In prospective human exploration of outer space the need to maintain a species over several generations under changed gravity conditions may arise. This paper reports the analysis of the third generation of fruit fly Drosophila melanogaster obtained during the 44.5-day space flight (Foton-M4 satellite 2014 Russia) followed by the fourth generation on Earth and the fifth generation under conditions of a 12-day space flight (2014 in the Russian Segment of the ISS). The obtained results show that it is possible to obtain the third-fifth generations of a complex multicellular Earth organism under changed gravity conditions (in the cycle weightlessness - Earth - weightlessness) which preserves fertility and normal development. However there were a number of changes in the expression levels and content of cytoskeletal proteins that are the key components of the spindle apparatus and the contractile ring of cells.

Genome-wide transcriptional profiling shows that reducing gravity levels in the International Space Station (ISS) causes important alterations in Drosophila gene expression. However simulation experiments on ground without space constraints show weaker effects than space environment. A global and integrative analysis using the gene expression dynamics inspector (GEDI) self-organizing maps reveals a subtle response of the transcriptome using different populations and microgravity and hypergravity simulation devices. These results suggest that in addition to behavioural responses that can be detected also at the gene expression level the transcriptome is finely tuned to normal gravity. The alteration of this constant parameter on Earth can have effects on gene expression that depends both on the environmental conditions and the ground based facility used to compensate the gravity vector. Alternative and commons effects of mechanical facilities like the Random Positioning Machine and a centrifuge and strong magnetic field ones like a cryogenically cooled superconductive magnet are discussed. We compare the effects over the gene expression profile of different gender/age Drosophila imagoes in 3-4 days-long experiments under altered gravity conditions into three GBF (Ground Based Facilities for micro/hyper- gravity simulation) using whole genome microarray platforms. Descriptions of different GBFs (treatments): LDC means Large Diameter Centrifuge. Samples can be placed under three conditions: inside LDC (at certain g level) at the LDC rotational control and at external 1g control (outside the LDC). RPM means Random Positioning Machine. Samples can be placed under two conditions: inside RPM (at nearly 0g Microgravity level) and at external 1g control (outside the RPM). At the magnet means INSIDE the Magnetic levitator (another GBF). Samples can be placed under four conditions: inside Magnet 0g* (at microgravity with magnetic field) inside Magnet at 1g* (internal control with magnetic field) or inside the magnet 2g* (at hypergravity with magnetic field) and at external 1g control (outside the magnet)

Genome-wide transcriptional profiling showed that reducing gravity levels in the International Space Station (ISS) causes important alterations in Drosophila gene expression intimately linked to imposed spaceflight-related environmental constrains during Drosophila metamorphosis. However simulation experiments on ground testing space-related environmental constraints show differential responses. Curiously although particular genes are not common in the different experiments the same GO groups including a large multigene family related with behavior stress response and organogenesis are over represented in them. A global and integrative analysis using the gene expression dynamics inspector (GEDI) self-organizing maps reveals different degrees in the responses of the transcriptome when using different environmental conditions or microgravity/hypergravity simulation devices. These results suggest that the transcriptome is finely tuned to normal gravity. In regular environmental conditions the alteration of this constant parameter on Earth can have mild effects on gene expression but when environmental conditions are far from optimal the gene expression is much more intense and consistent effects.

Drosophila samples were exposed to the levitation magnet inside a 25mm diameter tubes with 3 ml of yeast-based Drosophila food in the bottom and a chamber of only 5 mm of height over the food. This small region is needed in order to guarantee that all the flies were located in the effective g area so a maximum of 35 to 40 imagos or pupa can be exposed to each condition per experiment. All experiments were carried out with a parallel 1g external control in a temperature regulated incubator outside the magnet. Three experiments of different duration were performed inside the magnet system to analyze the effect of strong magnetic fields and magnetic levitation during different stages of the Drosophila development

Genome-wide transcriptional profiling showed that reducing gravity levels in the International Space Station (ISS) causes important alterations in Drosophila gene expression intimately linked to imposed spaceflight-related environmental constrains during Drosophila metamorphosis. However simulation experiments on ground testing space-related environmental constraints show differential responses. Curiously although particular genes are not common in the different experiments the same GO groups including a large multigene family related with behavior stress response and organogenesis are over represented in them. A global and integrative analysis using the gene expression dynamics inspector (GEDI) self-organizing maps reveals different degrees in the responses of the transcriptome when using different environmental conditions or microgravity/hypergravity simulation devices. These results suggest that the transcriptome is finely tuned to normal gravity. In regular environmental conditions the alteration of this constant parameter on Earth can have mild effects on gene expression but when environmental conditions are far from optimal the gene expression is much more intense and consistent effects.

