Graphical representation of the groups of larvae used for the microarray and qPCR analysis.

<p>Groups 1 and 2 (Canton-S and Ama-KTT/M/2) were not treated with α-amanitin, as symbolized by the yellow color. The larvae of group 3 (Ama-KTT/M/2) were treated with α-amanitin throughout their development, as indicated in red. Groups 1 and 2 were collected in five, and group 3 in six biological replicates (ten larvae in each replicate), as illustrated by the number of tubes and microarray chips.</p

Ama-KTT/M/2 is not less resistant to α-amanitin than Ama-KTT.

<p>Ten first-instar larvae were placed on each α-amanitin concentration. The dose response curve shows the percentage of hatching flies. Error bars indicate the s.e.m. of three replicates.</p

Genome enrichment analysis for genomic correlates.

<p>Genomic correlates are likely disrupted in Ama-KTT/M/2 versus Canton S (red) and Ama-KTT/M/2 on α-amanitin versus Ama-KTT on non-toxic food (blue). Colored lines above the gray line indicate significant enrichment of a genomic correlate. Of the five genomic correlates rising above the cutoff value, two genomic correlates are similar to those found in previous linkage studies on the Ama-KTT stock.</p

The qPCR results confirm the microarray data.

<p>A) Relative expression distribution (Y-axis) of ten selected genes is shown as a ratio comparing Ama-KTT/M/2 and Canton-S (group 2 versus group 1). Each measurement contains 15 replicates (3 replicates for each of the five biological controls of groups 1 and 2). B) Gene expression differences between Ama-KTT/M/2 treated with α-amanitin and Ama-KTT/M/2 (group 3 versus group 2) are compared. Group 3 contributes to 18 data points (three replicates for each of the six biological controls), while group 2 contributes to 15 data points, as previously mentioned. All comparisons were normalized with two reference genes, <i>Sucb</i> and <i>alpha-Tub84B</i>. Ratios above one indicate that a gene is up-regulated in the comparison.</p

The Mechanisms Underlying α-Amanitin Resistance in <i>Drosophila melanogaster</i>: A Microarray Analysis

<div><p>The rapid evolution of toxin resistance in animals has important consequences for the ecology of species and our economy. Pesticide resistance in insects has been a subject of intensive study; however, very little is known about how <i>Drosophila</i> species became resistant to natural toxins with ecological relevance, such as α-amanitin that is produced in deadly poisonous mushrooms. Here we performed a microarray study to elucidate the genes, chromosomal loci, molecular functions, biological processes, and cellular components that contribute to the α-amanitin resistance phenotype in <i>Drosophila melanogaster</i>. We suggest that toxin entry blockage through the cuticle, phase I and II detoxification, sequestration in lipid particles, and proteolytic cleavage of α-amanitin contribute in concert to this quantitative trait. We speculate that the resistance to mushroom toxins in <i>D. melanogaster</i> and perhaps in mycophagous <i>Drosophila</i> species has evolved as cross-resistance to pesticides, other xenobiotic substances, or environmental stress factors.</p></div

Single gene analysis for Ama-KTT/M/2 versus Canton-S on no toxin (group 2 versus 1).

<p>Type I and II detoxification, <i>Mdr</i>, and transcription factor genes with possible functions in detoxification processes are shown. The at least 2-fold differentially expressed genes are sorted by positive and negative fold-changes, followed by the genes that are not significantly differentially expressed. All p-values are corrected. The chromosomes, FlyBaseID, and probe ID numbers are presented.</p

Single gene analysis for Ama-KTT/M/2 on α-amanitin versus Ama-KTT/M/2 on no toxin (group 3 versus 2).

<p>Type I and II detoxification, <i>Mdr</i>, and transcription factor genes with possible functions in detoxification processes are shown, sorted by positive and negative fold-changes. The at least 2-fold differentially expressed genes are sorted by positive and negative fold-changes, followed by the genes that are not significantly differentially expressed. All p-values are corrected. The chromosomes, FlyBaseID, and probe ID numbers are presented.</p

Domain enrichment analysis for Ama-KTT/M/2 on α-amanitin versus Ama-KTT/M2 on no toxin (group 3 versus 2).

<p>This table shows the selected and significantly enriched domains in response to toxin treatment. “DEGs w/domain” are the differentially expressed genes that have a particular domain. “DEGs” is the number of all differentially expressed genes in this comparison. “Genes w/domain” is the total number of genes with a particular domain in the genome. “Genes” is the total number of genes in the genome. All p-values are corrected.</p

Domain enrichment analysis for Ama-KTT/M/2 versus Canton-S on no toxin (group 2 versus 1).

<p>This table shows the selected and significantly enriched domains without toxin treatment. “DEGs w/domain” are the differentially expressed genes that have a particular domain. “DEGs” is the number of all differentially expressed genes in this comparison. “Genes w/domain” is the total number of genes with a particular domain in the genome. “Genes” is the total number of genes in the genome. All p-values are corrected.</p

Crossing scheme for the generation of the isochromosomal lines.

<p>One highly resistant virgin female of each original Asian fly stock was mated with two males of the multi-balancer stock. F1 generation males that carried an Ama chromosome 2 balanced over CyO and an Ama chromosome 3 balanced over TM6B, <i>Tb</i> or MKRS were crossed back to one multi-balancer virgin female. F2 generation males carrying an Ama chromosome 2 balanced over CyO and an Ama chromosome 3 balanced over TM6B, <i>Tb</i> were back-crossed to one multi-balancer virgin female. Virgin siblings of the F3 generation were then crossed to produce the isochromosomal lines.</p