Genetic of Drosophila melanogaster

In 1909, Thomas Hunt Morgan, interested in understanding the Mendelian genetics much more deeply, built the first fruit fly laboratory at Columbia University. This ‘fly room’, as he called it, would be a milestone for the start of genetic studies in Drosophila, indeed, since each fly is a unique individual, its advantage over larger animal models was evident (Stephenson and Metcalfe, Reference Stephenson and Metcalfe2013). One example of these findings was the discovering of mutant flies with white eyes in large communities of red eyes flies (Moore, Reference Moore1983). It led to formulate the chromosome theory of inheritance discovering the mechanism by which gender-related genes are transmitted and the importance of the transmission on the behavior. Importantly, it gave the principles to understand how the body develops from a single cell to become the complex variety of tissues that built the adult organism and regulatory mechanisms involved at genetic level (Matthews et al., Reference Matthews, Kaufman and Gelbart2005). Although the complete genome of Drosophila melanogaster was only sequenced in 2000 and despite having a size of 180 Mb, which is 95% smaller than that of any average mammal (Adams et al., Reference Adams, Celniker, Holt, Evans, Gocayne, Amanatides, Scherer, Li, Hoskins, Galle, George, Lewis, Richards, Ashburner, Henderson, Sutton, Wortman, Yandell, Zhang, Chen, Brandon, Rogers, Blazej, Champe, Pfeiffer, Wan, Doyle, Baxter, Helt, Nelson, Gabor, Miklos, Abril, Agbayani, An, Andrews-Pfannkoch, Baldwin, Ballew, Basu, Baxendale, Bayraktaroglu, Beasley, Beeson, Benos, Berman, Bhandari, Bolshakov, Borkova, Botchan, Bouck, Brokstein, Brottier, Burtis, Busam, Butler, Cadieu, Center, Chandra, Cherry, Cawley, Dahlke, Davenport, Davies, de Pablos, Delcher, Deng, Mays, Dew, Dietz, Dodson, Doup, Downes, Dugan-Rocha, Dunkov, Dunn, Durbin, Evangelista, Ferraz, Ferriera, Fleischmann, Fosler, Gabrielian, Garg, Gelbart, Glasser, Glodek, Gong, Gorrell, Gu, Guan, Harris, Harris, Harvey, Heiman, Hernandez, Houck, Hostin, Houston, Howland, Wei, Ibegwam, Jalali, Kalush, Karpen, Ke, Kennison, Ketchum, Kimmel, Kodira, Kraft, Kravitz, Kulp, Lai, Lasko, Lei, Levitsky, Li, Li, Liang, Lin, Liu, Mattei, McIntosh, McLeod, McPherson, Merkulov, Milshina, Mobarry, Morris, Moshrefi, Mount, Moy, Murphy, Murphy, Muzny, Nelson, Nelson, Nelson, Nixon, Nusskern, Pacleb, Palazzolo, Pittman, Pan, Pollard, Puri, Reese, Reinert, Remington, Saunders, Scheeler, Shen, Shue, Sidén-Kiamos, Simpson, Skupski, Smith, Spier, Spradling, Stapleton, Strong, Sun, Svirskas, Tector, Turner, Venter, Wang, Wang, Wang, Wassarman, Weinstock, Weissenbach, Williams, Woodage, Worley, Wu, Yang, Yao, Ye, Yeh, Zaveri, Zhan, Zhang, Zhao, Zheng, Zheng, Zhong, Zhong, Zhou, Zhu, Zhu, Smith, Gibbs, Myers, Rubin and Venter2000), the alignment obtained a genetic homology rate of 60% with humans establishing that D. melanogaster could be a good experimental model for studying gene expression (Chien, Reference Chien2002; Yamamoto et al., Reference Yamamoto, Jaiswal, Charng, Gambin, Karaca, Mirzaa, Wiszniewski, Sandoval, Haelterman, Xiong, Zhang, Bayat, David, Li, Chen, Gala, Harel, Pehlivan, Penney, Vissers, de Ligt, Jhangiani, Xie, Tsang, Parman, Sivaci, Battaloglu, Muzny, Wan, Liu, Lin-Moore, Clark, Curry, Link, Schulze, Boerwinkle, Dobyns, Allikmets, Gibbs, Chen, Lupski, Wangler and Bellen2014; Mirzoyan et al., Reference Mirzoyan, Sollazzo, Allocca, Valenza, Grifoni and Bellosta2019). Importantly, 75% of the genes responsible for human diseases had homologues in D. melanogaster and thanks to the wide range of flies with genetic mutations available in laboratories, it became an animal model to investigate the genetic bases of diseases. For example in neurodegeneration, tumour development, immunity, muscle degeneration and cachexia, and intestinal dysfunction (Verheyen, Reference Verheyen2022). Since 1982, novel approaches have been employed to investigate genetic mechanisms, including site-directed mutagenesis in specific genes, oligonucleotide mutagenesis, P-element insertion mutagenesis, and, more recently, genomic editing with CRISPR-Cas9. The fruit fly, Drosophila, has been particularly useful for such studies due to its short life cycle and ability to produce a substantial number of offspring (Stephenson and Metcalfe, Reference Stephenson and Metcalfe2013). These experiments have yielded detailed insights into the regulation of numerous signalling pathways during early developmental stages in flies, and as a result, D. melanogaster has become one of the most popular experimental systems among eukaryotic species (Fig. 1) (Abbott et al., Reference Abbott, Hilgenfeld and Denell1992). Thanks to this model and the possibility of being able to generate a large number of repetitions, the contribution to topics related to Astrobiology that see adaptation processes of living organisms to space and the effects of radiation and microgravity have been enormous (Vernós et al., Reference Vernós, Carratalá, González-Jurado, Valverde, Calleja, Domingo, Vinós, Cervera and Marco1989a).

