Variability in the expressivity of the extra-vein phenotypes in the wings of the G2 <i>Dl*/Dl</i>^<i>+</i> flies.

<p>In the G2, paramutant <i>Dl*/Dl</i>^<i>+</i> flies exhibit a wide range of wing vein phenotypes. (A) An extra-vein in the inter-vein region together with a pcv gap (EVint+GAP). (B) An extra-vein perpendicular to the pcv together with a pcv gap (EVpcv+GAP). (C) An extra-vein perpendicular to the pcv (EVpcv), (D) An extra-vein in the inter-vein region (EVint), (E) A faint dot (arrow; DOT). (F) A percentage of wings are wild-type (WT).</p

Map of the <i>Dl</i> gene and phenotype of <i>Dl</i>^<i>05151</i><i>/TM3</i> wings.

<p>(A) The <i>Dl</i> gene is located at cytological position 92A1-92A2 and between nucleotides 19,302,731 and 19,326,217 of the 3R arm. It has three annotated transcripts transcribed from right to left and is flanked by transcripts CG44174 and CR44175. Green: gene extent of <i>Dl</i>. Beige: translated exons. Purple: transcribed exons. Horizontal black lines: introns. This map is extracted from Release R6.08 of the <i>Drosophila</i> genome sequence in FlyBase. The <i>Dl</i>^<i>05151</i> insertion (black triangle) is located 681 bp upstream from the <i>Dl</i> ATG and is transcribed from right to left. (B) Eye of a <i>ry</i>^<i>-</i><i>/ry</i>^<i>-</i> fly. (C) Left wing of a <i>ry</i>^<i>-</i><i>/ry</i>^<i>-</i> female. (D) Right wing of a <i>ry</i>^<i>-</i><i>/ry</i>^<i>-</i> male. (E) Eye of a heterozygous <i>Dl</i>^<i>05151</i><i>/TM3</i> fly. The transgene carrying the <i>ry</i>^<i>+</i> gene, the eye color is as in wild-type. (F) Right wing of a <i>Dl</i>^<i>05151</i><i>/TM3</i> female. Arrows point to expanded and extra-veins (EXP+EV). (G) Right wing of a <i>Dl</i>^<i>05151</i><i>/TM3</i> female presenting two extra-veins (EVs). (H) Wing of a <i>Dl</i>^<i>05151</i><i>/TM3</i> heterozygous female presenting one extra-vein perpendicular to the posterior cross vein (pcv) (EVpcv). (I) Example of an extra-vein in the L4-L5 inter-vein region (EVint). (J) Wild-type wing. Hereafter, wing orientations are according to their origin: left wings with distal tip to the left and right wings with distal tip to the right.</p

Quantification of the extra-vein phenotypes in the G0, G1 and G2.

<p>Quantification of the extra-vein phenotypes in the G0, G1 and G2.</p

Paramutation at the <i>Dl</i> locus.

<p>Eye (A) and wing (B) of a heterozygous G2 <i>Dl</i>^<i>05151</i>/<i>Dl</i>^<i>+</i> fly. The wing shown presents a frequent phenotype: two extra-veins near the pcv (arrows). Eye (C) and wing (D) of a <i>Dl</i>^<i>05151</i>, <i>ry</i>^<i>-</i><i>/Dl*</i>, <i>ry</i>^<i>-</i> (hereafter, <i>Dl</i>^<i>05151</i>/<i>Dl*</i>) fly. This G2 fly does not carry the <i>Dl</i>^<i>05151</i> insert, but shows an extra-vein perpendicular to the pcv (arrow) indicating that its <i>Dl</i>^<i>+</i> (<i>Dl</i>*) chromosome coming from the heterozygous <i>Dl</i>^<i>05151</i>/<i>Dl*</i> parent has been paramutated. (E) PCR on the genomic DNA of the fly carrying the wing shown in Fig 4D. The oligonucleotides used are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172780#pone.0172780.g002" target="_blank">Fig 2B</a>. In the fly carrying the transgene (<i>Dl</i>^<i>05151</i><i>/Dl</i>^<i>+</i>), oligonucleotides 003 and 011 (red arrow) and 004 and 006 (yellow arrow) amplify the 3’ and 5’ ends of the transgene, respectively. No amplification with these two couples of primers is seen for the paramutant <i>Dl*/Dl</i>^<i>+</i> fly (red and yellow circles, respectively). Control amplification with oligonucleotides 003 and 004 (green arrows) in the <i>Dl</i> gene occurs for both flies.</p

Perfect excision of the <i>Dl</i>^<i>05151</i> insert.

