Grand challenges in space synthetic biology

Space synthetic biology is a branch of biotechnology dedicated to engineering biological systems for space exploration, industry and science. There is significant public and private interest in designing robust and reliable organisms that can assist on long-duration astronaut missions. Recent work has also demonstrated that such synthetic biology is a feasible payload minimization and life support approach as well. This article identifies the challenges and opportunities that lie ahead in the field of space synthetic biology, while highlighting relevant progress. It also outlines anticipated broader benefits from this field, because space engineering advances will drive technological innovation on Earth.     

Effect of magnetically simulated zero-gravity and enhanced gravity on the walk of the common fruitfly

Understanding the effects of gravity on biological organisms is vital to the success of future space missions. Previous studies in Earth orbit have shown that the common fruitfly (Drosophila melanogaster) walks more quickly and more frequently in microgravity, compared with its motion on Earth. However, flight preparation procedures and forces endured on launch made it difficult to implement on the Earth''s surface a control that exposed flies to the same sequence of major physical and environmental changes. To address the uncertainties concerning these behavioural anomalies, we have studied the walking paths of D. melanogaster in a pseudo-weightless environment (0g*) in our Earth-based laboratory. We used a strong magnetic field, produced by a superconducting solenoid, to induce a diamagnetic force on the flies that balanced the force of gravity. Simultaneously, two other groups of flies were exposed to a pseudo-hypergravity environment (2g*) and a normal gravity environment (1g*) within the spatially varying field. The flies had a larger mean speed in 0g* than in 1g*, and smaller in 2g*. The mean square distance travelled by the flies grew more rapidly with time in 0g* than in 1g*, and slower in 2g*. We observed no other clear effects of the magnetic field, up to 16.5 T, on the walks of the flies. We compare the effect of diamagnetically simulated weightlessness with that of weightlessness in an orbiting spacecraft, and identify the cause of the anomalous behaviour as the altered effective gravity.     

Mechanistic analysis of the search behaviour of Caenorhabditis elegans

A central question in movement research is how animals use information and movement to promote encounter success. Current random search theory identifies reorientation patterns as key to the compromise between optimizing encounters for both nearby and faraway targets, but how the balance between intrinsic motor programmes and previous environmental experience determines the occurrence of these reorientation behaviours remains unknown. We used high-resolution tracking and imaging data to describe the complete motor behaviour of Caenorhabditis elegans when placed in a novel environment (one in which food is absent). Movement in C. elegans is structured around different reorientation behaviours, and we measured how these contributed to changing search strategies as worms became familiar with their new environment. This behavioural transition shows that different reorientation behaviours are governed by two processes: (i) an environmentally informed ‘extrinsic’ strategy that is influenced by recent experience and that controls for area-restricted search behaviour, and (ii) a time-independent, ‘intrinsic’ strategy that reduces spatial oversampling and improves random encounter success. Our results show how movement strategies arise from a balance between intrinsic and extrinsic mechanisms, that search behaviour in C. elegans is initially determined by expectations developed from previous environmental experiences, and which reorientation behaviours are modified as information is acquired from new environments.     

Responses of Microcrustaceans to Simulated Microgravity (2D-Clinorotation) - Preliminary Assessments for the Development of Bioregenerative Life Support Systems (BLSS)

Bioregenerative Life Support Systems (BLSS) are an endeavor to create environments able to maintain human life e.g. on future long-duration space missions like flights to Mars. Based on cyclic biological processes, these systems will be independent from material resupply (such as food, water and oxygen). Due to their central role in limnic ecosystems, herbivorous microcrustaceans could act as key player in aquatic BLSS as they link oxygen liberating, autotrophic producers like algae to higher trophic levels, such as fish. However, before such BLSS can be utilized in space, organisms inhabiting these systems have to be studied thoroughly to disclose the gravitational impact on the biological processes. This is possible in real microgravity, but requires high financial resources, is opportunity-limited or periods of microgravity are very short. Yet, cost-effective and almost permanently accessible tools for gravitational research are ground-based facilities (GBFs), providing simulated microgravity. Among those GBFs is the so called 2D-clinostat. In the present study we demonstrate, that rotation of clinostat tubes does not generate acceleration in form of (predator resembling) small scale turbulence, which can be perceived by Daphnia cucullata. Additionally, embryonal development is not disturbed in subitaneous eggs of Daphnia magna and resting eggs of the ostracod Heterocypris incongruens (besides through restrictions in space within the narrow clinostat tubes), just as subsequent hatching from the respective eggs. Hence, our results indicate that clinorotation is a suitable method to simulate microgravity for microcrustaceans.     

