Selection of Drosophila altered behaviour and aging strains for Microgravity Research.

Manuscript version accepted by the publisher.Some years ago Prof. Marco and his team discovered that Drosophila show noticeably different behaviour as well as accelerated aging in Space (1-4). It was also proven that by simulating these conditions on Earth, it was possible to replicate these phenomena (2,5,6). In order to demonstrate how closely connected the enhanced motility and the accelerated aging are, five Drosophila strains have been selected, each behaving differentially under altered gravity conditions. Three of these strains have been selected some years ago and reselected over the last six months according to their gravitropic response in a vertically-oriented ten-exit maze for 25 generations. The behaviour of flies at 1g resembles an exaggerated escape response to gravity, a neutral behaviour to gravity and a negative (attractive) behaviour to gravity, respectively. The other two strains have been selected over several decades according to the longevity of their parents to experimentally produce a short and a long-life strain. The five selected strains are scored against a gravitropic index, median survival and median motility in 1g conditions to evaluate their usability as microgravity research tools. Preliminary experiments have been performed at ESTEC/DESC altered gravity facilities (hypergravity and simulated microgravity). The establishment of these strains will provide science with an exciting new tool in Space Biology and their future utilization on space missions or long term stays in the International Space Station is promising

Cell cycle and nuclear changes under microgravity

50 p.-8 fig.-8 fig. supl.-2 tab.supl.Zero-gravity is an environmental challenge unknown to organisms throughout evolution on Earth. Nevertheless, plants are sensitive to altered gravity, as exemplified by changes in meristematic cell proliferation and growth. We found that synchronized Arabidopsis cultured cells exposed to simulated microgravity showed a shortened cell cycle, caused by a shorter G2/M phase and a slightly longer G1 phase. The analysis of selected marker genes and proteins by qPCR and flow cytometry in synchronic G1 and G2 subpopulations indicated changes in gene expression of core cell cycle regulators and chromatin-modifying factors, confirming that microgravity induced misregulation of G2/M and G1/S checkpoints and chromatin remodeling. Changes in chromatin-based regulation included higher DNA methylation and lower histone acetylation, increased chromatin condensation and overall depletion of nuclear transcription. Estimation of ribosome biogenesis rate using nucleolar parameters and selected nucleolar genes and proteins indicated reduced nucleolar activity under simulated microgravity, especially at G2/M. These results expand our knowledge of how meristematic cells are affected by real and simulated microgravity. Counteracting this cellular stress is necessary for plant culture in space exploration.This work was supported by the Spanish “Plan Estatal de Investigación Científica y Técnica y de Innovación” of the Ministry of Economy, Industry and Competitiveness [Grant numbers AYA2012-33982 and ESP2015-64323-R, co-funded by ERDF], by a predoctoral fellowship to [Kh.Y.K.] from CSIC, Spain [JAE-PreDoc Program, Ref. JAEPre_2010_01894] the ESA-ELIPS Program [ESA SEGMGSPE_Ph1 Project, contract number 4200022650], and ESA support via contract TEC-MMG / 2012/263.Peer reviewe

Gene Expression Variations During Drosophila Metamorphosis in Space. The GENE Experiment in the Spanish Cervantes Mission to the ISS

Manuscript long version, prior to reduction due to the mandatory final 2-pages published versionThe ISS expedition 8, a 10 days “taxi” flight Soyuz Mission to the International Space Station (ISS) to replace the two-member ISS crew, took place during October 2003. Within the Spanish Cervantes Scientific Mission, in this crew exchanging flight, some biological experiments were performed. The third member of the expedition, the Spanish born ESA astronaut Pedro Duque, returned with the Soyuz 7 capsule and the experiment containing transport box after 11 days on microgravity. In the GENE experiment, we intended to determine how microgravity affects the organism rebuilding processes that occurs during Drosophila metamorphosis. In addition to the ISS samples, some control experiments were performed including a 1g Ground control parallel to the ISS flight samples, a Random Position Machine microgravity si m u l a t e d c o n t r o l a n d a p a r a l l e l Hypergravity (10g) exposed samples experiment. We have used extracted RNA from these samples to test the differences among gene expression during Drosophila development with one of the current more powerful technology, a Drosophila complete genome microarray (version 1.0, AffymetrixTM). A preliminary analysis of the results indicates that around five hundred genes change their expression profiles being especially affected the mitochondrial ribosomal ones.Peer reviewe

Seed germination and seedling growth under simulated microgravity causes alterations in plant cell proliferation and ribosome biogenesis