Physical forces greatly influence the growth and function of an organism. Altered gravity can perturb normal development and induce corresponding changes in gene expression. Understanding this relationship between the physical and biological realms is important for NASA's space travel goals. We use combined RNA-Seq and qRT-PCR to profile changes in early Drosophila melanogaster pupae exposed to chronic hypergravity (3 g, three times Earth's gravity) to highlight gravity-dependent pathways and gene products. Robust transcriptional response was evident among the pupae developed in a hypergravity environment compared to control. 1,513 genes showed significantly (p less than 0.05) altered gene expression in the 3 g samples. These findings were supported with qRT-PCR data. Major biological processes affected include ion transport, redox homeostasis, immune and humoral stress response, proteolysis, and cuticle development.

Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen in parallel the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground as well as plus and minus hyperosmolar sodium chloride providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state suggesting mechanisms by which spaceflight may negatively affect cell fitness.

Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen in parallel the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground as well as plus and minus hyperosmolar sodium chloride providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state suggesting mechanisms by which spaceflight may negatively affect cell fitness.

Gene expression levels were determined in 3rd instar and adult Drosophila melanogaster reared during spaceflight to elucidate the genetic and molecular mechanisms underpinning the effects of microgravity on the immune system. The goal was to validate the Drosophila model for understanding alterations of innate immune responses in humans due to spaceflight. Five containers of flies with ten female and five male fruit flies in each container were housed and bred on the space shuttle (average orbit altitude of 330.35 km) for 12 days and 18.5 hours with a new generation reared in microgravity. RNA was extracted on the day of shuttle landing from whole body animals (3rd instar larvae and adults) hybridized to Drosophila 2.0 Affymetrix genome arrays and the expression level of all genes was normalized against the gene expression level from the corresponding developmental stage animals raised on ground. Spaceflight altered the expression of larval genes involved in the maturation of plasmatocytes (macrophages) and their phagocytic response as well as the level of constitutive expression of pattern recognition receptors and opsonins that specifically recognize bacteria and of lysozymes antimicrobial peptide pathway and immune stress genes hallmarks of humoral immunity. Larval microarrays (FL 6 samples) are based on RNA extracted from 6 independent sets of 50 mid 3rd instar larvae reared in microgravity and collected on the day of landing after 12 days and 18.5 hours on the space shuttle and the same number of control larvae raised on ground (GL 6 samples). Adults microarrays (F1 3 samples) are based on RNA from 3 sets of 20 adult females each that emerged during spaceflight and within 4 hours of landing and the same number of adult females from the corresponding ground control containers (G1 3 samples).

The spaceflight experiment was carried out using male C57BL/10J mice (8 weeks old at launch). Wild type mice (n=3) were launched by Space Shuttle Discovery and housed on the International Space Station (ISS) for 91 days. They returned to the Earth by Space Shuttle Atlantis. But only one mouse returned to the Earth alive. Whole brain was sampled from the mouse killed by inhalation of carbon dioxide at the Life Sciences Support Facility of Kennedy Space Center within 3-4 hours after landing. After the spaceflight experiment the on-ground experiment was also carried out at the Advanced Biotechnology Center in Genova Italy. A mouse with the same species sex and age was housed in mice drawer system (MDS) which was utilized for the spaceflight (SF) mice for 3 months as the ground control (GC). Another mouse was housed in normal vivarium cage as the laboratory control (LC). Amount of food and water supplementation and environmental conditions were simulated as the flight group. After 3 months brain was sampled from one mouse in group GC and LC respectively. Comprehensive analyses of gene expression were performed in the right brain. Total of 4,000 genes were analyzed. The expression levels of 60 genes significantly changed in response to SF compared with LC and/or GC. The 15 and 16 genes were up- (> 2 folds) and down-regulated (< 0.5 folds) respectively following SF vs. GC. The levels of 58 genes were significantly altered by housing in MDS in space and/or on the ground. Forty seven and 11 genes were significantly up- and down-regulated vs. LC. Twenty seven out of these genes responded to caging in MDS both in space and on the ground. Further 31 genes were influenced by housing in MDS on the Earth. Responses of the characteristics of brain to long-term gravitational unloading were investigated in mice.