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. 2007 May 30;2(5):e490.
doi: 10.1371/journal.pone.0000490.

Experimental selection for Drosophila survival in extremely low O(2) environment

Dan Zhou  1 , Jin Xue, Jianming Chen, Patrick Morcillo, J David Lambert, Kevin P White, Gabriel G Haddad
Affiliations

Experimental selection for Drosophila survival in extremely low O(2) environment

Dan Zhou et al. PLoS One. .

Abstract

Background: Cellular hypoxia, if severe enough, results usually in injury or cell death. Our research in this area has focused on the molecular mechanisms underlying hypoxic tissue injury to explore strategies to prevent injury or enhance tolerance. The current experiments were designed to determine the genetic basis for adaptation to long term low O(2) environments.

Methodology/principal findings: With long term experimental selection over many generations, we obtained a Drosophila melanogaster strain that can live perpetually in extremely low, normally lethal, O(2) condition (as low as 4% O(2)). This strain shows a dramatic phenotypic divergence from controls, including a decreased recovery time from anoxic stupor, a higher rate of O(2 )consumption in hypoxic conditions, and a decreased body size and mass due to decreased cell number and size. Expression arrays showed that about 4% of the Drosophila genome altered in expression and about half of the alteration was down-regulation. The contribution of some altered transcripts to hypoxia tolerance was examined by testing the survival of available corresponding P-element insertions (and their excisions) under extremely low O(2) conditions. We found that down-regulation of several candidate genes including Best1, broad, CG7102, dunce, lin19-like and sec6 conferred severe hypoxia tolerance in Drosophila.

Conclusions/significance: We have identified a number of genes that play an important role in the survival of a selected Drosophila strain in extremely low O(2) conditions, selected by decreasing O(2) availability over many generations. Because of conservation of pathways, we believe that such genes are critical in hypoxia adaptation in physiological or pathological conditions not only in Drosophila but also in mammals.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Phenotypic changes following long term hypoxia selection.
(A): Shortened recovery time of AF flies from anoxic stupor. Groups of 10 to 15 NF or AF flies were subjected to pure N2 for 5 min (anoxia) at room temperature. Recovery time of each fly from the time of anoxic stupor to that of arousal following reintroduction of room air was recorded. A significantly shortened recovery time was found in AF flies (p<0.01; NF: n = 166, AF: n = 114). (B): Increased O2 consumption rate of AF flies in hypoxic condition. O2 consumption rate was measured in a sealed testing jar at room temperature under normoxia (21% O2, Baseline) and hypoxia (3% O2, Hypoxia), respectively. Groups of 600 to 800 NF or AF flies were used in each test. Although both AF and NF decreased their O2 consumption when switched to hypoxic condition, AF flies reduced less than NF (p<0.01, n = 5). (C and D): Decreased body size and weight in AF flies. AF flies had decreased body weight and size. Body weight of a group of 100 NF or AF male flies were measured at 16th and 17th generation (n = 6) following hypoxia selection. A significant decrease at body weight was found in AF flies (lower panel, p<0.01). Data were presented as mean ± SEM, and the statistical significance was analyzed by student's t-test.
Figure 2
Figure 2. Decreased cell number and size in AF flies.
The wing was used as a model organ to determine whether the weight and size reduction in AF flies could be due to either a reduction in cell number, a reduction in cell size (i.e. increase in cell density) or both. Panel A is representative pictures of wings from an AF (blue) and a NF (red) fly (bar = 250 µm). There was more than 20% reduction in wing area in AF (B; n = 16, p<0.01), that is not able to be fully compensated by a corresponding increase in cell density (C and D; n = 16, p<0.01). Further estimation of the total number of cells in the wing by multiplying the area by the cell density resulted in a total 10.2% cell loss in AF flies. Therefore, the reduction of the fly size is due to decrease of both cell size and cell number. Data were presented as mean ± SEM. The statistical significance was calculated by student's t-test.
Figure 3
Figure 3. Distinct expression clusters between AF and NF samples revealed by microarrays.
cDNA Microarray results were clustered according to the levels of expression of the hypoxia selected and the control cross adult fly samples using GeneCluster . Differentially expressed genes were sharply distinguished between NF and AF flies. Upper panel: a representative subgroup of up-regulated genes. Lower panel: a representative subgroup of down-regulated genes. Yellow color represents relative high levels of expression while blue represents low levels of expression. The brightest color is 1.5-fold or greater differential from the reference black.
Figure 4
Figure 4. Survival of P-element insertion alleles of candidate genes in severe O2 environment.
Survival of single P-element insertion lines for specific candidate genes. Embryos from each P-element insertion line were collected and cultured at 5% O2 condition. Total and eclosed pupae were counted and the ratio of eclosion for each allele was compared to that of yw and NF controls. Each bar represents the average of at least three tests of individual P-element insertion lines. The total number of scored pupae was indicated over each bar. The statistical significance was obtained when p values were <0.001 (Chi-squared test).
Figure 5
Figure 5. Alteration of target gene expression by P-element insertion.
(A–F): Genomic localization of P-element insertions within or around gene Best1, broad, CG7102, dunce, lin19 and sec6. (G): Effect of P-element insertion on target gene expression was determined by sq-RT-PCR. Each open-bar represents the mean value of sqRT-PCR of a P-element allele for the target gene.
Figure 6
Figure 6. Precise-excision of P-elements in genomic region of gene sec6 reverses hypoxia tolerance.
Survival of precise excision lines for P-element insertion targeting gene sec6 was determined in hypoxic condition. Embryos from each precise excision line were collected and cultured in 5% O2. Total and eclosed pupae were counted and the ratio of eclosion for each allele was compared to that of yw, NF controls and original P-element alleles. Each bar represents the average of at least three tests of individual excision line. The total number of scored pupae was indicated over each bar. The statistical significance was obtained when p values were <0.001 (Chi-squared test).
See this image and copyright information in PMC

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