Towards synthetic biological approaches to resource utilization on space missions

This paper demonstrates the significant utility of deploying non-traditional biological techniques to harness available volatiles and waste resources on manned missions to explore the Moon and Mars. Compared with anticipated non-biological approaches, it is determined that for 916 day Martian missions: 205 days of high-quality methane and oxygen Mars bioproduction with Methanobacterium thermoautotrophicum can reduce the mass of a Martian fuel-manufacture plant by 56%; 496 days of biomass generation with Arthrospira platensis and Arthrospira maxima on Mars can decrease the shipped wet-food mixed-menu mass for a Mars stay and a one-way voyage by 38%; 202 days of Mars polyhydroxybutyrate synthesis with Cupriavidus necator can lower the shipped mass to three-dimensional print a 120 m³ six-person habitat by 85% and a few days of acetaminophen production with engineered Synechocystis sp. PCC 6803 can completely replenish expired or irradiated stocks of the pharmaceutical, thereby providing independence from unmanned resupply spacecraft that take up to 210 days to arrive. Analogous outcomes are included for lunar missions. Because of the benign assumptions involved, the results provide a glimpse of the intriguing potential of ‘space synthetic biology’, and help focus related efforts for immediate, near-term impact.     

Apparent power-law distributions in animal movements can arise from intraspecific interactions

Lévy flights have gained prominence for analysis of animal movement. In a Lévy flight, step-lengths are drawn from a heavy-tailed distribution such as a power law (PL), and a large number of empirical demonstrations have been published. Others, however, have suggested that animal movement is ill fit by PL distributions or contend a state-switching process better explains apparent Lévy flight movement patterns. We used a mix of direct behavioural observations and GPS tracking to understand step-length patterns in females of two related butterflies. We initially found movement in one species (Euphydryas editha taylori) was best fit by a bounded PL, evidence of a Lévy flight, while the other (Euphydryas phaeton) was best fit by an exponential distribution. Subsequent analyses introduced additional candidate models and used behavioural observations to sort steps based on intraspecific interactions (interactions were rare in E. phaeton but common in E. e. taylori). These analyses showed a mixed-exponential is favoured over the bounded PL for E. e. taylori and that when step-lengths were sorted into states based on the influence of harassing conspecific males, both states were best fit by simple exponential distributions. The direct behavioural observations allowed us to infer the underlying behavioural mechanism is a state-switching process driven by intraspecific interactions rather than a Lévy flight.     

Experiments and theory of undulatory locomotion in a simple structured medium

Undulatory locomotion of micro-organisms through geometrically complex, fluidic environments is ubiquitous in nature and requires the organism to negotiate both hydrodynamic effects and geometrical constraints. To understand locomotion through such media, we experimentally investigate swimming of the nematode Caenorhabditis elegans through fluid-filled arrays of micro-pillars and conduct numerical simulations based on a mechanical model of the worm that incorporates hydrodynamic and contact interactions with the lattice. We show that the nematode''s path, speed and gait are significantly altered by the presence of the obstacles and depend strongly on lattice spacing. These changes and their dependence on lattice spacing are captured, both qualitatively and quantitatively, by our purely mechanical model. Using the model, we demonstrate that purely mechanical interactions between the swimmer and obstacles can produce complex trajectories, gait changes and velocity fluctuations, yielding some of the life-like dynamics exhibited by the real nematode. Our results show that mechanics, rather than biological sensing and behaviour, can explain some of the observed changes in the worm''s locomotory dynamics.     