Figure 1.

Timeline contrasting the major milestones in biologic research and space research on Drosophila melanogaster in normal and microgravity conditions.

Drosophila as an experimental model in Space radiobiology

The biological consequences of ionizing radiation on humans participating in Space missions are studied by the multidisciplinary field of Space radiobiology (Radiology (ACR), 2022). The environment of space radiation is a complex mixture of radiation species dominated by highly penetrating charged particles from various sources such as galactic cosmic rays (GCRs) that originate outside the solar system, particles trapped in the Earth's magnetic field (i.e. Van Allen Belts), and particles emitted by our Sun (SPE) as a result of solar activity (Ohnishi and Ohnishi, Reference Ohnishi and Ohnishi2004; Onorato et al., Reference Onorato, Di Schiavi and Di Cunto2020). This is why dose-effect models of radioactive species are essential to understand and minimize damage to biological systems, including humans. In this sense, D. melanogaster debuted as a model to examine the effects of radiation at the molecular level, since Morgan and his students managed to identify ionizing radiation as a causal agent of genetic mutations (Stephenson and Metcalfe, Reference Stephenson and Metcalfe2013). These findings were instrumental in designing experiments that employed genes marked with mutations induced by ionizing radiation to investigate phenomena such as translocations and population dynamics (Muller, Reference Muller1927; Muller and Altenburg, Reference Muller and Altenburg1930). Indeed, during the first experiments on Drosophila existed the hypotheses that mutations were directly linked to chromosomal rearrangements. However, after Muller and Altenburg (Reference Muller and Altenburg1930) measured the frequency of translocations among Drosophila's four pairs of chromosomes, he found a number of translocations occurring between Chromosomes C1, C2 and C3 only after exposure to X-rays. Several years later, Demerec and Fano (Reference Demerec and Fano1944) proposed a similar study irradiating D. melanogaster sperm with different intensities of X-rays trying to test this hypothesis. Their results confirmed that the link between mutations and chromosome rearrangements did not exist (Muller and Altenburg, Reference Muller and Altenburg1930; Demerec and Fano, Reference Demerec and Fano1944). So, the use of the fruit fly in different life stages was so successful that, based on the availability of the technology, three types of studies were conducted as a model to examine the effects of ionizing radiation on living organisms: (i) research exposing flies to X, gamma, or beta radiation using radioactive isotopes; (ii) research employing balloons or exposing flies to suborbital radiation levels in peaks; and (iii) research exposing flies to cosmic radiation in the low-Earth orbit radiation environment. Experiments in this sense allowed to expose larvae of D. melanogaster to high-frequency X-ray radiation revealing that exposure to ionizing radiation extended the duration of their prepupal stage. Furthermore, the researchers found that this effect was more pronounced when the larvae were exposed to radiation during later stages of development (Hussey et al., Reference Hussey, Thompson, Tennant and Campbell1932). As the researchers continued their investigations, they ratified that ground radiation was negligible in the generation of spontaneous mutations, prompting them to investigate the impact of cosmic radiation on genes (Muller, Reference Muller1927; Jollos, Reference Jollos1937).