5 páginas -- PAGS nros. 169-174The study of the modifications induced by altered gravity in functions of plant cells is a valuable tool for the objective of the survival of terrestrial organisms in conditions different from those of the Earth. We have used the system “cell proliferation–ribosome biogenesis”, two inter-related essential cellular processes, with the purpose of studying these modifications. Arabidopsis seedlings belonging to a transformed line containing the reporter gene GUS under the control of the promoter of the cyclin gene CYCB1, a cell cycle regulator, were grown in a Random Positioning Machine, a device known to accurately simulate microgravity. Samples were taken at 2, 4 and 8 days after germination and subjected to biometrical analysis and cellular morphometrical, ultrastructural and immunocytochemical studies in order to know the rates of cell proliferation and ribosome biogenesis, plus the estimation of the expression of the cyclin gene, as an indication of the state of cell cycle regulation. Our results show that cells divide more in simulated microgravity in a Random Positioning Machine than in control gravity, but the cell cycle appears significantly altered as early as 2 days after germination. Furthermore, higher proliferation is not accompanied by an increase in ribosome synthesis, as is the rule on Earth, but the functional markers of this process appear depleted in simulated microgravity-grown samples. Therefore, the alteration of the gravitational environmental conditions results in a considerable stress for plant cells, including those not specialized in gravity perceptionPeer reviewe

The “Gene” Experiment in the Spanish Soyuz Mission to the International Space Station. II. Effects of oxygen concentration constrain

This is a manuscript version. The Final online version of this article (http://www.springerlink.com/content/m21585633538t8q3/) contains supplementary material, which is available to authorized users.In the GENE experiment performed during an 11-day Soyuz Mission to the International Space Station (ISS), we intended to determine if microgravity affects Drosophila metamorphosis processes. Control experiments were performed including a 1g ground control parallel to the ISS flight samples and a Random Position Machine microgravity simulated control. A preliminary analysis of the results indicates that five hundred to one thousand genes change their expression profiles depending on the cut-off levels selected. Especially affected among them are the mitochondrial ones (an example with the respiratory chain is presented). We show here that there is a synergic effect of the constraints introduced to meet the requirements of the space experiment (mainly, a cold step and the use of hermetically closed Type-I containers). The cold transport step to the launch site was introduced to slow down the pupal development. The hermetically closed Type I containers were required to ensure the containment of the fixative (acetone) in the experiment. As shown here, the oxygen concentration inside the container was not optimal but fully compatible with pupal development. It is highly likely that such combined environmental effects will become a common finding in these types of studies as they become more complicated and extensive. They could open the way to understand how the gene expression patterns and the actual phenotypes can adjust to the environment. These findings indicate the importance of a vigorous ground based program in support of real microgravity experiments. Only then we can utilize the ISS in order to understand the consequences of the modified environment in outer space on living organisms.Peer reviewe

Drosophila Behaviour & Gene expression in altered gravity conditions: Comparison between Space and ground facilities

Manuscript versionPrevious experiments in space (unmanned satellites, space shuttle and the International Space Station, ISS), have shown that adult Drosophila flies change their motile behaviour in microgravity. A consistent increase in motility in space was found in these experiments, but mature flies (two weeks old) showed less increase than recently hatched flies. In the case of relatively long exposure to microgravity, the aging of male flies measured upon return to Earth was increased, with flies dying earlier than the corresponding in-flight 1g centrifuge or ground controls. The older flies, which experienced a smaller increase in motility, did not show this acceleration in the aging process. More recently we have performed comparative experiments using ground simulation facilities. Preliminary experiments using a random positioning machine (RPM) indicate that the effects of this simulation approach on the behavior of Drosophi l a a r e o f s m a l l e r m a g n i t u d e t h a n t h e corresponding exposure to real microgravity. Further experiments are in progress to confirm this effect. However, when exposed to magnetic levitation, flies exposed to simulated weightlessness increased markedly their motile behavior compared with 1g controls both inside and outside the magnet. This altered gravity-related increase in motility was also less pronounced in more mature flies. This motility effect at the levitation position reproduces the results in real microgravity indicating the interest for space science of this simulation approach. Similar experiments are being performed in the Larger Diameter Centrifuge (LDC) located in ESTEC (the Netherlands) and indicate that 6g, 12g and 20g are key points in the hypergravity response in flies. Our experiments have shown that developmental processes from embryo to adult proceeded normally in the magnet, the RPM and the LDC. In terms of gene expression, preliminary results i n d i c a t e t h a t t h e a f f e c t e d s e t o f g e n e s u n d e r hypergravity responds in general in an opposite direction than that induced by the real or simulated microgravity exposure. The interest in conducting comparative parallel experiments in the complete spectrum of ground simulation methods is shown in the above studies and will be achieved in the near future.Peer reviewe

Which precocial rodent species is more suitable as the experimental model of microgravity influence on prenatal musculosketal development on international space station?

