Effect of the down-regulation of the transcription factor <i>dorsal-1A</i> by RNAi on DWV replication in bees.

<p>(<b>A</b>) <i>Dorsal-1A</i> transcript level in bees fed for different times with a sucrose/protein solution, containing dsRNA of honeybee <i>dorsal-1A</i> (dsRNA Dorsal) or dsRNA of Green Fluorescent Protein (dsRNA GFP) as a control. (<b>B</b>) Deformed wing virus genome copies in bees treated as above. The error bars indicate the standard deviation. The significant rate (H = 7.00, df = 1: <i>P</i> = 0.008) of silencing of the target gene triggered a significant increase (H = 9.61, df = 1: <i>P</i> = 0.002) of viral replication.</p

Seasonal dynamics of bees in colonies with low and high levels of mite infestation.

<p>(<b>A</b>) Estimated bee numbers recorded in each hive in October, when a sudden decrease of bee population was observed in highly infested colonies. (<b>B</b>) Bee mortality over time. The error bars indicate the standard deviation; mean values significantly different are denoted with asterisks (*<i>P</i>≤0.05; **<i>P</i>≤0.01). Bee population in highly infested colonies reached minimum levels in October, because of a marked increase of bee mortality.</p

<i>Varroa</i> infestation and DWV genome copies in infested bees and the effect of viral load on bee survival.

<p>(<b>A</b>) Number of DWV genome copies in honeybees larvae artificially infested with different numbers of <i>V. destructor</i> mites, for different time intervals; the error bars indicate the standard error. (<b>B</b>) Survival of honeybees larvae injected with two different dilutions (1∶1,000 and 1∶100,000) of a whole body lysate of bees with deformed wings (DW) and of bees with normal wings as control (NW). Infestation by the <i>Varroa</i> mite caused increasing number of DWV genome copies in infected bees, this significantly affected bee longevity.</p

Accelerating or ‘threshold’ immuno-suppression by DWV can create bistable DWV dynamics.

<p>The stable (solid line) and unstable (dotted line) equilibrium level of DWV (arbitrary scale) are calculated from equations S4, S5, and plotted as a function of increasing levels of immune depletion (<i>y</i>). Below the dotted line, the virus can be efficiently regulated by the immune-system to some intermediate (potentially cryptic) density, represented by the solid line. Above the dotted line (and for high <i>y</i>, any point to right of intersection with solid line), the virus cannot be efficiently regulated and a viral explosion ensues. Any factor such as mite feeding that depletes the immune system (increasing <i>y</i>) will first cause a gradual increase in copy number, <i>V</i> (moving right along the solid line), and then at a defined point (intersection of solid and dotted lines), a viral explosion will ensue. Parameters are <i>x</i> = 0.09 (<i>y>x</i> ensures that the virus can invade from rare) and <i>z</i> = 0.4.</p

Dorsal expression in virus free and virus infected bees.

<p>Dorsal copies in virus free and virus infected honeybee larvae, either infested or not with one <i>Varroa</i> mite, 12 days after cell sealing; the error bars indicate the standard deviation. Average viral load in infected bee larvae, uninfested or infested by the <i>Varroa</i> mite, was 2.40E+10 and 3.22E+12, respectively. Dorsal expression was significantly reduced in virus infected bees compared to virus free bees, while <i>Varroa</i> infestation did not affect gene expression.</p

Schematic diagram of within-host viral copy number (<i>V</i>) and immune currency (<i>I</i>) dynamics.

<p>The bold lines represent dynamical processes captured explicitly in equations S4, S5. In this model, the viral population dynamics are governed by two antagonistic processes, replication and control (by the immune system). The immune dynamics are in turn governed by three processes; maintenance (increasing immune stocks), stressors (depleting immune stocks) and a specific impact of virally-mediated immune modification (ranging from excitatory to suppressive). The dotted lines represent processes that are external to the model: 1) over-growth of the virus directly leads to increased bee mortality and collapse of the colony (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002735#ppat-1002735-g001" target="_blank">Figures 1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002735#ppat-1002735-g002" target="_blank">2</a>); 2) despite impending collapse within a focal colony, the virus can escape its host via horizontal transmission facilitated by its mite symbiont <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002735#ppat.1002735-Rosenkranz1" target="_blank">[21]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002735#ppat.1002735-Greatti1" target="_blank">[73]</a>; 3) the mite may gain further advantages from its association with an immuno-suppressive virus, as the suppression will further release immunological control of mite feeding; 4) the mite can affect honeybee survival <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002735#ppat.1002735-Rosenkranz1" target="_blank">[21]</a>.</p

Honeybee immune genes showing significant differences (<i>P</i>≤0.05) of their transcription level, as affected by different mite infestation densities.