Later studies led by King et al. (Reference King, Rubinson and Smith1956; King, Reference King1957) focused on studying the effects of gamma radiation on the reproductive capabilities of D. melanogaster. The application of radiation on eggs of different strains showed no significant effects on hatchability, but the irradiation on females flies produced the apparition of lethal mutations in their offspring, and several morphological effects on their tissues, including the degeneration of nurse cells and the cytolysis of the ovaric chambers (King et al., Reference King, Rubinson and Smith1956; King, Reference King1957). Other studies related to reproductive abilities showed stronger correlations between the appearance of lethal mutations in sex chromosomes (Ives, Reference Ives1959) and the opposite effects of high and low doses of radiation that included advantages in heterozygous populations (Blaylock and Shugart, Reference Blaylock and Shugart1972). These authors irradiated homozygous populations of D. melanogaster and D. simulans with lethal doses of 5000 mGy, and observed greater viability in the heterozygous populations of both flies due to chromosomal mixing, in addition to obtaining a greater number of individuals for 20 generations of flies exposed to low doses of radiation (Ives, Reference Ives1959; Mukai et al., Reference Mukai, Yoshikawa and San1966; Blaylock and Shugart, Reference Blaylock and Shugart1972). Interestingly, genetic models with the fruit fly throughout history have also shown that the changes observed due to radiation are not necessarily linear to the radiation dose. As mentioned previously, this is due to the various variables that affect the biological systems (eco-evo-devo). Importantly, a recent study which analysed some stress-sensitive genes subjected to gamma radiation in D. melanogaster showed nonlinear effects of low dose radiation versus sex-specific radio-resistance of the post-mitotic cell state of the imago. This meant that the observed positive changes in life expectancy (hormesis) were not related to the changes of organism physiological functions after the exposure to low doses of ionizing radiation (Zhikrevetskaya et al., Reference Zhikrevetskaya, Peregudova, Danilov, Plyusnina, Krasnov, Dmitriev, Kudryavtseva, Shaposhnikov and Moskalev2015).

On the other hand, altitude studies referred to exposition to suborbital cosmic radiation were started with Jollos (Reference Jollos1937). The researcher analysed the radiation exposure of fruit flies at more than 4000 m above sea level (more than 14 000 feet) for 44 days, finding that there was an increase in sex-linked mutations in comparison to control. Two years later, the same author discovered that the presence of metals, such as lead, increased the amount of radiation, due to secondary radiation, which was perceived by the flies leading to a greater number of mutations (Jollos, Reference Jollos1937, Reference Jollos1939). These studies were also important to enhance the nascent high altitude medicine and the effect of radiation on populations that live in high mountains such as the Andes or the Alps. Then, in the search to a better understanding of recessive mutations and translocations due to radiation, several studies were initiated with balloons that can reach the stratosphere containing D. melanogaster flies. Since cosmic radiation is 5 times greater in the stratosphere than on the sea level, Reddi and Sanjeeva (Reference Reddi and Sanjeeva1964) analysed the effects of the exposure of male flies divided to test for mutations by breeding in six successive batches for 3 days in stratospheric balloons at 32 km altitude. To test for dominant lethality, each male was tested individually in mating and a reduction in hatchability relative to controls was determined. They found no evidence of recessive lethal mutations or translocations, but found a constant reduction in hatchability which they explained as an effect of the production of dominant lethal mutations mainly by chromosome breakage (Reddi and Sanjeeva, Reference Reddi and Sanjeeva1964).

Although the study of the effects of radiation on organisms was very popular in the eras prior to the installation of the ISS, after this, the radiation studies were mixed with the search for the effects of the almost absence of gravity known as microgravity (as we will describe later) (See Table 1). A relevant study analysed two types of mutations, one sex-linked recessive lethal mutations induced in male reproductive cells and other somatic mutations which give rise to morphological changes in hairs growing on the surface of wing epidermal cells. For this research the authors used a wild-type strain of D. melanogaster and another radiation-sensitive called mei-41 loaded on the US Space Shuttle Endeavour. It's interesting to note that the research revealed that while the frequencies of somatic wing-hair mutations were no significantly different in both the flight and control groups, the frequencies of sex-linked recessive fatal mutations in flight were greater than those in ground control groups. These findings raised the prospect that specific cell types, including male reproductive cells, may have undergone alterations as a result of the space environment (Hara, Reference Hara1994) (Fig. 2).

Table 1.

Space Radiation studies with Drosophila melanogaster

Figure 2.

