In situ resource utilization (ISRU) on Mars is a critical component to enabling humans on Mars to both establish long-term outposts and become self-reliant. This article focuses on a mission architecture using the SpaceX Starship as cargo and crew vehicles for the journey to Mars. The first Starships flown to Mars will be uncrewed and will provide unprecedented opportunities to deliver ∼100 metric tons of cargo to the martian surface per mission and conduct robotic precursor work to enable a sustained and self-reliant human presence on Mars. We propose that the highest priority activities for early uncrewed Starships include pre-placement of supplies, developing infrastructure, testing of key technologies, and conducting resource prospecting to map and characterize water ice for future ISRU purposes.A main goal of human space exploration is to develop humanity into a multi-planet species where civilization extends beyond planet Earth. Establishing a self-sustaining human presence on Mars is key to achieving this goal.  Extraction and processing of critical in situ resources for use such as propellants, life support, power generation, and radiation and rocket exhaust shielding, among other applications, can significantly reduce the required launch mass, risk, and cost of human and robotic space exploration.7–14The fourth and final condition will require sustained 15–23 Water will be required for uses such as life support and agriculture, and it will also be utilized through electrolysis to produce hydrogen and oxygen for use in fuel cells and rocket engines. Each kilogram of consumable material transported from Earth incurs a penalty of about 200 kg of propellant to launch, transfer, and land on Mars, depending on vehicle specifics. The ISRU of water ice facilitates the development of a self-sustaining civilization since the launch mass penalty for transporting water from Earth to Mars is eliminated.24The most valuable resource on Mars for ISRU is the vast deposits of martian water ice.Because of the importance of martian water, the characterization of the ice resource is a top priority for near-term robotic flights to Mars in preparation for human exploration. This paper, thus, focuses on an assessment of initial uncrewed flights to Mars aimed at characterizing the ice resource availability plus the methods and equipment required to extract and process the ice to support future human exploration. In addition, sustained human travel to and survival on Mars is a challenging goal that will require expertise on various topics such as on-site construction, infrastructure planning and development, power systems, communications capabilities, and human health and safety considerations, etc., which must also be addressed as early as the first uncrewed missions.This article presents a mission architecture assuming use of the SpaceX Starship vehicle  at one landing site or can land in separate regions of Mars if in situ site reconnaissance is warranted in different locations to select the final landing site for the crewed missions. The arrival of Starships at Mars could also be staggered by ∼1–2 months such that landing site assessment can be performed with data returned from the first Starship before deciding on the final landing site for the subsequent Starship vehicle(s) during a given launch window. These first uncrewed Starships should remain on the surface of Mars indefinitely and serve as infrastructure for building up the human base.We assume that initially, at least two uncrewed Starships will be launched to Mars.25–27 and thus the next wave of Starships can be launched at the next launch opportunity. This second wave of launches would include two or more uncrewed vehicles plus at least two crewed Starships; all of these vehicles can land at the preferred landing site for construction of the human Mars base. Starship launches then continue at each subsequent launch opportunity. An optimal plan is for the total number of landed vehicles to double at a minimum with each consecutive opportunity, with a to-be-determined split between crewed and uncrewed vehicles. These landings should be focused at the site of the human Mars base.Launch windows from Earth to Mars occur approximately every 26 months when the Earth and Mars are optimally aligned for interplanetary travel with maximum speed and minimal propulsion costs,29 This two-stage vehicle (Super Heavy first stage and Starship as the second stage) is fully reusable and can provide transport to the Earth orbit and the Moon, as well as Mars.30 Starship will serve as the lander for both uncrewed and crewed missions, customizing the payload volume depending on the mission.Starship will be launched by a SpaceX Super Heavy Booster.3 Musk1 outlines how SpaceX missions to Mars will utilize in-space propellant transfer. In this case, the Booster launches Starship into the Earth orbit where it is refilled with CH4 and O2 by additional tanker flights from Earth (tankers are Starships that carry only propellant as payload); the Boosters and tankers return to the launch site for reuse. The refilled Starship vehicle then travels to the surface of Mars.28 Refilling Starship in orbit effectively resets the rocket equation, allowing for large payloads to be transported to the Moon and Mars.Given its expected payload capacity, Starship is able to transport the necessary equipment to support sustained human exploration as well as the crews, enabling the eventual establishment of cities on Mars.3 and can utilize both forward and aft storage capacity.30 Starship is 9 m in diameter and 50 m in length. Nominal payload deployment doors are assumed to be 3 m × 3 m and can be further customized if necessary for specific payloads. Initial payloads will require significant autonomy for deployment to the surface and operations, whereas future payloads will have more crew oversight once a human presence has been established on the planet.We utilize the expected capabilities and performance of Starship for this analysis. Starship will be capable of delivering 100 metric tons of payload to the martian surface3 Starship then launches from Mars and conducts a direct return to Earth.28Starship is also capable of returning crew and cargo from Mars to Earth. The vehicle is refilled with propellants on Mars by using local resources processed through a surface propellant production plant.3 forward space, most of which will be pressurized for human habitation,30 an 800 m3 LOX tank, and a 600 m3 methane tank with a stainless steel primary structure.Starship flights carrying the first humans to Mars are optimally planned for the Mars launch window after the launch of the first two (or more) uncrewed Starship vehicles. Therefore, on human arrival on Mars, there will already be at least two cargo Starships on the surface. This