Genome-wide transcriptional profiling in flies exposed to a reduced gravity level in the International Space Station (ISS), or in simulated microgravity (using a Random Positioning Machine, RPM), is severely altered . These important alterations in Drosophila gene expression are intimately linked to imposed spaceflight-related environmental constraints (i.e. uncontrollable temperature during transport, launch and travel to the ISS plus other spaceflight hardware container constraints, such as limited amount of oxygen, light or humidity supply) during Drosophila metamorphosis; the alterations do not appear when similar experiments are performed in optimal environmental conditions .
Two experimental approaches can be used to evaluate the effects of altered gravity. The first of these is to perform experiments in orbit, where the g force is reduced by three orders of magnitude compared to the force of gravity on the ground. However, access to spaceflight opportunities is problematic, expensive and scientifically constrained. The second approach is to use a Ground Based Facility (GBF) which balances the force of gravity, or otherwise neutralizes the effects of gravity on the organism [2, 3]. The effects of Earth’s surface gravity on an organism can be lessened or neutralized by means of a mechanical device that constantly changes the direction of the effective g force (g*) with respect to the sample, i.e. using a classical horizontal 2D clinostat or a random positioning machine (RPM); the latter is a 3D version of the clinostat that continuously randomizes the orientation and speed of rotation [4, 5]. To enhance our understanding of altered gravity effects, we also employ mechanical simulation of a hypergravity environment, i.e., a centrifuge with a large enough radius that shear forces in the sample chamber are reduced to an acceptable level [6, 7]. An example of such a GBF is the Large Diameter Centrifuge (LDC) located at ESA research center in The Netherlands (ESTEC) .
A different kind of reduced gravity simulator, free of the rotational, mechanical and inertial forces generated by spinning simulators and with the advantage of acting at the molecular level, is based on diamagnetic levitation [9–13]. Diamagnetic material, such as water, is repelled from a magnetic field. Since the composition of the majority of biological tissues is largely water, this technique can also levitate living organisms (0g*), with exposures to hypergravity (2g*) and magnetic field control conditions (1g*) simultaneously and in the same environment .
Here, we study the effects of altered gravity conditions on the gene expression profile of Drosophila melanogaster during metamorphosis (3–4 day-long experiments), using three GBFs and whole genome microarray platforms. In addition, we study the effect of applying two environmental constraints to the system, a cold step of three days at 12°C (ΔT) and a containment of the samples in a chamber that reduces the amount of available oxygen (↓O2), both of them not reaching deleterious doses [15, 16]. Both environmental parameters were found to be crucial in previous studies in real and simulated space environments  and in a preliminary attempt at using magnetic levitation as a microgravity simulator with Drosophila. The main and novel conclusion reported here is that Drosophila responses to altered gravity environments are variable, being greatly dependent on environmental conditions and the type of GBF used, despite some common stress response and behavioral effects confirmed by altered expression of related genes.