Experiments to test the effects of cosmic radiation and microgravity measured at different altitudes. Over time it has been possible to study these conditions separately with the development of more sophisticated methods.

By the time the ISS was launched into low Earth orbit (LEO), the relevance of studying Drosophila melanogaster as a model organism was already recognized, and a specific module, named ‘the insect hab’ was installed for this purpose (Santos, Reference Santos2001) allowing to better resolve several questions that came to light from the research carried out previously. For example, studies by Ikenaga et al. (Reference Ikenaga, Yoshikawa, Kojo, Ayaki, Ryo, Ishizaki, Kato, Yamamoto and Hara1997), a few years before the ISS, suggested that microgravity could induce an increase in mutation rates caused by radiation. Ohnishi et al. (Reference Ohnishi, Takahashi and Ohnishi2002) began a more in-depth study to corroborate these hypotheses by analysing the role of DNA repair systems, antioxidants and protective proteins in fruit flies on the ISS. Although other organism models such as bacteria, fungi, and cell cultures gave contradictory results and effects. Drosophila was able to demonstrate sustained increased sensitivity to changes during space travel and suggested that space radiation may depress the recovery of DNA damage induced by space radiation. This was one of the pioneering results in seeing biological effects of the interaction between microgravity and radiation. Another study using D. melanogaster also suggested that radiation is important for certain normal functions at the genetic level. The authors showed that flies placed in an underground laboratory in Russia had significant deficiencies in various molecular and cellular functions (Zarubin et al., Reference Zarubin, Gangapshev, Gavriljuk, Kazalov and Kravchenko2021). These results could be related to previous findings where experiments conducted at Fruit Fly Lab-01 (FFL-01) revealed that chronic low-dose radiation (16.7 mGy h⁻¹ exposure dose rate) caused behavioural changes in D. melanogaster. Indeed, despite impairing climbing activity, exploratory movement, effects on development (pupation and hatching rates), some changes in the expression of genes involved in the regulation of metabolism resulted in a surprising extension of lifespan of life (Kim et al., Reference Kim, Seong, Lee, Lee, Yang, Kim and Nam2015).

For other types of radiation, Alexandrov et al. (Reference Alexandrov, Alexandrova and Afanasyeva2022) studied the nature of hereditary recessive genetic mutations induced by ionizing radiation (γ rays and neutrons) examining five sex-linked genes on chromosome 2 and discovered that neutrons were 2.5 times more effective than γ-radiation in causing mutations. Conversely, they discovered that, in contrast to neutrons, γ-radiation frequently caused clusters of changes in DNA, sometimes even in a single double-stranded helix. Curiously, up to fourteen genetic alterations at the chromosomal and intragenic level were present in mutants with the same visible phenotype. This phenomenon is known as cryptic genetic variation in which they do not produce phenotypic differences under normal conditions but during periods of stress or environmental changes, they could express (Gilbert et al., Reference Gilbert, Bosch and Ledón-Rettig2015).

Drosophila melanogaster, due to its versatility in handling for experimentation, has proven to be very useful for the study of radiation and its effects on living beings. The different forms of radiation and the different variables in the environment in the various experiments with fruit flies were tried to control to understand parts of the phenomenon in biological systems. The different experiences presented and summarized here aim in some way to expose the importance of Drosophila in achieving milestones in the field of mutations linked to sex chromosomes, which are without a doubt, together with the analysis of radiation-sensitive genes, the findings most important aspects of using this model.

Drosophila and microgravity

Microgravity refers to conditions in which the gravitational acceleration acting on an object is much lower compared to Earth's gravity, which is approximately 9.8 m s⁻² at the Earth's surface (1 G) (National Research Council, 2000). Physics originally predicted that the acceleration due to gravity would not be enough to produce any change in living things; however, studies in many living organisms and tissues have shown the opposite, showing effects on early life cycle, tissue-cell interactions, and gene expression (Morey-Holton, Reference Morey-Holton, Rothschild and Lister2003; Kiss et al., Reference Kiss, Millar and Edelmann2012).

The experiments conducted by Roberto Marco and his team on the ISS have shown that Drosophila alters its behaviour when exposed to microgravity, resulting in developmental disruptions during oogenesis and embryogenesis (Abbott et al., Reference Abbott, Hilgenfeld and Denell1992; de Juan et al., Reference de Juan, Benguría, Villa, Leandro, Herranz, Duque, Horn, Medina, van Loon and Marco2007; Herranz et al., Reference Herranz, Laván, Dijkstra, Larkin, Davey, van Loon JJWA, Medina, Marco and Schiller2008). Specifically, hatched flies exhibited increased motility in comparison to adult flies, which in turn experienced reduced motility by the time they reach two weeks of age. In addition, the experiment included a hypergravity control via centrifugation (2 G). Notably, Drosophila's locomotor activity increased in microgravity, but was repressed under 2 G hypergravity, with both conditions having detrimental effects on the overall survival of the flies (Serrano et al., Reference Serrano, van Loon, Manzano, Francisco Javier and Herranz2010).