second wave of missions can include two Starships carrying crew plus additional uncrewed/cargo Starships. The human Starships will have on order of 1,100 mThe LOX and methane tanks could later become pressurized living space on the surface of Mars. We recommend that these first crewed Starships each have about 10–20 total people onboard with an additional 100+ metric tons of available cargo mass per Starship. Cargo carried on these flights will necessarily include additional equipment required for human health and productivity during the transit to Mars and on the martian surface. These vehicles will also carry fully operational hardware needed to support the human Mars base, which is likely to include equipment for power production, water extraction, pre-prepared landing pads, radiation shielding, dust control equipment, and exterior shelters for humans and equipment.Humans will likely live on Starship for the first few years on Mars until additional habitats are constructed, so the radiation risk must be assessed and mitigated accordingly and equipment planned to support this initial infrastructure. The first wave of uncrewed Starship vehicles can also be relocated and/or repurposed as needed to support the people on the surface. These vehicles will be valuable assets for storage, habitation, and as a source of refined metal and components.As a sustained presence of humans on Mars is predicated on ISRU, a key objective for the first uncrewed Starship missions is to confirm the presence of water ice (and other desired resources) and characterize these resource deposits. This work would serve to either (1) validate the selection of the initial landing site as satisfactory for subsequent human landing or (2) provide valuable information to consider moving the human landing site to a different location. Here, we describe multiple uses of water for ISRU and a suggested payload for characterizing the distribution and properties of near-surface water ice for ISRU.31–34 This estimate will be higher for a longer-term base on Mars with fewer restrictions on water use such as using more water for personal hygiene compared with current International Space Station (ISS) protocols, and/or allowing more water for activities such as laundry and cleaning dishes.An estimate of requirements for water consumption in the first 5 to 7 years of human habitation is needed to create a notional water budget for an initial Mars base. Based on space station experience, the amount of water required (without recycling) is estimated at 0.6 kg/h/person, which includes water for consumption, hygiene, and everyday living.e.g., regolith processing, manufacturing, construction, perchlorate remediation, plant growth, habitat maintenance, etc). When designing water treatment and distribution systems for Mars, it is important to recognize that there will be various uses of water, which will each have different requirements for purity .The amount of water required per person will also increase as the Mars community grows and eventually includes activities that use water in addition to personal hygiene and hydration  ratio of ∼3.5. With its 1,200 metric ton propellant capacity, Starship requires on order of 933 metric tons of oxygen and 267 metric tons of methane for refilling on Mars. Through the use of water electrolysis and Sabatier reactions, the relevant net ISRU reaction to produce this oxygen and methane is COThis net reaction produces a mass ratio of O/F of 4 and therefore excess oxygen will be produced through this process, which can be used for breathing. The total quantity of water needed to refill one Starship through these processes is then on the order of 600 metric tons, and equivalent to an ice cube about 9 m on the side.There are multiple options for obtaining this water to support return flights to Earth for the first few crews on Mars. Water can be transported to Mars from Earth within Starship cargo vehicles to ensure the availability of necessary water for life support and propellant production. This approach will be especially important for ensuring life support needs for the first few human crews on Mars, before ISRU of water ice is fully functional and reliable.Bringing water from the Earth for propellant production is also likely possible in an emergency situation but is challenging, requiring the expensive delivery of water from Earth. Ultimately, the required water will be mined as a natural resource on Mars to support the expanding base and provide propellant for routine return trips to the Earth.As a resource required for survival and growth of a human civilization on Mars, the location and ease of access of water ice are key drivers for the landing site selection. The location of the Mars base, adjacent to the landing site, is one of the most critical decisions to be made as all other mission architecture planning and trade studies must be copacetic with the characteristics of this site.in situ at and near the first uncrewed Starship landing sites must, therefore, be developed. This on-the-ground reconnaissance is critical, because we must validate assumptions based on orbital and remote-sensing data, coupled with geological modeling. A top reason for characterizing the near-surface ice is to inform the development of ISRU systems. Knowing the form and purity of the ice  as well as its distribution  is important for planning the ice excavation operations and ISRU processing.An architecture to characterize the ice Understanding the distribution and quantity of the ice in the vicinity of the landing location may be especially important, as orbital observations have relatively large footprints, often averaging data over many square kilometers, and local variations may be significant. These uncrewed reconnaissance missions will not only demonstrate whether it is safe and how to land Starships at the site, but also provide data to determine whether there is, indeed, a viable ice resource in the vicinity of the landing site.in situ characterization will provide the ground truth needed to assess the viability of the ice resource at the chosen landing site and inform the final decision for the human landing location on Mars.Characterization of the near-surface ice resource will be accomplished by analyzing data collected using robots and rovers delivered by the first uncrewed Starship missions. This in situ measurements are required to characterize the ice resource. In addition to the composition, distribution, and form of the ice, the deposit's overburden must be understood. Depth and material properties of the overburden will influence the design and operation of the ISRU system needed to penetrate to the ice itself. Local geology and trafficability of the site are also important for general site knowledge and for planning of