<p>The gene expression values, as Reads Per Kilobase of exon model per Million mapped reads (RPKM) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002735#ppat.1002735-Mortazavi1" target="_blank">[84]</a>, scored on bees from low infested and highly infested colonies are reported. The “fold change” represents the ratio between the average gene expression value of highly infested colonies and that of low infested ones; values smaller than one indicate a significant transcriptional down-regulation, while those higher than 1 indicate up-regulation. An asterisk marks genes whose differential expression was confirmed by Quantitative Real-Time RT-PCR (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002735#ppat.1002735.s002" target="_blank">Figures S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002735#ppat.1002735.s003" target="_blank">S3</a>). A significant down-regulation of several immune genes was observed in bees from highly infested colonies; the most marked effect was recorded for <i>dorsal-1A</i>, a member of the NF-κB gene family.</p

Mites and DWV in low and highly infested colonies.

<p>(<b>A</b>) Number of mites per 1,000 bees. (<b>B</b>) Seasonal prevalence of Deformed wing virus (DWV) in low and highly infested colonies. (<b>C</b>) Number of DWV genome copies in infected honeybees, collected in September and October from low and highly infested colonies. The error bars indicate the standard deviation; mean values significantly different are denoted with asterisks (*<i>P</i>≤0.05; **<i>P</i>≤0.01). Mite population steadily increased along the season in untreated colonies; DWV prevalence approached 100% at the end of the season both in low and highly infested colonies, but the number of genome copies was much higher in highly infested colonies.</p

A Virulence Factor Encoded by a Polydnavirus Confers Tolerance to Transgenic Tobacco Plants against Lepidopteran Larvae, by Impairing Nutrient Absorption

<div><p>The biological control of insect pests is based on the use of natural enemies. However, the growing information on the molecular mechanisms underpinning the interactions between insects and their natural antagonists can be exploited to develop “bio-inspired” pest control strategies, mimicking suppression mechanisms shaped by long co-evolutionary processes. Here we focus on a virulence factor encoded by the polydnavirus associated with the braconid wasp <i>Toxoneuron nigriceps</i> (<i>Tn</i>BV), an endophagous parasitoid of noctuid moth larvae. This virulence factor (<i>Tn</i>BVANK1) is a member of the viral ankyrin (ANK) protein family, and appears to be involved both in immunosuppression and endocrine alterations of the host. Transgenic tobacco plants expressing <i>Tn</i>BVANK1 showed insecticide activity and caused developmental delay in <i>Spodoptera littoralis</i> larvae feeding on them. This effect was more evident in a transgenic line showing a higher number of transcripts of the viral gene. However, this effect was not associated with evidence of translocation into the haemocoel of the entire protein, where the receptors of <i>Tn</i>BVANK1 are putatively located. Indeed, immunolocalization experiments evidenced the accumulation of this viral protein in the midgut, where it formed a thick layer coating the brush border of epithelial cells. <i>In vitro</i> transport experiments demonstrated that the presence of recombinant <i>Tn</i>BVANK1 exerted a dose-dependent negative impact on amino acid transport. These results open new perspectives for insect control and stimulate additional research efforts to pursue the development of novel bioinsecticides, encoded by parasitoid-derived genes. However, future work will have to carefully evaluate any effect that these molecules may have on beneficial insects and on non-target organisms.</p></div

Immunolocalization of <i>Tn</i>BVANK1 in the midgut of <i>S. littoralis</i> larvae.

<p>In samples obtained from larvae fed on control plants, only a faint hybridization signal is visible (A and B), while an evident positive signal is present on the brush border lining the larval midgut epithelium (C and D) of larvae fed on ANK1 Line 1 plants. Bars: A, C 20 µm; B, D 10 µm.</p