<p>(A) Crosses carried out to obtain perfect excisions of the <i>Dl</i>^<i>05151</i> transgene. <i>Δ2–3</i> is the chromosome carrying the transposase, <i>Vno</i> and <i>Ly</i> are dominant wing markers, {ry⁺} indicates that the flies have ry⁺ eyes because they carry the <i>Dl</i>^<i>05151</i> insertion, while {ry^-} indicates that the flies have ry^- eyes because they are devoid of the <i>Dl</i>^<i>05151</i> transgene. <i>Dl</i>^{<i>Δ05151</i>} are the chromosomes in which an excision of <i>Dl</i>^<i>05151</i> occurred. At least seven excised hemizygous viable and rescued stocks we obtained. (B) Sizes and map of the amplicons obtained by PCR analysis. Oligonucleotides 003 and 004 are downstream and upstream of the <i>Dl</i>^<i>05151</i> transgene, respectively. Oligonucleotides 006 and 011 are in the 5’ and 3’ of the transgene, respectively. (C) 168m1 and 169m2 are two independent hemizygous viable <i>ry</i>^<i>-</i> lines in which excision of <i>Dl</i>^<i>05151</i> occurred through Δ2–3 transposase-mediated excision. As shown by the absence of amplification with the couples of oligonucleotides at the 3’ (red line) and 5’ (yellow line) ends of the transgene, these two strains do not carry the transgene. Sequencing of the <i>Dl</i> amplicons (green line) shows that the excisions are perfect. (D-G) Wild-type wings of <i>Dl</i>^{<i>Δ05151</i>}<i>/ry</i>^<i>-</i> females for the two stocks 168m1 and 169m2 as indicated.</p

Wing phenotypes of heterozygous <i>Dl</i>^<i>05151</i>/<i>Dl*</i> females.

<p>Wings of the <i>Dl</i>^<i>05151</i><i>/Dl</i>^<i>+</i> progeny of an out cross of <i>Dl</i>^<i>05151</i><i>/TM3</i> males to <i>ry</i>^<i>-</i><i>/ry</i>^<i>-</i> females. A whole spectrum of extra-vein phenotypes is found. (A) Expanded and extra-veins (EXP+EVs). (B) Two extra-veins (EVs). (C) One extra-vein emerging from the pcv (EVpcv). (D) An extra-vein in the inter-vein region (EVint). (E) A dot in the inter-vein region (arrow; DOT). (F) An anterior gap of the pcv (GAP). The spectrum of phenotypes is very similar to that of the <i>Dl</i>^<i>05151</i><i>/TM3</i> stock.</p

Environment Exploration and Colonization Behavior of the Pea Aphid Associated with the Expression of the <i>foraging</i> Gene

<div><p>Aphids respond to specific environmental cues by producing alternative morphs, a phenomenon called polyphenism, but also by modulating their individual behavior even within the same morph. This complex plasticity allows a rapid adaptation of individuals to fluctuating environmental conditions, but the underlying genetic and molecular mechanisms remain largely unknown. The <i>foraging</i> gene is known to be associated with behavior in various species and has been shown to mediate the behavioral shift induced by environmental changes in some insects. In this study, we investigated the function of this gene in the clonal forms of the pea aphid <i>Acyrthosiphon pisum</i> by identifying and cloning cDNA variants, as well as analyzing their expression levels in developmental morphs and behavioral variants. Our results indicate that the expression of <i>foraging</i> changes at key steps of the aphid development. This gene is also highly expressed in sedentary wingless adult morphs reared under crowded conditions, probably just before they start walking and foraging. The cGMP-dependent protein kinase (PKG) enzyme activity measured in the behavioral variants correlates with the level of <i>foraging</i> expression. Altogether, our results suggest that <i>foraging</i> could act to promote the shift from a sedentary to an exploratory behavior, being thus involved in the behavioral plasticity of the pea aphid.</p></div

Structure of the <i>Apfor1</i> and <i>Apfor2</i> transcripts.

<p>Exons 3–16 are identical, only exons 1 and 2 differs as the result of alternative splicing. Scaffold 409 containing the two transcripts is represented at the top. Exons are indicated by grey boxes and introns by dotted lines. White triangles indicate the position of start codons, black triangles indicate the position of the stop codon.</p

Relative expression of <i>Apfor1</i> and <i>Apfor2</i> among behavioral variants of adults pea aphids.

<p>(A), Relative expression of <i>Apfor1</i>. (B), Relative expression of <i>Apfor2</i>. VW, winged viviparous adults reared under high population density; VWL, wingless viviparous adults reared under low population density; VWLc, wingless viviparous adults reared under high population density; VWLf, wingless viviparous adults foragers reared under high population density. Errors bars represent the standard errors converted to the same arbitrary scale as the means. Relative expression is normalized to VW. A one-way ANOVA followed by a Fisher's PLSD test was performed. The statistically significant differences between groups are denoted by different letters (<i>P</i><0,01 in case of <i>Apfor1</i> and <i>P</i><0,05 in case of <i>Apfor2</i>).</p

PKG enzyme activity among behavioral variants of adults pea aphids.

<p>(A) PKG enzyme activity in whole bodies. (B) PKG enzyme activity in heads. PKG enzyme activity is expressed as the OD for 5 µg of total proteins for each behavioral variant. Error bars represent the standard errors converted to the same arbitrary scale as the means. A one-way ANOVA followed by a Fisher's PLSD test was performed. The statistically significant differences between groups denoted by different letters (<i>P</i><0,05).</p