larger-scale ISRU processing and resource transportation systems.A number of 35To accomplish these objectives, a planetary instrument suite is proposed that has already been selected for flight to the Moon by NASA. The VIPER (Volatiles Investigating Polar Exploration Rover) mission is slated for flight in the mid-2020s to characterize the lateral and vertical distribution of volatiles on the Moon with the goal of helping to inform lunar ISRU architectures.35 The situation is similar for Mars, in that orbital data coupled with numerical modeling are suggestive of near-surface ice, but in situ data at higher confidence and at finer spatial scales are important to verify the potential for ISRU and required for optimizing the engineering solution for obtaining the ice.Although orbital observations of volatiles provide valuable information, missions such as VIPER will provide measurements on centimeter-scale resolution over distances of kilometers that are needed to generate adequate “volatile mineral models” for use in evaluating lunar resource potential.36 Each payload element is discussed here.The VIPER payload recommended for flight to Mars consists of several instruments integrated with a mobility platform  and subsurface drill  to characterize near-surface ice  provides (1) estimated hydrogen abundance via epithermal neutron flux, and (2) bulk regolith chemical information via thermal-to-epithermal neutron flux.The NSS measures both thermal and epithermal neutrons and is active during roving and drilling operations. The instrument operates by measuring the changes in the leakage flux of low energy neutrons out of the regolith. These neutrons are produced by galactic cosmic rays, which are so energetic that they shatter the nuclei in surface materials. The neutrons interact with other nuclei and lose energy, becoming thermalized in the process.Hydrogen is the most efficient at thermalizing neutrons because of their similarity in mass to protons. Water will necessarily have a hydrogen signature, though a hydrogen signal by itself does not uniquely identify the presence of water.38 The instrument operates both by viewing the surface underneath a rover while driving and also by viewing subsurface drill samples. Using different wavelengths of light to illuminate the surface, NIRVSS surveys the surface and excavation site for water and other volatiles, providing surface and regolith mineral context.The Near-InfraRed Volatiles Spectrometer System (NIRVSS) measures volatile composition, mineralogy, thermophysical properties, and fine-scale geomorphology.® MPH high-performance quadrupole mass spectrometer. The instrument analyzes volatiles released from the regolith during traverses as well as subsurface regolith extraction via drilling. MSolo can detect and differentiate low-molecular-weight volatiles between 1 and 100 atomic mass units. This capability allows for the estimation of water (H2O) abundance, and the identification of and relative intensities of various volatile species .The Mass Spectrometer Observing Lunar Operations (MSolo) instrument is a modified commercial off-the-shelf instrument based on INFICON's TranspectorThe Regolith and Ice Drill for Exploring New Terrain (TRIDENT) is a 1-m rotary-percussive drill developed by Honeybee Robotics and designed to deliver dry and volatile-rich regolith to the surface for inspection by NIRVSS and MSolo. TRIDENT relies on a full-faced drill bit with carbide cutters at the end of a 1 m auger to break up regolith and convey cuttings to the surface. The auger is split into two sections: The bottom 10 cm  has deep flutes at shallow angle which are ideal for retention of material; the upper 90 cm section has shallow flutes at steep angle which are suited for efficient material removal.40This design allows for a “bite sampling” approach that preserves subsurface stratigraphy. To reduce drilling power, TRIDENT delivers subsurface regolith in ten 10 cm “bites” all the way to 1 m depth (or deeper depending on drill string length).42 Both surface and airborne deployed instrumentation can be considered depending on the trafficability of the landing site area on Mars.To detect deeper ice within the upper few 10s of meters of the martian surface, a geophysics payload may also be considered. Shallower ice is preferred for ease of access to the resource, whereas deeper ice may still be valuable if it has higher purity or other beneficial characteristics. In all cases, also understanding the ice characteristics with depth and the total ice thickness is important for resource utilization planning. Multiple geophysical techniques are used terrestrially to detect subsurface ice, including ground penetrating radar, DC resistivity sounding, seismic refraction, and electromagnetic surveys.43 Each type of site likely requires a different style of mobility platform. The northern plains ice could easily be trafficked by a typical rover given the relatively flat terrain and ability to navigate around any local obstacles.Mobility systems are key to enabling these measurements of ice distribution. There are two different types of potential ice-rich landing sites on Mars for Starship: (1) subsurface ice in the northern hemisphere plains and (2) lobate debris aprons (LDAs), or rock glaciers with ice buried beneath a surface layer of rock and debris.Data collection on an LDA is likely more complex given the complicated topography often associated with these geologic features. Unless a particularly smooth area with suitable geotechnical properties is found, a typical wheeled rover likely could not traverse across an LDA given the expected instability of the surface and the sizes and frequency of rocks and boulders. In this case, airborne assets, vehicles with enhanced mobility capabilities, and/or instrumented impactors may be required to access and measure the LDA.e.g., if the LDA is actively flowing or not) will also affect the location of maximum ice concentration . An option is to access ice horizontally along the glacier margins (instead of from the top of the LDA) if the accumulation of rocks is not prohibitive near the toe or margins of the glacier. Before investigating an LDA, additional site selection work would be required to determine the local trafficability and site access as well as to identify locations within the LDA with the highest probability of hosting accessible ice.The current state of the LDA .The mobility vehicles can be made robust and able to navigate significant terrain challenges since mass need not be minimized given Starship's large payload capability. Easy access from the landed vehicle to the martian surface is possible from within the payload pods at the base of Starship. On landing, these vehicles can be deployed directly on the surface without the need for complex unfolding or