Other studies about the development of D. melanogaster and Carasius morosus exposed to microgravity at low orbit at 2000 m.a.s.l at the ISS, showed changes on the embryonic hatching rates and a delay in the development of larvae and adult insects after a 7 days flight (Vernós et al., Reference Vernós, Carratalá, González-Jurado, Valverde, Calleja, Domingo, Vinós, Cervera and Marco1989a, Reference Vernós, Gonzáles-Jurado, Calleja and Marco’1989b). The authors observed stimulation of the oogenesis and a decreased number of hatching of larvae for D. melanogaster exposed to microgravity. Following their return to Earth, several Drosophila from these tests showed delayed larval and adult development as well as a decline in the number of flies that reached maturity (~25%) (Fig. 3).

Figure 3.

Workflow of gene expression after microgravity experiments.

As previously described, since 1937, Drosophila has been one of the most studied multicellular organisms (on Earth and Space). Scientists have been able to investigate the impact of weightlessness on living things, particularly in terms of genetic regulation, thanks to technological advancements. For gene expression profile of fruit flies subjected to microgravity circumstances was thoroughly analysed by the researchers using next-generation sequencing techniques. These methods allow the investigation of thousands of genes at once and offer complete details on how their activity varies in response to microgravity. On the other hand, the precise monitoring of morphological and structural changes in Drosophila's cells under microgravity has also been possible thanks to advances in microscopy. It is possible to examine how the absence of gravity affects the expression of important genes and proteins in various tissues and organs, thanks to confocal and fluorescence microscopes, which allow viewing and following biological activities in real time.

Several studies related to understand the phenotype under microgravity conditions were developed on the ISS. For example, the Fruit Fly Lab-02 had the objective to study physiologic and genetic changes in the development of the heart in Drosophila under microgravity. Although the fruit fly cardiovascular system is less complex than that of mammals, there are shared structural, functional, and genetic similarities with flies that help researchers to understand the possible effects and risks of new and stressful environments associated with long-duration Space missions (Keller, Reference Keller2017). The hearts of flies grown under these conditions showed exhibited myofibrillar remodelling, constricted heart and in the isolated hearts, RNA sequencing (by RNA-seq) indicated decreased sarcomeric/ECM gene expression and dramatically elevated proteasomal gene expression, which was consistent with the damaged and smaller hearts, suggesting aberrant proteostasis (Walls et al., Reference Walls, Diop, Birse, Elmen, Gan, Kalvakuri, Pineda, Reddy, Taylor, Trinh, Vogler, Zarndt, McCulloch, Lee, Bhattacharya, Bodmer and Ocorr2020). Another interesting example was the Fruit Fly Lab-03. This mission aboard the ISS investigated the effects on the innate immune response against parasites (Keller, Reference Keller2018). Since the immune system dysfunction and infections are major risks for astronauts on long-duration space exploration missions, the model parasite-host in Drosophila was important to determine changes in this interaction and determine whether spaceflight alters the ability of the parasite to infect the host. The results of these experiments and others are summarized in Table 2.

Table 2.

