Suppression of <i>Inr</i> Expression in <i>Inr^E19/Inr^GC25</i> Flies Affects Developmental Time and Adult Size

<div><p>(A) Developmental time and adult wing size of <i>Inr^E19/Inr^GC25</i> females switched from 17 °C to 24 °C increasingly late in development, expressed as percentage of developmental time and adult wing size of <i>Inr^E19/TM3</i> females maintained under identical thermal conditions. Temperature-control flies were maintained at 17 °C throughout development. TSPs of female <i>Inr^E19/Inr^GC25</i> for wing area and delayed eclosion can be seen as regions of the chart where switching from 17 °C to 24 °C increasingly early in development results in increasingly abnormal phenotypes (that is, where the gradient of the relationship between switch day and phenotype is non-zero). For delayed adult eclosion, the TSP of female <i>Inr^E19/Inr^GC25</i> is before the ninth day of development at 17 °C. For reduced wing size, the TSP of female <i>Inr^E19/Inr^GC25</i> is between the ninth and approximately the 20th day of development at 17 °C.</p> <p>(B) The stages of development of <i>Inr^E19/Inr^GC25</i> flies at 17 °C (A, adult; E, embryo; L1, first instar; L2, second instar; L3, third instar; P, pupae). The point at which suppression of the insulin pathway changes from delaying adult development to reducing adult wing size occurs approximately 40% into the third instar (vertical gray bar)</p> <p>(C) Dry mass of <i>Inr^E19/Inr^GC25</i> males switched from 17 °C to 24 °C at different points in development, expressed as percentage of dry mass of <i>Inr^InrE19/TM3</i> males maintained under identical thermal conditions. The TSP of male <i>Inr^E19/Inr^GC25</i> for reduced adult mass is after the ninth day of development but before pupariation.</p> <p>(D) Proportion of 17 °C <i>Inr^E19/Inr^GC25</i> larvae pupariating when completely starved at different points in development. The point at which 50% of larvae pupariate in the absence of food marks the critical size. The critical size is reached approximately 40% through the third instar and coincides with the end of the TSP for delayed eclosion and the beginning of the TSP for reduced wing size and adult dry mass (vertical gray bar). All pupariating larvae successfully completed metamorphosis and eclosed as adults.</p></div

Different Organs Respond Differently to Suppression of <i>Inr</i> Activity

<p>Bars show organ area in <i>Inr^E19/Inr^GC25</i> males as a percentage of area in <i>Inr^E19/TM3</i> males, to control for temperature effects. Bars with different letters indicate organs that differ: A, B, and C are significantly different at α = 0.05 (Tukey-Kramer pairwise comparison). Mean areas of all organs given in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030289#st001" target="_blank">Table S1</a>. s.e., standard error.</p

A Reduction in Inr Activity Affects Cell Size and Cell Number Independently

<div><p>(A) At 17 °C, the difference in wing area between <i>Inr^E19/Inr^GC25</i> and <i>Inr^E19/TM3</i> flies is due to a difference in cell size, whereas at 24 °C the difference is due to an additional difference in cell number. Bars show wing area, cell area, and cell number in <i>Inr</i>^E19/<i>Inr</i>^GC25 flies as a percentage of area or number in <i>Inr^E19/TM3</i> flies.</p> <p>(B) At 17 °C the reduced <i>Inr</i> activity in <i>Inr^E19/Inr^GC25</i> flies reduces cell area to approximately 85% the area in <i>Inr^E19/TM3</i> flies, whereas at 24 °C there is no further reduction in cell area, but there is a reduction in cell number to approximately 75% of the number in <i>Inr^E19/TM3</i> flies. Mean wing and cell area, and cell number are given in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030289#st002" target="_blank">Table S2</a>.</p></div

A Model of the Insulin-Signaling Regulation of Growth and Development

<div><p>(A) Under normal conditions, imaginal discs grow to a critical size, which initiates an increase in the ecdysteroid titer. When ecdysteroid levels rise above a maximum threshold, the discs cease cell proliferation and undergo differentiation, fixing their final size. A, adult; E, embryo; L1–L3, first to third larval instar; P, pupa.</p> <p>(B) In <i>Inr</i> mutants, growth of imaginal discs to critical size is slowed, retarding development. When critical size is reached, the ecdysteroid titer again increases, rising above the maximum threshold for cell proliferation in the imaginal discs. Temporal changes in the ecdysteroid titer are unaffected by insulin signaling. Because the rate of cell proliferation is slowed, the imaginal discs are smaller when they begin to differentiate, reducing final organ size. Different discs have different thresholds of sensitivity to ecdysteroid and so cease cell proliferation at different times. Hormones other than ecdysteroids may also be involved.</p></div