deployment activities as have been used on previous missions.Additional vehicles can also be stowed and deployed from the main (forward) cargo area. The vehicles must be robust enough to traverse the martian terrain near the landing site but should not be overdesigned or optimized at high cost since more risk tolerance is allowed by deploying many assets at once. If one or several vehicles are lost or malfunction, the remaining fleet can accomplish the task of mapping the water ice. For the relatively benign topography of the plains ice site with its easy trafficability, vehicles similar to those used in many terrestrial environments could serve as mobility platforms to characterize the subsurface ice.44To understand the resource potential of the martian ice, the spatially distributed resource prospecting data must be synthesized to determine optimal locations for resource extraction and utilization. Resource assessments are common practice in terrestrial mining operations and provide a framework for making decisions under conditions of uncertainty by first filling in data at locations that were not sampled and then supplying information about resources in terms of potential occurrence, distribution, type, quality, amount, value, and certainty in assessment results.i.e., areas that are the most likely to contain, in this case, concentrations of water ice).45 The ability to geostatistically analyze ice prospecting datasets, determine cross-correlation relationships among in situ remote sensing datasets, and create predictive maps regarding water ice for ISRU (including areas where ground-truth data are not available) will be critical components of the prospecting process.Mineral Prospectivity Mapping (MPM) is a geospatial mathematical technique for predicting the location and likelihood for the presence or absence of a mineral (or resource) accumulation. The result of mineral potential modeling is a collection of prospectivity maps highlighting areas as not-permissive, permissive, favorable, and prospective  of the layer to ensure suitable drilling and/or excavation techniques are used to access the buried ice.The first step to enable ISRU is to characterize and verify the presence of the resource. The distribution  of the potential reserve must be mapped, and the form and concentration of the resource (ice) in the subsurface must be determined. Surface ice is largely not stable in the mid to low latitudes on the surface of Mars in the present-day climate,e.g., pore ice vapor deposited from the atmosphere, massive ice remnant from a relict glacier, frozen flood waters or snowpacks). Depending on the site, the overburden material may be wind-blown sands and fines, regolith and/or rock resultant from ice sublimation over geologic time, glacial depositions of larger rocks and boulders, headwall erosion, etc.Understanding the local geologic setting is also important for characterizing the subsurface ice itself as well as the overburden properties. Understanding the local geology and history of the site will inform the type of ice present in the subsurface  technology is a terrestrial method for extracting water in polar environments that is promising for martian application. The Rodwell was first designed and tested for use in Greenland and Antarctica.51 and has successfully provided tens of millions of liters of fresh water.52 The lifespan of a South Pole Rodwell is ∼7 years, which would be ideal for Mars since this timeframe would cover multiple launch opportunities to support an initial base of humans.Rodwell systems are robust and still in routine use in polar regions on Earth. A Rodwell has been used at the U.S. South Pole Station in Antarctica since 1995 to supply liquid water to the station53 This system is capable of mining water at up to 25 m depth and through up to 20 m of overburden. RedWater implements two proven terrestrial technologies: coiled tubing (CT) for drilling and Rodwell for water extraction. The RedWater components are shown in A Rodwell system dubbed “RedWater” is under design specifically for use in the martian environment by Honeybee Robotics.54 and heaters. On reaching an ice layer, the drill continues for another ∼3 m and then stops advancing forward, but the bit continues spinning. The drill deploys a flexible packer to seal off the hole and subsequently, the heaters are turned on to melt the surrounding ice. After melting a section of ice, the borehole is further pressurized, and a valve opens to allow water to flow to the surface tank, as in terrestrial geysers.Once the hole is made, the CT is left in the hole and used as a conduit for water extraction. The BHA contains a rotary-percussive drill subsystem similar to the one used in Honeybee Robotics Deep DrillA prototype RedWater system was designed and tested in a −25°C block of ice at the same temperature  to maintain the base for a significant amount of time at once, and then storing that water for future use. Regardless, growth and maintenance must be considered when designing each asset and component of the martian water distribution system.in situ sources on Mars must be tested for contaminants and potential biological presence and purified accordingly.55 The level of tolerable impurities must be identified for different uses of the water resource, for example, requirements for propellant purity will differ from requirements for drinking water. However, the fraction of water required to sustain the human presence on Mars will be relatively small compared with the water required to refill the Starship vehicles. As is routine on the ISS, 90% of water can be recycled; this would also be the case on Mars.Water extracted from 56–59 In the design of water analysis technology for Mars, avoidance of consumables is ideal to minimize the types and amounts of specific materials that must be transported from the Earth. A priori knowledge of ice characteristics will help inform the design of a suitable water analysis system.The first step in the water purification process is a laboratory analysis to characterize the impurities and guide the treatment processes. System reliability must be high for this testing, as chemical or biological contamination may significantly affect the health of the crew.60 For these methodologies, chemical consumables are generally not utilized but maintenance and replacement of hardware components is often required.There are multiple methodologies that can be employed for water and/or ice purification. Physical treatment technologies utilize phase separation of constituents in water through density differential , phase changes , selective physical barrier (filtration), reverse osmosis, or differential surface chemistry .in situ and the byproducts are benign.55 Numerous chemical purification methods exist, each with advantages and