilies 7a and 7b

<p>C, K and X = cysteine, lysine and any amino acid other than C, respectively.</p>*<p>Consensus sequence. In subfamily 7a this motif is present exclusively at C-termini of type 2 submodules, which in turn exclusively constitute the C-termini of modules.</p>**<p>To the extent reported so far: <i>MTT1</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000291#pone.0000291-Boldrin1" target="_blank">[20]</a>; this work); <i>MTT3</i> and <i>MTT5</i> (this work); <i>TpigMT-1</i> and <i>TpigMT-2</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000291#pone.0000291-Shang1" target="_blank">[19]</a>; <i>TpMT1</i> and <i>TpMT2</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000291#pone.0000291-Piccinni2" target="_blank">[18]</a>; <i>MTT2</i> and <i>MTT4</i> (L-F Feng and W. Miao, personal communication).</p

Hierarchical modular structure of the DNA sequence in the <i>MTT5</i> 5′ flanking region.

<p>The two major tandem 416-bp repeats are delimited by blank lines; a three-bp gap (dashed) has been introduced in the first repeat in order to facilitate visual detection of the two repeats. Grey highlight: MTCM1 motif; underlined: degraded versions of MTCM1. Aqua (TCA) and yellow (TGA) shading within MTCM1: elements of the AP1 binding site-like heptanucleotide TGA(A/T)TCA, and of a TCATGA palindromic hexanucleotide that overlaps the AP1-like site; this shading is included to facilitate visual identification of the orientation of various MTCM1 copies. Nucleotide substitutions between the two repeats are boxed. Underlined ATG at the end of the sequence: <i>MTT5</i> start codon.</p

Prevalence of pox-like lesions by land-use type in the province of Huelva in 2013 and 2014.

<p>Number over bars indicate sample size, number inside bars indicate number of birds with cutaneous lesions.</p

Bayesian phylogeny of DNA sequences from a 448 bp fragment of the 4b core proteins for 66 unique Avipox strains, showing posterior probability values.

<p>Avipoxvirus clades A-C following Jarmin <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168690#pone.0168690.ref008" target="_blank">8</a>] and Gyuranecz <i>et al</i>.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168690#pone.0168690.ref007" target="_blank">7</a>].</p

Avipox strain diversity in <i>Passer domesticus</i>, introducing a simple descriptive nomenclature.

<p>Avipox strain diversity in <i>Passer domesticus</i>, introducing a simple descriptive nomenclature.</p

Distribution of 2013 and 2014 sampling, pox-like lesions and genotypes (CNPV-PD1, dark red; CNPV-PD2, red) in the province of Huelva.

<p>Geographical data are downloaded from <a href="http://www.ideandalucia.es/portal/web/ideandalucia" target="_blank">http://www.ideandalucia.es/portal/web/ideandalucia</a>.</p

Summary of total pox-like lesions, type of samples and molecular analysis of APV strains.

<p>Summary of total pox-like lesions, type of samples and molecular analysis of APV strains.</p

TCS haplotype network of the nine unique Avipox sequences detected in <i>Passer domesticus</i>.

<p>The maximum coverage for all nine sequences is 357 nt, though coverage for seven haplotypes is 538 nt. Canarypoxvirus (CNPV) strains are shown in red; Fowlpoxvirus (FWPV) strains are blue. Number of nucleotide substitutions are marked on the line by a solid circle, or shown in parenthesis (when numerous). The continent and avian order of detection, and number of known host species (<i>N</i> = <i>x</i>), are shown below each strain. Note that FWPV-PD1 and PD2 are identical in the 357 nt sequence, but differ at five nt sites in the extended 538 nt sequence (resolution determined only from the extended 538 nt sequence, is indicated by a dotted line). Geographic and host taxonomic information associated with these sequences is based on the extended 538 nt sequence.</p