The International Space Station (ISS) has the possibility to perform experiments regarding rodent reproduction in microgravity. The musculoskeletal system at birth in precocial rodent species more resembles the human than that of altricial rodent species. For precocial rodent species with body weight ≤ 500 g (limit of ISS) determined were: adult body mass, newborn body mass, head-body length, tail length, existing variants (wild, domesticated, laboratory), single/group housing, dry food consumption/24 h, water intake/24 h, basal metabolic rate mlO2/g/h, environmental temperature, sand baths, urine output ml/24 h, fecal output g/24 h, size of fecal droplet, hair length, life span, length of oestrus cycle, duration of pregnancy, building nest, litter size, stage of musculoskeletal maturity at birth, and the duration of weaning. Characteristics were obtained by searching SCOPUS as well as the World Wide Web with key words for each of the species in English, Latin and, local language name. These characteristics were compared in order to find most appropriate species. Twelve precocial rodent species were identified. There is not enough data for Common yellow-toothed cavy, and Eastern spiny mouse. Inappropriate species were: Gundis, Dassie rat are a more demanding species for appropriate tending, litter size is small; Octodon degus requires sand baths as well as a nest during the first two weeks after delivery; muscle maturity of Spiny mouse at birth (myotubular stage), does not correspond to the human (late histochemical stage); Chinchilla requires separately housing, daily sand baths, has upper limit of weight. Possibility of keeping Southern mountain cavy as pet animal, short estrus, large litter size, absence of the need for nest and sand baths, makes this species the most promising candidates for experiments on ISS. If an experiment is planned with exposing gravid animals before term of the birth, then they might be kept together in the existing Rodent Habitat (USA). If an experiment with birth in microgravity is planned on ISS, the existing habitats do not provide conditions for such an experiment. It is necessary to develop habitats for separate keeping of pregnant animals to enable the following: 1. undisturbed delivery 2. prevent the possibility of hurting the newborns 3. ensure adequate post-partum maternal care and nursing

Novel, Moon and Mars, partial gravity simulation paradigms and their effects on the balance between cell growth and cell proliferation during early plant development

11 p.-8 fig.-1 tab.Clinostats and Random Positioning Machine (RPM) are used to simulate microgravity, but, for space exploration, we need to know the response of living systems to fractional levels of gravity (partial gravity) as they exist on Moon and Mars. We have developed and compared two different paradigms to simulate partial gravity using the RPM, one by implementing a centrifuge on the RPM(RPMHW), the other by applying specific software protocols to driving the RPM motors (RPMSW). The effects of the simulated partialgravity were tested in plant root meristematic cells, a system with known response to real and simulated microgravity. Seeds of Arabidopsis thaliana were germinated under simulated Moon (0.17 g) and Mars (0.38 g) gravity. In parallel, seeds germinated under simulated microgravity (RPM), or at 1 g control conditions. Fixed root meristematic cells from 4-day grown seedlings were analyzed for cell proliferation rate and rate of ribosome biogenesis using morphometrical methods and molecular markers of the regulation of cell cycle and nucleolar activity. Cell proliferation appeared increased and cell growth was depleted under Moon gravity,compared with the 1g control. The effects were even higher at the Moon level than at simulated microgravity, indicating that meristematic competence (balance between cell growth and proliferation) is also affected at this gravity level. However, the results at the simulated Mars level were close to the 1g static control. This suggests that the threshold for sensing and responding to gravity alteration in the root would be at a level intermediate between Moon and Mars gravity. Both partial g simulation strategies seem valid and show similar results at Moon g levels, but further research is needed, in space flight and simulation facilities,especially around and beyond Mars g levels to better understand more precisely the differences and constrains in the use of these facilities for the space biology community.Funding: for [JvL]: Grant ALW-GO-MG/10-07 from the Netherlands Organization for Scientific (NWO) Research Earth and Life Sciences (ALW)via the Netherlands Space Office (NSO) and the ESA contract 4000107455/12/NL/PA.For [FJM]: Grant ESP2015-64323-R from the Spanish National Plan for Research and Development (MINECO-ERDF co-funding). For [RH]: ESA-ELIPS Program ESA GIA Project, contract number 4000105761. [AM] was recipient of a fellowship of the Spanish National Program for Young Researchers Training (MINECO, Ref. BES-2013- 063933).Peer reviewe