disadvantages as well as varying applicability to a long-duration space flight.55Chemical treatment options can remove unwanted contaminants as well as disinfect a water supply. These methods are generally rapid and reliable, although they typically consume reactants that must be resupplied and can produce byproducts that must be considered for waste management. Mars missions should prioritize chemical treatment technologies where reactants can be produced 62 Although the kinetics of biological processes are typically lower (hours to days) compared with physical or chemical processes (seconds to minutes), biological processes are capable of auto-regeneration and may provide a sustainable purification system within a bioreactor on Mars. Bioreactors have become common practice on the Earth63 and can consist of aerobic, anaerobic, or phototrophic systems. Trade studies must be conducted to determine the optimal combination of physical, chemical, and/or biologic water purification systems to support the Mars base.Biologic treatment options can also be considered to determine the optimal combination of techniques for water purification on Mars.Once water has been extracted from the martian subsurface, it must be distributed to end users or stored until needed. On Mars, water can be stored in either the frozen (ice) or liquid (water) state. The most efficient means for distributing water will greatly depend on the state in which it is stored. Trade studies should be conducted to determine the most efficient means for storing the water. Some considerations are outlined here.If a Rodwell system is used continuously to pump liquid water from the martian subsurface, then the water will be extracted and may be stored as a liquid. For safety, the base should always maintain some liquid water for crew use. Depending on production rates, some water would likely be stored for longer periods of time possibly as ice, which would remain frozen in the ambient martian environment but would need to be protected against sublimation.2 and its collection and processing must be integrated with the associated water production.The option of frozen storage may apply to the production of propellant for Starship return trips to the Earth, which occur only every 2 years. Trade studies are required regarding the options to constantly produce propellant at the base to minimize any surges in power needs versus producing propellant on demand in a batch process. Batch processing would reduce the cryogenic cooling requirements for longer term storage but would likely be difficult due to the power requirements to generate propellant all at once. Propellant production also requires COe.g., Sabatier).60 Electrolysis converts four water molecules into four H2 and two O2 molecules (4H2O → 4H2 + 2O2). Methanation uses a catalyst to convert carbon dioxide from the atmosphere with hydrogen from the water electrolysis to produce methane and water .Once martian water is extracted, further processing requires a combination of electrolysis and the methanation process  needed to perform for long periods of time can be tested to measure reliability. It is relatively easy for a human to perform maintenance activities such as changing out end effectors for drills and other hardware components, whereas such maintenance can be difficult to automate.During this time, it would demonstrate the long-term, high cycle operation of the COTherefore, long-duration performance testing hardware should be conducted before crews arrive, whereas more complex tasks should take advantage of the availability of human problem-solving skills once they arrive. However, in terms of enabling martian ISRU for support of the human base, the highest priority activity for the first uncrewed Starship missions is to characterize the Mars ice resource to finalize designs of the water extraction, purification, and processing systems for ISRU and begin testing out some generic high cycle systems in the martian environment.In addition to ISRU considerations, we outline additional high-priority objectives for the first two uncrewed Starship missions to optimize development of the human base on Mars. These tasks include deploying initial power infrastructure elements, characterizing the local environment at the landing site, implementing initial radiation shielding, food growth experiments, autonomous regolith excavation and drilling demonstrations, pre-positioning of supplies, and assessing hazards to human habitability during surface operations.The successful deployment and operation of a power infrastructure is of highest priority for both uncrewed and crewed missions to the martian surface. Since the availability of power is critical to surface operations, an initial deployment and test of power systems is a priority for the first Starship vehicles that land on Mars. For the landed missions (both with and without crew), power will initially be provided by battery power, followed by solar arrays once they are deployed and verified. A backup power source is also required that can be obtained via battery, chemical, and/or propellant sources.Solar power is likely an early power option beyond just batteries, but with the regular occurrence of global dust storms, nuclear power will be the long-term solution. Nuclear reactors can, thus, also be used for early crews if available. Although initial demands may be smaller, Starship's payload capacity will allow deployment of very large nuclear power facilities. As an example, the Hyperion reactor design weighs 20 metric tons and produces 25 MW of power.Significant uses of power include propellant generation and storage on the martian surface, Starship vehicle support, storage of water and food, and support for crew extravehicular activities away from the main base. Power will likely be allocated in a distributed manner with a number of networked assets that can access and harness the required power where and when needed. Contingency power systems are also required to ensure stay-alive power levels. If needed, non-essential activities such as propellant production and resource exploration activities could be curtailed in emergencies.A key technology demonstration that could be conducted on the first uncrewed Starship missions is the autonomous deployment and operation of a solar power system. Deployment methodologies as well as solar panels and array designs should be tested. For example, solar panels on space missions are typically optimized for high performance. Trades in solar panel design and manufacture for lower performance may be offset by simply deploying a larger array on Mars. For example, terrestrial solar panels are often mounted on heavy support structures that track the Sun to increase power production over time.The sheer areal extent of solar arrays required