Comment on Rohrscheib et al. 2016 "Intensity of mutualism breakdown is determined by temperature not amplification of Wolbachia genes"

Rohrscheib et al. (PLOS Pathogens, 2016) [1] discuss the interaction between the pathogenicity of the wMel variant wMelPop, temperature and Octomom copy number. The effect of temperature on wMelPop pathogenicity was already reported in the original work on wMelPop [2]. The absence of pathogenicity at low temperatures was also shown before [3]. We have recently demonstrated, in Chrostek and Teixeira 2015, that Octomom copy number determines wMelPop pathogenicity [4]. Rohrscheib et al. claim to provide evidence that our main conclusions were wrong. We disagree and our reasoning is outlined in the points below. 1) In the first set of experiments Rohrscheib et al. show that wMelPop pathogenicity in Drosophila melanogaster varies with temperature, being stronger at higher temperatures. This confirms previous results [2,3], which were acknowledged and applied in our work (Fig. 3E and Fig. S5H of Chrostek and Teixeira [4]). The authors also show that Wolbachia titers vary with temperature, being higher at higher temperature. Finally, Rohrscheib et al. also analyze Octomom copy numbers in flies kept at different temperatures. Based on a combination of these analyses the authors conclude that wMelPop pathology or levels are not dependent on Octomom copy number. We disagree with the argument presented to justify the approach outlined above to study the interaction between Octomom copy number and wMelPop pathology: “(. . .), if the Octomom copy number determines Wolbachia density, and consequently pathology, we would expect to observe a decrease in Octomom copy number as the extrinsic temperature decreases”. Temperature interacting with pathogenicity does not imply that temperature influences a genetic determinant of pathogenicity—Octomom copy number. Environment can influence phenotype without affecting the genotype, as is the case in many examples of phenotypic plasticity. The setup presented by Rohrscheib et al. does not test the influence of Octomom copy number on wMelPop pathogenicity correctly. Temperature is a confounding factor already known to influence both Drosophila lifespan and wMelPop pathogenicity. To directly test the effect of one variable (Octomom copy number) over a phenotype (pathogenicity or Wolbachia levels), the variable should be manipulated and tested under stable environmental conditions in which the pathogenicity is known to be expressed. Using this approach we have shown that Octomom copy number determines wMelPop pathogenicity at two different temperatures (25 ̊C and 29 ̊C). 2) Rohrscheib et al. show that Octomom copy numbers change throughout life of the host. We have previously shown that Octomom copy number is highly variable between flies. In