Reviewer 2's Review

“The manuscript by Zhou et al addresses the interesting problem of genetic factors essential for the response of organisms and eventual adaptation to long term hypoxia. Therefore, the authors tackle a multigene-regulated physiological process and demonstrate its genetic plasticity in the context of a drastic environmental change, but also identify at least some of the genes behind this change. The authors have utilized two powerful approaches to find such genes. First, a successful genetic selection scheme which clearly demonstrates that strains that can grow and reproduce under extremely low oxygen (4%) conditions. Second, a microarray approach complemented by analysis of mutants in genes identified in this screen. The combined results firmly establish the genetic basis of resistance to hypoxia and identified a number of genes involved in the process (es). Because this reviewer considers the findings of this manuscript of broad interest a number of changes and clarifications in the manuscript are recommended.
A. Methods and results
1. Was there a method to ascertain that the F1 produced by crossing the individual DMN lines represented progeny from these isogenic lines? It is curious that DMN 6, DMN12 and especially DMN20 exhibit eclosion rates under 5% O2 with out selection as some mutant lines do. Most notably, KG04048 and EY0062 of the Broad complex, both dnc alleles and two of the four lin19 alleles (BG02329 and EY11668). Could it be that the genome of selected hypoxia resistant line(s) ia derivative mostly of those of the DMN6, DMN12 and DMN20 lines? Do DMN6, DMN12 and DMN20 already carry mutations in any the genes identified in the mutant screen? This reviewer thinks that the authors should minimally comment on these questions.
2. The size of males flies (Figure 1C) is shown to be reduced (but the rationale of using male flies for this is not presented) and we are led to assume that females follow suit. Is that so? Is the size of females more variable and if so why?
3. There is no mention of the source of the microarrays in the manuscript.
4. The rationale of starting the selection at 8% O2 is not clear
5. It is unclear what recovery time from anoxic stupor (Figure 1A) measures, other than altered physiology. Also the higher rate of oxygen consumption under hypoxia (Figure 1B) appears marginal to this reviewer and warrants commenting upon by the authors.
6. Pg 11 line 15. The authors state that the density of the tracheal vessels is increased, but it is not clear this is something they hypothesize is a consequence of a compact body size, or an actual observation they made. If the former is true, it is unclear why a smaller body would not have less or smaller (in diameter) tracheae. This is a parameter that can be investigated with relative ease and may prove quite pertinent to the mechanism of hypoxia resistance.
7. Figure 5. Although the transcripts of Best1,lin19 and sec6 appear reduced with respect to the yw control, their levels in the various mutant alleles of each gene do not appear to correspond at least with the eclosion phenotypes shown in Figure 4. For example, the lin19BG02329 appears to contain very little of the transcript and exhibits a 30% eclosion in contrast to lin195-HA-1051 which appears to contain a lot more transcript, but exhibits a much higher eclosion rate. The authors should comment on these observations as well as on the number of independent experiments that the RT-PCR results on that figure represent. Quantitative PCR results would likely resolve these issues quite nicely and provide a much more precise estimate of the mRNA levels from these genes in the mutant flies.
B. Discussion.
In general this reviewer thinks that the findings of the microarray experiments and the genetic tests that followed are not discussed adequately.
1. What is the relationship (if any) of the genes that regulate the response to acute anoxia (references 6, 7, 8, 9) to the ones identified here as factors of adaptability to long term hypoxia?
2. The genes with respect to their possible function and potential linkage to the phenotype under study of the up-regulated class are not discussed at all. What kinds of genes are represented by the 25% of mutants in genes of this class that do not die under hypoxic conditions? (page 13 line 6).
3. Similarly, the authors must comment on the nature of sec6 and how they envision that a synaptic vesicle protein might affect hypoxia tolerance. The same is true for genes like dnc, br, Best1 etc... Do the authors envision that high cAMP (in the dnc mutant) is essential for hypoxia tolerance?”

n.b. These are the general comments made by the reviewer when reviewing this paper in light of which the manuscript was revised. Specific points addressed during revision of the paper are not shown.