on Mars may prohibit the use of tracking devices, and the first missions can assess the merits and drawbacks of deploying the arrays on the ground or above ground, with or without tracking capabilities. For example, simpler deployment techniques such as pressurizing ribs to uncoil large rolls of solar arrays should be tested. In addition, maintenance of the arrays  must be tested to determine optimal long-term solutions.The first uncrewed Starships provide an unprecedented opportunity to collect environmental data at the landing site which will be valuable for the design and construction of future equipment and infrastructure to be flown to Mars. A basic weather station to measure atmospheric and surface temperatures, wind speed and direction, atmospheric dust content, atmospheric pressure, and radiation levels is necessary to minimize uncertainty regarding the environmental conditions expected for future missions. Surface properties such as trafficability, geomorphology, and geotechnical properties of the surrounding landscape should be assessed to optimize future infrastructure development such as roads, landing pads, and habitats.The flight of two uncrewed Starship vehicles to the martian surface also provides the opportunity to test several high-risk items before human arrival. Operations that may be particularly sensitive to unknowns in the martian environment and require long duration testing have the highest priority on the first Starship missions.Radiation shielding is a high-priority requirement to ensure human safety on the surface. Astronauts will require adequate radiation shielding while working and living within the Starship vehicle on Mars and will also require radiation mitigation when working outside of the Starship. For example, outdoor protected areas to conduct maintenance activities, laboratory work, and for transit between different Starships and/or facilities are required.65 along with numerous radiation models to predict expected radiation levels.67 However, existing surface radiation measurements on Mars are at discrete locations not coincident with the expected landing sites of Starship and, thus, the applicability of these measurements to different locations is unknown. Radiation models also include multiple assumptions and uncertainties and must be validated with surface measurements at the Starship landing site. Therefore, a long-duration radiation monitoring station on the surface at the Starship landing site along with different materials and designs for radiation shielding testing are envisioned, including the potential use of local Mars materials for radiation shielding purposes.Limited measurements of radiation levels on the martian surface are available68 Food production and processing is highly dependent on environmental conditions , which are variable between sites.69–71 Plant growth experimentation is necessary to assess the effects of various conditions such as high carbon dioxide levels, low light, low water and nutrient levels, pollination efficiency, and the effect of low pressure and magnetic fields on plant growth and crop productivity.72–75Food growth experiments are an opportunistic activity to conduct on early Starship landers. The ability for humans to grow their own food on Mars will be key to becoming self-sustaining.77 Hydroponics will also be assessed on Mars, building on hydroponic food production research on the ISS with the Vegetable Production System (VEGGIE).78 In addition, fresh plants and crops are expected to have a positive impact on the psychological health of the crew.75 Food production can be tested to a limited extent on robotic missions, whereas human-tended plant growth activities can potentially be conducted with less operational complexity.Most of these martian conditions are not believed to be limiting for crop productivity and can be compensated for within a greenhouse environment.79–81Additional experiments to be considered for the first uncrewed Starship landers are centered on the processes of excavation, drilling, and building by using autonomous operations. Excavation and drilling are key activities to enable long-term survival of humans on Mars. These activities are critical for ISRU as well as civil engineering applications such as building berms, roads, trenches, and landing pads.Much of this work can be done robotically, and the first Starship missions can be utilized to test and demonstrate autonomous techniques. These initial construction activities will inform variations on future designs for equipment to be flown to Mars, and to develop as much infrastructure as feasible before the first human landings.The substantial cargo capacity (100 metric tons) of the Starship vehicle provides ample opportunity to deliver and pre-position key supplies on Mars in advance of human arrival. The Starship vehicles themselves will serve as initial infrastructure at the Mars base. The large interior volume of Starship (both pressurized and unpressurized) provides significant space to serve as shelter in terms of habitation quarters, laboratory and work space for humans, and storage space for equipment and supplies.The first humans to Mars should be able to live within the Starship vehicles that flew them from Earth. However, in the event of any off-nominal issues, the human crew also has the original uncrewed Starship vehicles that first arrived on Mars as backup sources of habitation and storage space as well as spare parts and supplies.The first uncrewed Starships can also pre-position stockpiles of food and water on Mars before human arrival. This approach will provide redundancy and ensure adequate supplies to support the humans on the martian surface, as the crewed Starships will also bring food and water supplies. Food is key to human sustenance, and water will be important not only for human use and consumption but also to enable ISRU on Mars. In the event that water cannot be harvested from Mars in the time or quantities required to support ISRU activities, contingency rescue scenarios, including delivery of water from Earth via Starship, can be used to save the crew if needed.Communication systems from the first uncrewed Starships will be prepositioned and tested before human arrival on Mars. These systems will be established and tested for communication between Earth and the uncrewed Starships on Mars. Successful communication is required to ensure that the humans will have the ability to communicate with the Earth, even in the event of any off-nominal issues with the communications system in the human-tended Starships.The first uncrewed Starship will also preposition additional equipment such as rovers to enable mobility of equipment and humans. Rovers should ideally be equipped with a modular ability to complete multiple tasks and maintain energy