Mapping the stereotyped behaviour of freely moving fruit flies

A frequent assumption in behavioural science is that most of an animal''s activities can be described in terms of a small set of stereotyped motifs. Here, we introduce a method for mapping an animal''s actions, relying only upon the underlying structure of postural movement data to organize and classify behaviours. Applying this method to the ground-based behaviour of the fruit fly, Drosophila melanogaster, we find that flies perform stereotyped actions roughly 50% of the time, discovering over 100 distinguishable, stereotyped behavioural states. These include multiple modes of locomotion and grooming. We use the resulting measurements as the basis for identifying subtle sex-specific behavioural differences and revealing the low-dimensional nature of animal motions.     

Searching for motifs in the behaviour of larval Drosophila melanogaster and Caenorhabditis elegans reveals continuity between behavioural states

We present a novel method for the unsupervised discovery of behavioural motifs in larval Drosophila melanogaster and Caenorhabditis elegans. A motif is defined as a particular sequence of postures that recurs frequently. The animal''s changing posture is represented by an eigenshape time series, and we look for motifs in this time series. To find motifs, the eigenshape time series is segmented, and the segments clustered using spline regression. Unlike previous approaches, our method can classify sequences of unequal duration as the same motif. The behavioural motifs are used as the basis of a probabilistic behavioural annotator, the eigenshape annotator (ESA). Probabilistic annotation avoids rigid threshold values and allows classification uncertainty to be quantified. We apply eigenshape annotation to both larval Drosophila and C. elegans and produce a good match to hand annotation of behavioural states. However, we find many behavioural events cannot be unambiguously classified. By comparing the results with ESA of an artificial agent''s behaviour, we argue that the ambiguity is due to greater continuity between behavioural states than is generally assumed for these organisms.     

Life History Responses and Feeding Behavior of Microcrustacea in Altered Gravity – Applicability in Bioregenerative Life Support Systems (BLSS)

Manned space missions, as for example to the planet Mars, are a current objective in space exploration. During such long-lasting missions, aquatic bioregenerative life support systems (BLSS) could facilitate independence of resupply from Earth by regenerating the atmosphere, purifying water, producing food and processing waste. In such BLSS, microcrustaceans could, according to their natural role in aquatic ecosystems, link oxygen liberating, autotrophic algae and higher trophic levels, such as fish. However, organisms employed in BLSS will be exposed to high acceleration (hyper- g) during launch of spacecrafts as well as to microgravity (μg) during space travel. It is thus essential that these organisms survive, perform and reproduce under altered gravity conditions. In this study we present the first data in this regard for the microcrustaceas Daphnia magna and Heterocypris incongruens. We found that after hyper-g exposure (centrifugation) approximately one third of the D. magna population died within one week (generally indicating that possible belated effects have to be considered when conducting and interpreting experiments during which hyper-g occurs). However, suchlike and even higher losses could be countervailed by the surviving daphnids’ unaltered high reproductive capacity. Furthermore, we can show that foraging and feeding behavior of D. magna (drop tower) and H. incongruens (parabolic flights) are rarely altered in μg. Our results thus indicate that both species are suitable candidates for BLSS utilized in space.     

The Human Centrifuge

Life on Earth has developed at unit gravity, 9.81 m/s², which was a major factor especially when vertebrates emerged from water onto land in the late Devonian, some 375 million years ago. But how would nature have evolved on a larger planet? We are able to address this question simply in experiments using centrifuges. Based on these studies we have gained valuable insights in the physiological process in plants and animals. They adapt to a new steady state suitable for the high-g environments applied. Information on mammalian adaptations to hyper-g is interesting or may be even vital for human space exploration programs. It has been shown in long duration animal hypergravity studies, ranging from snails, rats to primates, that various structures like muscles, bones, neuro-vestibular, or the cardio-vascular system are affected. However, humans have never been exposed to a hyper-g environment for long durations. Centrifuge studies involving humans are mostly in the order of hours. The current work on human centrifuges are all focused on short arm systems to apply short periods of artificial gravity in support of long duration space missions in ISS or to Mars. In this paper we will address the possible usefulness of a large human centrifuge on Earth. In such a centrifuge a group of humans can be exposed to hypergravity for, in principle, an unlimited period of time like living on a larger planet. The input from a survey under scientists working in the field of gravitational physiology, but also other disciplines, will be discussed.     

