Cell-cell fusions occur in <i>par-5</i> loss-of-function mutants.

<p>Time-lapse images of a <i>par-5(it55)</i> embryo show multiple cell-cell fusion events, as seen in 100% of observed embryos (n = 3). A cytoplasmic reporter, <i>lbp-1p</i>::<i>gfp</i>, is specific to a subset of hypodermal cells that fuse to form hyp7. Three noticeable fusions events (t = 0–10, t = 60–70, t = 80–90) are apparent by a decrease in GFP fluorescence (solid arrow) of a brighter cell (dashed arrow) fusing with darker neighbors. Timepoints 110–130 reveal an adjacent bright cell (t = 110, dashed arrow) and dark cell that fuse with each other (t = 120, solid arrow and asterisk) to form a binucleated cell (dashed bracket). This binucleate cell subsequently dims while fusing with other neighboring cells (t = 130, solid bracket, decrease in fluorescence). Shortly after, one additional fusion event occurs (t = 130–140). Images shown are maximum intensity Z-projections of 27 one-micron-spaced confocal optical sections through the entire embryo, captured at 10-minute intervals. Posterior is lower-left and dorsal is lower-right. Scalebar = 10 μm.</p

<i>elt-3p</i>::<i>yfp</i> reporter of cell-cell fusions.

<p>Time-lapse images of a wild-type embryo (top panel) expressing the <i>elt-3p</i>::<i>yfp</i> cytoplasmic hypodermis-specific reporter show cell-cell fusions in the developing epidermis. Fusion pores are revealed by diffusion of YFP from labeled cells to neighboring unlabeled cells. Both white and yellow arrows denote the anterior and posterior limits of each successively expanded multinucleated cell during the stepwise fusion events that create the large hyp6 and hyp7 syncytia. Yellow arrows show specific fusion events that were monitored in optical-section time-lapse recordings of mutant and rescued genotypes in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146874#pone.0146874.g005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146874#pone.0146874.g007" target="_blank">7</a>. The <i>elt-3p</i>::<i>yfp</i> pattern of cytoplasmic and nuclear fluorescence in the dorsal hypodermis of the <i>eff-1</i> mutant (bottom panel) remains variegated throughout embryonic elongation. Images are maximum intensity projections of 27 one-micron-spaced confocal optical sections through the entire embryo shown at 5-minute intervals. Scalebar = 5 μm.</p

EFF-1(S632/634/654A)::GFP localization patterns are similar to wild-type EFF-1::GFP <i>in vivo</i>.

<p>Time-lapse images of EFF-1(S632/634/654A)::GFP show spatiotemporal localization to fusion competent ventral (A) cell borders (arrow) in the absence of phosphorylatable residues within putative 14-3-3 binding sites compared to wild-type EFF-1::GFP (B). One-micron-spaced image stacks were captured every 2.5 minutes using widefield (A) or confocal (B) microscopy, and maximum intensity Z-projections of the ventral surface were rendered. In 100% of the mutant embryos (n = 3), the same pattern of junctional localization is seen as for wild-type EFF-1::GFP (B) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146874#pone.0146874.ref038" target="_blank">38</a>]. Anterior is up and posterior is down. Times shown are in minutes.</p

Larval hypodermal cell fusions occur normally in 14-3-3 double mutants.

<p>Double-knockdown mutant phenotype was generated using <i>par-5-</i>specific RNAi on <i>ftt-2(n4426Δ)</i> null mutant animals. Cells fated to fuse into the hyp7 syncytium of L4 larvae are labeled with <i>elt-3p</i>::<i>yfp</i> in three different genotypes: wild-type (A), 14-3-3 double knockdown (B), and <i>eff-1(zz10)</i> null mutant (C). In panels A and B, fields of syncytial hyp7 cytoplasm and nuclei display even and continuous distribution of YFP (arrows), as seen in 100% of observed larvae (n = 12 and 4 respectively). In the <i>eff-1</i> null larva in panel C, arrows indicate labeled hypodermal cells that have failed to fuse with hyp7 (arrowheads), as seen in in 100% of observed larvae (n = 6). Scalebar = 10 μm.</p

AJM-1::GFP reporter of cell-cell fusions.

<p>Time-lapse images of a wild-type embryo expressing a sub-adherens junction marker, AJM-1::GFP, show the disappearance of borders between fused cells (arrows). An <i>eff-1</i> mutant embryo (inset) shows no cell fusions at a timepoint past that at which most fusions are completed in wild-type embryos. Anterior is left and dorsal is facing the viewer (t = 380–410) or oriented up (t = 420–460). Images shown are maximum intensity Z-projections of 27 one-micron-spaced confocal optical sections through the entire embryo, captured at 10-minute intervals. Scalebar = 10 μm.</p

Cell-cell fusions in the absence of FTT-2.