Microgravity Studies with Drosophila melanogaster in the space

It is commendable for these investigations with Drosophila that they allowed the phenotypic results to be extended to other insects and animals on the ISS. Later studies evaluated the development and changes in morphology based on these antecedents found, and therefore made an enormous leap in understanding several processes that generated the bases of Space Biology. However, given that the research carried out was almost 80% more related to the phenotype than that carried out for the genotype, perhaps due to technological difficulties in their initial stages, the studies only focused on evaluating specific genes or mutations due to radiation. These points came full circle when new studies began to show that microgravity alone caused changes at the genetic level. Therefore, this led to studies evaluating genomics in a more comprehensive manner, which is evident in the low production of studies only focused on genetic effects due to radiation. Studies analysing the joint effects of microgravity and radiation began to be carried out on missions to the ISS, and new base-ground tools on Earth such as magnetic systems or microgravity simulators made their appearance with greater participation in this research field. In this regards, Leandro et al. (Reference Leandro, Szewczyk, Benguría, Herranz, Laván, Medina, Gasset, van Loon, Conley and Marco2007) performed microarray analysis for the transcriptome on flies (D. melanogaster) and nematodes (Caenorhabditis elegans) grown in the ISS, concluded that there were significant differences in gene expression levels in response to spaceflight (Leandro et al., Reference Leandro, Szewczyk, Benguría, Herranz, Laván, Medina, Gasset, van Loon, Conley and Marco2007). They found that the set of genes with changes in their expression levels were different between these species. Studies like this brought others related to analysing which genes changed, and seeing if these were related to important biological functions that included aging (de Juan et al., Reference de Juan, Benguría, Villa, Leandro, Herranz, Duque, Horn, Medina, van Loon and Marco2007; Herranz et al., Reference Herranz, Laván, Dijkstra, Larkin, Davey, van Loon JJWA, Medina, Marco and Schiller2008). For instance, Herranz et al. (Reference Herranz, Benguría, Laván, López-Vidriero, Gasset, Javier Medina, Van Loon and Marco2010) discovered notable changes in gene expression in comparison to 1 g controls when examining the impact of microgravity (experiments conducted on the ISS) on the pupal development of Drosophila. It was shown that decreasing gravity levels significantly impacted gene expression, that non-ideal environmental conditions on the ISS and spaceflight preparation processes entail additional pressures that also impacted gene expression. In turn, experiments performed on the ground under ideal simulated gravity conditions using the random position machine (RPM) showed more subtle effects on gene expression, But by repeating the ground experiments under conditions that replicated the additional environmental stresses of spaceflight procedures, 79% of the differentially expressed genes (DEGs) detected on the ISS were reproduced in the RPM experiment. The genes that underwent variation were related to breathing, developmental processes and stress-related changes. In turn, hypergravity simulation induced a similar transcriptome response but in the opposite direction, where genes promoted in microgravity were generally suppressed in hypergravity. From which they deduced that the transcriptome of pupal-stage Drosophila is finely tuned to normal gravity and that microgravity, together with the environmental constraints associated with space experiments, can have profound effects on gene expression (Herranz et al., Reference Herranz, Benguría, Laván, López-Vidriero, Gasset, Javier Medina, Van Loon and Marco2010).

Other investigations reported about genes encoding proteins involved in morphogenesis, cytoskeletal organization, cell motility, and transcriptional regulation, cuticle genes, and genes involved in proteolytic processes, which changed from microgravity conditions to Earth standard gravity. The genes Cp11B, Cpr92F, Cpr56F, TwdlF, Cpr67Fb that codify cuticular proteins are overexpressed leading to a higher percentage of chitin in the larva compared to regular flies (Herranz et al., Reference Herranz, Hill, Dijkstra, Eaves, Van Loon and Medina2013). Importantly, these studies argue the complexity of epigenetic factors in the final results that must be better studied.

Base-ground studies in microgravity

The difficulty of transferring systems to the ISS, either due to the costs related to transportation or due to the limited availability of access and space in its laboratories, the microgravity simulator systems on Earth sought to somewhat equate the research in this field. Different strategies were designed for the study of short or prolonged exposition to microgravity conditions such as parabolic flights, which allow to observe the effects of the exposition to short periods of time to microgravity followed by hypergravity periods; powerful electromagnets or superconducting solenoids provided to cause a repulsive diamagnetic force on water that could balance the weight of several samples and cause it to float them (diamagnetic levitation) (Herranz et al., Reference Herranz, Larkin, Dijkstra, Hill, Anthony, Davey, Eaves, van Loon, Medina and Marco2012); rotating wall vessels (RWV) bioreactors where cells are suspended in medium that is constantly rotating; or/and clinostats and random positioning machines (RPM) which consists of two or three axis that rotate generating a simulated microgravity environment at their centre (Blaber et al., Reference Blaber, Marçal and Burns2010; Laván et al., Reference Laván, Diego, Valdivia-Silva, Gabriela, Agapito, Castillo-Cara, Olga, Maroto, Enrrique, Loon and Roberto2015) (See Table 3). Importantly, low orbit facilities and parabolic flights are been associated with numerous stressful factors that can affect the organism in study. Because of that these based-ground experiments minimized key factors related to the environment such as temperature, pH, pressure, oxygen, CO₂ concentration, etc., which require to be controlled all the time in the other processes. However, some researchers disagree with this argument, suggesting that there are too many variables to control in any case. Another debate caused by studies on Earth with microgravity simulators versus those carried out in real environments like the ISS is the representativeness and significant similarity of genetic processes in both environments. This fact, although it has given controversial results, has shown that in the case of D. melanogaster, the genes and mechanisms that were detected in studies on the ISS could be partially or almost completely repeated in the simulators on Earth. For example, using a clinostat to simulate microgravity on Schneider S-1 cells originating from Drosophila, it was observed that there were changes in the mitochondrial structure and cristae morphology as a consequence of microtubule disruption. All these results matched those attained by exposure to cultured human lymphocyte (Jurkat) cells during spaceflights (Schatten et al., Reference Schatten, Lewis and Chakrabarti2001).