A consistent muscle activation strategy underlies crawling and swimming in Caenorhabditis elegans

Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower. Theoretical treatment of observed behaviour has suggested a large change in force–posture relationships at different viscosities, but analysis of bend propagation suggests that short-range proprioceptive feedback is used to control and generate body bends. How muscles could be activated in a way consistent with both these results is unclear. We therefore combined automated worm tracking with calcium imaging to determine muscle activation strategy in a variety of external substrates. Remarkably, we observed that across locomotion patterns spanning a threefold change in wavelength, peak muscle activation occurs approximately 45° (1/8th of a cycle) ahead of peak midline curvature. Although the location of peak force is predicted to vary widely, the activation pattern is consistent with required force in a model incorporating putative length- and velocity-dependence of muscle strength. Furthermore, a linear combination of local curvature and velocity can match the pattern of activation. This suggests that proprioception can enable the worm to swim effectively while working within the limitations of muscle biomechanics and neural control.     

The “<Emphasis Type="Italic">Daphnia</Emphasis>” Lynx Mark I Suborbital Flight Experiment: Hardware Qualification at the Drop Tower Bremen

The Drop Tower Bremen, a ground-based facility enabling research under real microgravity conditions, is an excellent platform for testing new types of experimental hardware to ensure full performance when deployed in costly and rare flight opportunities such as suborbital flights. Here we describe the “Daphnia” experiment which will fly on XCOR Aerospace Lynx Mark I and our experience from the hardware tests with the catapult system at the drop tower. The aim of the “Daphnia” experiment is to obtain data on the biological performance of daphnids and predator-prey interactions in microgravity, which are important for the development of aquatic bioregenerative life support systems (BLSS). The experiment consists of two subunits: The first unit is dedicated to predator-prey interactions, where behavioural analysis should reveal if microgravity interfere with prey (Daphnia) detection or feeding and therefore may interrupt the trophic cascade. The functioning of such an artificial food web is indispensable for a long-lasting BLSS suitable for long-duration manned space missions or Earth-based explorations to extreme habitats. The second unit is designed to investigate the impact of microgravity on gene expression and the cytoskeleton in Daphnia. Next to data collection, the real microgravity conditions at the drop tower have helped to identify the weak points of the “Daphnia” experimental hardware and lead to further improvement. Hence, the drop tower is ideal for testing new experimental hardware which is indispensable before the implementation in suborbital flights.     

Drifts exhibited by cryogenically cooled InSb infrared filtered detectors and their importance to the ATSR-2 and Landsat-5 Earth observation missions

The spectral responsivity of commercially available InSb detectors with low-pass cold filters attached to their cold shields for optimum operation in the 1.6-2.6 microm wavelength range is observed to drift slowly with time. These drifts are shown to arise because of a thin film of water-ice deposited on the cold low-pass filters mounted on the cold shields of the detectors. The temporal characteristics of these drifts are shown to strongly depend on wavelength. A model is proposed for the behavior of the water present in the Dewar vacuum, which can explain and predict the temporal characteristics of the observed drifts for all wavelengths. These observations are particularly relevant to space instruments that use cryogenically cooled IR filter radiometers for Earth observation. The temporal profile of drifts observed in missions such as Landsat-5 is identical to that observed in cryogenically cooled filtered InSb detectors during laboratory measurements. This study confirms that the deposition of a thin film of a material such as ice on the cold bandpass filters and windows is therefore the most likely source of the oscillatory drifts observed in the response of some of the channels of the ATSR-2, Landsat-4, and Landsat-5 Earth observation missions.     