efficiency. Rovers conceivably would be used for such varied tasks as deployment of solar arrays, excavation of regolith for construction and ISRU, moving cargo and material to different areas of the base site, and transport of equipment to field sites. Depending on the Starship landing site locations and the size of the human base, rovers may be needed to transport people and/or cargo between Starships and more remote locations .82Early Starships will land on native martian terrain with no landing site preparation available; however, operational capabilities can be enhanced for subsequent flights through the development of dedicated landing areas. Landing pads must not only withstand the heat and pressure of the engine thrusters, but also berms surrounding the pads must mitigate regolith sandblasting of the surrounding area.83 Multiple surface stabilization techniques have been proposed for the construction and evaluation of landing pads such as use of in situ materials, 3D printing, and freezing of landing areas.85 Multiple options could be assessed on Mars by producing multiple “landing coupons” using equipment delivered in the first uncrewed Starship and testing under subsequent uncrewed Starship landings.These protective measures are important to allow buildup of a human base with infrastructure in close proximity to the pads. The construction of landing pads will also prevent lander exhaust plumes from excavating holes under the lander, possibly melting subsurface ice and/or tipping the lander and causing lander damage.To enhance human health and safety, early uncrewed Starships could also include a payload(s) to assess the hazard potential to operations on the surface, including characterizing materials that humans are likely to encounter for potential biological, chemical, and mechanical hazards. This would include characterization of the water ice and dust along with refining our understanding of perchlorates in the soil. Instruments that could characterize these aspects of the martian environment and evaluate the biological potential in the area of interest would help inform mitigation measures and risk evaluation for initial crews traveling to Mars.86 Near-surface ice likely had water activity levels high enough to support life at higher obliquities, and ground ice is an especially efficient means for preserving organic biomarkers for extended periods of time.87–89 Multiple energy sources to support martian life exist in the form of carbon dioxide and nitrogen in the atmosphere, nitrates in the soil, and/or perchlorate combined with basaltic rock.86 This combination of parameters  leads to a reasonable possibility of extinct and/or extant life on Mars, which cannot be ruled out at the present time.Regarding the potential for life on Mars, recent work has examined the issue of martian habitability, which suggests that Mars could have harbored life within the past 5 million years.90–94 These investigations can provide baseline measurements of the biologic potential for native martian life in the near-surface environment on the uncrewed missions in preparation for the much more thorough search for life studies that will be enabled by having astronauts on the surface to conduct more sophisticated experiments and access subsurface environments.The instrumentation for biological hazard assessments could, thus, also provide additional scientific data in the field of astrobiology to help determine whether life ever arose on the Red Planet, a fundamental scientific question driving the exploration of Mars.1–3 The first steps of developing a self-sustaining human civilization on Mars utilizing planned SpaceX missions to the Red Planet have been outlined. The first Starships flown to Mars will be uncrewed and provide unprecedented opportunities to each deliver ∼100 metric tons of cargo and conduct precursor work to enable martian ISRU for a sustained and self-reliant human presence.3The SpaceX Starship vehicle fundamentally changes the paradigm for human exploration of space and enables humans to develop into a multi-planet species.Highest priority activities for the uncrewed Starships will be pre-emplacement of supplies and infrastructure, testing of key technologies, and conducting resource prospecting to map and characterize water ice.Starship missions to the lunar surface may be an important stepping stone for reaching Mars both technically and programmatically. The Moon can be a testbed and demonstration platform for ISRU technologies as well as Starship operations. Regardless of destination, this article highlights the importance of early master planning to enable development of a sustained human presence on another world.ISRU is a key component for enabling humans to decrease reliance on the Earth and instead develop a self-sustaining presence off-planet. The development and expansion of an initial Mars base will allow more humans to live on the Red Planet, and thereby accomplish the historic and unprecedented task of making humanity a multi-planet species.


Drosophila. We use embryos that greatly differ in length and, importantly, possess distinct length-scaling characteristics of the Bicoid (Bcd) gradient. We systematically analyze the dynamic movements of gap gene expression boundaries in relation to both embryo length and Bcd input as a function of time. We document the process through which such dynamic movements drive both an emergence of a global scaling landscape and evolution of boundary-specific scaling characteristics. We show that, despite initial differences in pattern scaling characteristics that mimic those of Bcd in the anterior, such characteristics of final patterns converge. Our study thus partitions the contributions of Bcd input and regulatory dynamics inherent to the AP patterning network in shaping embryonic pattern's scaling characteristics.How patterns are formed to scale with tissue size remains an unresolved problem. Here we investigate embryonic patterns of gap gene expression along the anterior-posterior (AP) axis in  PatternDrosophila, the maternally-derived morphogenetic protein Bicoid (Bcd) forms a concentration gradient along the AP axis to instruct embryonic patterning .Prof Jun Ma was supported by National Key R&D Program of China [2021YFC2700403 & 2018YFC1003203], National Key R&D Program of China [2021YFC2700403 & 2018YFA0800102], 10.13039/501100001809National Natural Science Foundation of China [31871249].Prof Feng He was supported by Data will be made available on request.The authors declare no competing interests.