Increasing the Temperature of <i>Inr^E19/Inr^GC25</i> Flies Suppresses the Insulin-Signaling Pathway

<div><p>The dFOXO panel shows localization of dFOXO protein in the fat body, the propidium iodide panel shows the position of the nuclei, and the merge panel clarifies the degree of dFOXO localization to the nuclei.</p> <p>(A) Endogenous dFOXO in the fat body of <i>Inr^E19/Inrv^GC25</i> third instar larvae has weak nuclear localization at 17 °C.</p> <p>(B) Increase in rearing temperature causes a decrease in cytoplasmic distribution and an increase in nuclear localization of dFOXO, consistent with a decrease in the level of insulin signaling</p> <p>(C and D) Temperature has no detectable effect on dFOXO localization in <i>Inr^E19/TM3</i> control flies.</p> <p>(E) GPH membrane localization reveals high levels of insulin signaling in the fat body of <i>Inr^E19/Inr^GC25</i> second instar larvae reared at 15 °C. GPH is in green, DNA is stained blue. (F) This localization is lost when the larvae are moved to 25 °C for 12 h, consistent with a decrease in the level of insulin signaling.</p> <p>(G and H) Temperature has no detectable effect on GPH membrane localization in <i>Inr^E19</i>/TM3 control flies.</p></div

Temperature Sensitivity in <i>Inr^E19/Inr^GC25</i> Flies

<p>Increasing the rearing temperature of <i>Inr</i>^E19/<i>Inr^GC25</i> females from 18 °C to 25 °C causes a reduction in wing area from approximately wild-type (Oregon-R [Ore-R]) to that of an insulin pathway mutant <i>(chico)</i>. Wing size is expressed as percentage area of Oregon-R female wing at 25 °C. No <i>Inr^E19/Inr^GC25</i> flies survived rearing at 29 °C. The standard errors are smaller than the markers.</p

F2 data

This data file contains phenotype, genotype and linkage data for 550 F2 individuals resulting from an interspecific cross between C. grandiflora and C. rubella. The individuals were phenotyped in the greenhouse

Ancestry probabilities for scaffold 7

This folder contains ancestry probabilities from MSG v0.3 for markers on scaffold 7. Each file contains ancestry probabilities for 7075 markers for each of 550 F2 individuals. File par1_sc7.tsv contains probabilities for being homozygous C. rubella, par12_sc7.tsv contains probabilities for being heterozygous and par2_sc7.tsv contains probabilities for being homozygous C. grandiflora. Individual order is as in the F2 data file, and these ancestry probabilities were used to fine-map self-compatibility

Elimination of B. aphidicola by Treatment with Antibiotics Has No Effect on the Determination and Maintenance of the Bacteriocyte Cell Fate in A. pisum

<div><p>(A–C) Confocal micrographs of control embryos stained with anti-Dll antibody (red) show expression of Dll, as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000021#pbio-0000021-g001" target="_blank">Figure 1</a>. Enlarged views of the bacteria within the broken white boxes in each embryo are shown in (A′)–(C′).</p> <p>(D–F) Embryos within aposymbiotic aphids at comparable stages as the controls in (A)–(C) express Dll in bacteriocyte nuclei. No bacteria are observed within these embryos, as seen in the enlarged views of (D′)–(F′).</p></div

The Second Wave of Bacteriocyte Determination

<div><p>In (A)–(D), the embryos, which are normally folded in upon themselves in a pretzel shape within the ovariole (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000021#pbio-0000021-Miura1" target="_blank">Miura et al. 2003</a>), have been dissected flat, stained with anti-Dll antibody (red) and phalloidin (green), and examined with a confocal microscope.</p> <p>(A) Dll expression (red) in a stage 14 embryo is detected in the labrum (La) and all developing limbs on the ventral surface except the mandibular segment (Mn). (Other abbreviations: An, antenna; Mx, maxilla; Lb, labium; T1, T2, T3, first, second, and third thoracic leg, respectively.) The dorsal surface of the abdomen of the same embryo is shown illustrating Dll expression in the original bacteriocytes (white arrow) and in a more posterior population of nuclei or cells (blue arrow). Germ cells (gc) are labeled.</p> <p>(B) Dll expression is first observed in the new bacteriocyte nuclei at stage 13.</p> <p>(C) By stage 15, many of the new bacteriocytes have migrated to and begun intercalating between the original bacteriocytes.</p> <p>(D) By stage 16, all of the new bacteriocytes have intercalated between the original bacteriocytes.</p> <p>(E) The migration of the new bacteriocytes is seen in a confocal section of an undissected stage 14 embryo.</p> <p>Embryos in (A)–(D) are oriented with the anterior of the germband towards the left.</p></div