<p><i>Ftt-2</i> loss-of-function mutants show no disruption in the reproducible timing, position or orientation of cell-cell fusions in the developing epidermis. (A) Time-lapse images of a <i>ftt-2(n4426Δ)</i> null embryo expressing <i>elt-3p</i>::<i>yfp</i> show a hallmark of cell-cell fusion, the diffusion of YFP from labeled cells (solid arrows) to neighboring unlabeled cells (dashed arrows). Pattern observed in 100% of embryos (n = 5). (B) Time-lapse images of a <i>ftt-2</i> null embryo expressing a sub-adherens junction marker, AJM-1::GFP, show the disappearance of borders between fused cells (arrows). Pattern observed in 100% of embryos (n = 2). Anterior is left and dorsal is facing the viewer (t = 380–420) or oriented up (t = 430–450). Images shown are maximum intensity Z-projections of 27 one-micron-spaced confocal optical sections through the entire embryo, captured at 10-minute intervals. Scalebar = 10 μm.</p

<i>eff-1(zz1)</i> mutants display a growth defect and are delayed in embryonic hyp7 cell fusions.

<p>(A) Normalized body-length measurements from 96-hour-old adult worms carrying distinct mutations in <i>eff-1</i>. Genotype-matched control strains used for each normalization were: N2 (Bristol) for alleles <i>oj55</i>, <i>hy21</i>, <i>zz10</i>, and <i>ku433</i>; C55584 (<i>mIs12 II)</i> for <i>zz1</i> and <i>zz8</i>; SU93 (<i>jcIs1)</i> for <i>zz7</i>. Mean and standard deviation are shown (n = 17–76). (B) Confocal volume projection of a 1.5-fold <i>eff-1(zz1)</i> mutant embryo expressing AJM-1::GFP. Arrows show sites in hyp7 where AJM-1::GFP has disappeared, indicating the final stages of fusion between 2 pairs of cells. Arrowheads show intact cell junctions between hyp7 cells that are fusion-delayed. A wild-type AJM-1::GFP embryo at an equivalent embryonic stage is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146874#pone.0146874.g004" target="_blank">Fig 4</a> (t = 450) where arrows show fused junctions. Anterior is left and dorsal is up. Scalebar = 10 μm. (C) Average number of fused hyp7 cell borders seen before the beginning of embryonic movement (~1.5 fold stage) in wild-type (n = 5) and <i>eff-1(zz1)</i> (n = 13) embryos. Error bars show standard error of the mean. *p<0.001 in an independent t-test.</p

Erythritol, at insecticidal doses, has harmful effects on two common agricultural crop plants

<div><p>Erythritol, a non-nutritive polyol, is the main component of the artificial sweetener Truvia^<b>®</b>. Recent research has indicated that erythritol may have potential as an organic insecticide, given its harmful effects on several insects but apparent safety for mammals. However, for erythritol to have practical use as an insecticide in agricultural settings, it must have neutral to positive effects on crop plants and other non-target organisms. We examined the dose-dependent effects of erythritol (0, 5, 50, 500, 1000, and 2000 mM) on corn (<i>Zea mays</i>) and tomato (<i>Solanum lycopersicum</i>) seedling growth and seed germination. Erythritol caused significant reductions in both belowground (root) and aboveground (shoot) dry weight at and above the typical minimum insecticidal dose (500 mM erythritol) in tomato plants, but not in corn plants. Both corn and tomato seed germination was inhibited by erythritol but the tomato seeds appeared to be more sensitive, responding at concentrations as low as 50 mM erythritol (in contrast to a minimum damaging dose of 1000 mM erythritol for corn seeds). Our results suggest erythritol may have damaging non-target effects on certain plant crops when used daily at the typical doses needed to kill insect pests. Furthermore, if erythritol’s damaging effects extend to certain weed species, it also may have potential as an organic herbicide.</p></div

Seedling growth (measured as dry weight) and seed germination of corn and tomato exposed to 5 different erythritol treatments and a deionized water control.

<p>Dry weight: n = 11 replicates per treatment group for each species; seed germination: n = 3 replicate petri dishes (with 4 seeds per petri dish) per treatment group for each species.</p

Erythritol delays seed germination in corn.

<p>Mean (± 1 SE) number of days to seed germination for A) corn and B) tomato over 18 days at 6 different treatment concentrations of erythritol. For each species, seeds were treated in petri dishes (4 seeds per dish; 3 dishes per treatment group) and the mean days to germination was calculated for each petri dish. Note that no seeds germinated in the 2000 mM treatment group for corn and the 500 mM, 1000 mM, and 2000 mM treatment groups for tomato, so these treatment groups were not included in the analysis. Means with the same letter are not significantly different from each other (Tukey HSD test, p < 0.05).</p