Table 3.

Types of study of Microgravity on ground

Although gravity modification appears to have minimal influence on gene expression under ideal environmental conditions, when those criteria are far from optimal, gene expression must be effectively controlled and the effects become more predominant. This could be due either to the lack of experience that animals have with new ecosystems, such as those that do not have gravitational attraction or perhaps the opposite, that the organisms spent an indeterminate amount of time under microgravity and therefore the return to those previous conditions a biological response was generated. Either way, research in this field using D. melanogaster has led us to better understand these effects (Herranz et al., Reference Herranz, Hill, Dijkstra, Eaves, Van Loon and Medina2013).

Although radiation is a factor considered mutagenic for inducing genetic alterations that lead to a greater risk of the appearance of malignant tumours (carcinogenesis), and despite the number of studies that show that radiation seems to worsen its effects if microgravity coexists, Other studies such as those presented by Grimm's group in human cancer cells suggest that the mechanisms are more complex than it seems (Grimm, Reference Grimm2021). This point allows us to emphasize here that in this review we focus on the contributions of the fruit fly to space research and that the extrapolation of these processes and mechanisms in humans should not be so direct but rather be used as guides for a better understanding of the variables studied. Indeed, the effects of microgravity on human cancer cells had controversial results showing pro- or anti-tumoural effects in different studies. Importantly, at the beginning the objective to analyse tumours in Space was generate 3D models capable to explain the mechanisms of invasion and metastasis in cancer. The initial studies on this were also developed in Drosophila melanogaster models (Becker and Souza, Reference Becker and Souza2013). Finally, to understand the mechanisms at the molecular level, studies by our group suggested the presence of genetic sensors that, to our understanding, could be the most sensitive in determining gravitational waves (Laván et al., Reference Laván, Diego, Valdivia-Silva, Gabriela, Agapito, Castillo-Cara, Olga, Maroto, Enrrique, Loon and Roberto2015). Six genes found in our study showed significant different patterns of expression against microgravity, compared to the thousands of genes analysed molecularly. In addition, these genes suggested to be microgravity ‘sensors’ in Drosophila have orthologous genes in humans very related to those found in the main types of cancers (Valdivia-Silva et al., Reference Valdivia-Silva, Laván, Diego, Gabriela and Andres2015).