Facilities for Simulation of Microgravity in the ESA Ground-Based Facility Programme

Knowledge of the role of gravity in fundamental biological processes and, consequently, the impact of exposure to microgravity conditions provide insight into the basics of the development of life as well as enabling long-term space exploration missions. However, experimentation in real microgravity is expensive and scarcely available; thus, a variety of platforms have been developed to provide, on Earth, an experimental condition comparable to real microgravity. With the aim of simulating microgravity conditions, different ground-based facilities (GBF) have been constructed such as clinostats and random positioning machines as well as magnets for magnetic levitation. Here, we give an overview of ground-based facilities for the simulation of microgravity which were used in the frame of an ESA ground-based research programme dedicated to providing scientists access to these experimental capabilities in order to prepare their space experiments.     

Logic programming to predict cell fate patterns and retrodict genotypes in organogenesis

Caenorhabditis elegans vulval development is a paradigm system for understanding cell differentiation in the process of organogenesis. Through temporal and spatial controls, the fate pattern of six cells is determined by the competition of the LET-23 and the Notch signalling pathways. Modelling cell fate determination in vulval development using state-based models, coupled with formal analysis techniques, has been established as a powerful approach in predicting the outcome of combinations of mutations. However, computing the outcomes of complex and highly concurrent models can become prohibitive. Here, we show how logic programs derived from state machines describing the differentiation of C. elegans vulval precursor cells can increase the speed of prediction by four orders of magnitude relative to previous approaches. Moreover, this increase in speed allows us to infer, or ‘retrodict’, compatible genomes from cell fate patterns. We exploit this technique to predict highly variable cell fate patterns resulting from dig-1 reduced-function mutations and let-23 mosaics. In addition to the new insights offered, we propose our technique as a platform for aiding the design and analysis of experimental data.     

Assessing the impact of marine wind farms on birds through movement modelling

Advances in technology and engineering, along with European Union renewable energy targets, have stimulated a rapid growth of the wind power sector. Wind farms contribute to carbon emission reductions, but there is a need to ensure that these structures do not adversely impact the populations that interact with them, particularly birds. We developed movement models based on observed avoidance responses of common eider Somateria mollissima to wind farms to predict, and identify potential measures to reduce, impacts. Flight trajectory data that were  collected post-construction of the Danish Nysted offshore wind farm were used to parameterize competing models of bird movements around turbines. The model most closely fitting the observed data incorporated individual variation in the minimum distance at which birds responded to the turbines. We show how such models can contribute to the spatial planning of wind farms by assessing their extent, turbine spacing and configurations on the probability of birds passing between the turbines. Avian movement models can make new contributions to environmental assessments of wind farm developments, and provide insights into how to reduce impacts that can be identified at the planning stage.     

The importance of accurate muscle modelling for biomechanical analyses: a case study with a lizard skull

Computer-based simulation techniques such as multi-body dynamics analysis are becoming increasingly popular in the field of skull mechanics. Multi-body models can be used for studying the relationships between skull architecture, muscle morphology and feeding performance. However, to be confident in the modelling results, models need to be validated against experimental data, and the effects of uncertainties or inaccuracies in the chosen model attributes need to be assessed with sensitivity analyses. Here, we compare the bite forces predicted by a multi-body model of a lizard (Tupinambis merianae) with in vivo measurements, using anatomical data collected from the same specimen. This subject-specific model predicts bite forces that are very close to the in vivo measurements and also shows a consistent increase in bite force as the bite position is moved posteriorly on the jaw. However, the model is very sensitive to changes in muscle attributes such as fibre length, intrinsic muscle strength and force orientation, with bite force predictions varying considerably when these three variables are altered. We conclude that accurate muscle measurements are crucial to building realistic multi-body models and that subject-specific data should be used whenever possible.     

Chemical methods for searching for evidence of extra-terrestrial life

This paper describes the chemical concepts used for the purpose of detecting life in extra-terrestrial situations. These methods, developed initially within the oil industry, have been used to determine when life began on Earth and for investigating the Moon and Mars via space missions. In the case of Mars, the Viking missions led to the realization that we had meteorites from Mars on Earth. The study of Martian meteorites in the laboratory provides tantalizing clues for life on Mars in both the ancient and recent past. Meteorite analyses led to the launch of the Beagle 2 spacecraft, which was designed to prove that life-detection results obtained on Earth were authentic and not confused by terrestrial contamination. Some suggestions are made for future work.