Implications of Drosophila research in Space human biology

Fruit flies and humans have 60% similarity in the genome, and around 75% of human disease-causing genes have Drosophila homologous (orthologous) (Ugur et al., Reference Ugur, Chen and Bellen2016; Verheyen, Reference Verheyen2022). Due to, several of the fundamental biological mechanisms and pathways that regulate development and survival have been conserved throughout evolution in these two species (Jennings, Reference Jennings2011), These characteristics, along with the fruit fly's quick generation time, minimal care requirements, and accessibility to potent genetic tools, make it possible to explore intricate processes important to biological studies, including aerospace biology (Mirzoyan et al., Reference Mirzoyan, Sollazzo, Allocca, Valenza, Grifoni and Bellosta2019). Indeed, the studies of the effects of ionizing radiation in D. melanogaster initiated in the first half of the 20th century and the possibility of extrapolating the results to humans achieved its maximum potential when the fly genome sequence was published in 2000 (Adams et al., Reference Adams, Celniker, Holt, Evans, Gocayne, Amanatides, Scherer, Li, Hoskins, Galle, George, Lewis, Richards, Ashburner, Henderson, Sutton, Wortman, Yandell, Zhang, Chen, Brandon, Rogers, Blazej, Champe, Pfeiffer, Wan, Doyle, Baxter, Helt, Nelson, Gabor, Miklos, Abril, Agbayani, An, Andrews-Pfannkoch, Baldwin, Ballew, Basu, Baxendale, Bayraktaroglu, Beasley, Beeson, Benos, Berman, Bhandari, Bolshakov, Borkova, Botchan, Bouck, Brokstein, Brottier, Burtis, Busam, Butler, Cadieu, Center, Chandra, Cherry, Cawley, Dahlke, Davenport, Davies, de Pablos, Delcher, Deng, Mays, Dew, Dietz, Dodson, Doup, Downes, Dugan-Rocha, Dunkov, Dunn, Durbin, Evangelista, Ferraz, Ferriera, Fleischmann, Fosler, Gabrielian, Garg, Gelbart, Glasser, Glodek, Gong, Gorrell, Gu, Guan, Harris, Harris, Harvey, Heiman, Hernandez, Houck, Hostin, Houston, Howland, Wei, Ibegwam, Jalali, Kalush, Karpen, Ke, Kennison, Ketchum, Kimmel, Kodira, Kraft, Kravitz, Kulp, Lai, Lasko, Lei, Levitsky, Li, Li, Liang, Lin, Liu, Mattei, McIntosh, McLeod, McPherson, Merkulov, Milshina, Mobarry, Morris, Moshrefi, Mount, Moy, Murphy, Murphy, Muzny, Nelson, Nelson, Nelson, Nixon, Nusskern, Pacleb, Palazzolo, Pittman, Pan, Pollard, Puri, Reese, Reinert, Remington, Saunders, Scheeler, Shen, Shue, Sidén-Kiamos, Simpson, Skupski, Smith, Spier, Spradling, Stapleton, Strong, Sun, Svirskas, Tector, Turner, Venter, Wang, Wang, Wang, Wassarman, Weinstock, Weissenbach, Williams, Woodage, Worley, Wu, Yang, Yao, Ye, Yeh, Zaveri, Zhan, Zhang, Zhao, Zheng, Zheng, Zhong, Zhong, Zhou, Zhu, Zhu, Smith, Gibbs, Myers, Rubin and Venter2000).

The effect of radiation reported by Blaylock and Shugart (Reference Blaylock and Shugart1972) in a homozygous population of D. melanogaster could be of importance for the future establishment of humans on celestial bodies such as Mars, where a small population may give rise to a group of Martians with poor genetic pool because of inbreeding effects. In accordance with these studies, there would be a radiation threshold where the generation of mutations could be beneficial to the population, because it would generate genetic diversity.

Studies focused on germ-line cells found that spontaneous mutation rates per generation are similar among D. melanogaster and humans and these rates are not related to time (Drost and Lee, Reference Drost and Lee1995) In the case of D. melanogaster, the average limit number of cell divisions throughout its life is 19, with no major difference between sexes. It has been seen that the average mutation rate in humans is approximately 2.5⁻⁸ mutations per nucleotide site or 175 mutations per diploid genome per generation and it has been estimated that the genomic deleterious mutation rate (U) is at least 3, a higher value than that estimated for Drosophila (U = 0.02 – 1) (Nachman and Crowell, Reference Nachman and Crowell2000). These data about comparative mutation rates are important to validate the use of Drosophila melanogaster as a model to study the effects of the Space environment at the genetic and molecular level. Potentially, the results obtained in Drosophila about possible genetic damage caused by cosmic radiation or microgravity could mean that in humans, those effects could be similar or worse. In addition, the base substitution mutation rates are substantially higher in males than in females and rise with paternal age, this is explained as male germline cells divide often (Crow, Reference Crow1997). In this sense, Drosophila could also be used for evaluating spatial effects on the germline genome, due to the similarities in mutation rates and the conservation of several metabolic pathways (Drost and Lee, Reference Drost and Lee1995). Of course, the research in the line of reproduction is very important to future projects of other planets or satellites colonization, and Drosophila can help us predict possible harmful effects on humans in Space.

Laván et al. (Reference Laván, Diego, Valdivia-Silva, Gabriela, Agapito, Castillo-Cara, Olga, Maroto, Enrrique, Loon and Roberto2015) showed interesting implications for understanding the microgravity effects at the transcriptomic level. They found a group of six genes of D. melanogaster which are inhibited in real and simulated microgravity and probably could be the keys to understanding the roots of multiple effects on cells. These genes and their human orthologues are currently the focus of research in our group as gravity sensors which would imply an important advance to correlate physiological or pathophysiological processes in living organisms with changes in gravitational waves (Laván et al., Reference Laván, Diego, Valdivia-Silva, Gabriela, Agapito, Castillo-Cara, Olga, Maroto, Enrrique, Loon and Roberto2015).