Cyclura cychlura inornata and Cyclura cychlura figginsi). We studied populations within and outside the reproductive season and quantified tourist numbers during sample collection. Specifically, we measured clutch size, body condition, plasma energy metabolites, reactive oxygen species, baseline corticosterone concentrations and immune function of male and female iguanas from each population to address whether (i) disparate physiologies are emerging across a gradient of tourism and feeding, (ii) both subspecies respond similarly and (iii) responses vary with season/reproductive condition. We found significant effects of tourism level, season and their interaction on the physiology of both C. c. inornata and C. c. figginsi, supporting the idea that tourism is leading to the divergence of phenotypes. Specifically, we found elevated plasma energy metabolites, oxidative stress and a measure of innate immunity , but reduced corticosterone concentrations with increasing tourism in both subspecies of rock iguanas. These physiological metrics differ according to the level of tourism in both subspecies and persist across seasons despite variation with natural seasonal and reproductive changes. These findings suggest that anthropogenic disturbance results in disparate physiologies in northern Bahamian rock iguanas.To promote survival and fitness, organisms use a suite of physiological systems to respond to both predictable and unpredictable changes in the environment. These physiological responses are also influenced by changes in life history state. The continued activation of physiological systems stemming from persistent environmental perturbations enable animals to cope with these challenges but may over time lead to significant effects on the health of wildlife. In the present study, we tested how varying environmental perturbations driven by tourism and associated supplemental feeding affects the energetics, corticosterone and immunity of six discrete populations of the northern Bahamian rock iguana ( An organism’s physiology is inextricably linked to its environment, whereby an animal’s physiology enables it to cope with a variable landscape of perturbations . There iThe continued activation of physiological systems that enable animals to cope with environmental challenges may lead to disparate physiological phenotypes both in terms of circulating hormone levels and their downstream effects. As a result, differing phenotypes or eventually genetically distinct ecotypes may emerge whereby we see consistent differences in morphology, behaviour and/or physiology across variable environmental landscapes . A classxref ref-type="bibr" rid="ref76"Smith and Iverson, 2015/xref). For example, a long-term demography study in Bahamian rock Iguanas showed that individual growth rates increased and population density increased over time on tourist-visited cays  and changes in survival  in The Bahamas offer an exemplary system to address the effects of anthropogenic pressures on physiology by testing whether long-standing anthropogenic influences can induce physiological changes in free-ranging animals. In this model system, tourist visitation and supplemental feeding of iguanas is consistently different on adjacent cays that are otherwise similar. Animals at high tourist sites are visited and fed by hundreds of people daily, whereas moderate tourist sites only received approximately 18 visitors a day on average, and iguanas from unvisited sites are not fed by humans at all. Tourists primarily feed fruit on skewers, with grapes being to most frequent food item. This provides an ideal natural experiment to study the impact of disturbance and altered diet on physiology and reproduction, especially since altered diet and resource availability have been shown to have profound effects on other wildlife , except the introduced population on Alligator Cay (C. c. figginsi), making the results valuable to enhance our fundamental understanding of iguana health, its implications for affected wildlife and to inform conservation management. We assessed current visitation rates and specifically measured clutch size, body condition, plasma energy metabolites, reactive oxygen species, baseline corticosterone concentrations and innate immunity of male and female iguanas from each population to address the following questions: (i) Are disparate physiologies emerging across a gradient of tourism and feeding? (ii) Are both subspecies responding similarly? (iii) How do the responses vary/interact with season/reproductive condition? We predicted that physiological measures across populations would correspond to the degree of tourism and supplemental feeding and differences would be replicated in the northern and southern subspecies. We also predicted that these responses would shift as a result of reproductive activity, leading to interactions between season and tourism exposure.We tested how varying tourism levels and associated supplemental feeding affect the energetics, stress and immunity of six discrete populations of Bahamian rock iguanas within and outside the reproductive season. Our study sites represent all known reproducing populations of the northern subspecies of northern Bahamian rock iguanas .C. c. inornata) and a high-, moderate- and no-tourism site located in the southern Exuma Islands (C. c. figginsi) in The Commonwealth of The Bahamas. All pursuits, captures and times to blood collection were recorded for each animal. The majority of capture and bleed times were under 3 minutes and so we saw no effect of capture time on physiological parameters measured  via capture by hand, dipnet or snare connected to a retractable pole. We sampled within the reproductive season from 18–24 May  to 20–26 June  and outside the reproductive season from 3 to 11 September  from a high-, moderate- and no-tourism site located in the northern Exuma Islands , ‘moderate tourism’  or ‘no tourism’ (no tourists) based initially on our estimation of the intensity of human presence on each of the cays and quantified during our visits in 2019 as the number of visitors per day averaged over the sampling time frame. These ranking were based on both volume of tourism and regularity of visits, whereby high-tourism sites experience consistent daily visits from multiple tour companies with large numbers of people, weather permitting. Moderate-tourism sites experience less organized tourist visits and are visited intermittently by significantly fewer numbers of people from private yachts or smaller, personalized tour groups. To our knowledge, no-tourism sites experience no visitors due to the extreme difficulty of cay access (no landing beaches). This was substantiated by the skittish behaviour of the iguanas on these cays. These rankings are further supported by over 25 years of annual personal observations at each site.r2 < 0.06). We calculated body condition as the residual from a snout–vent length–mass ordinary least squares regression (We collected blood samples following the procedures described in We measured two lipid metabolites (triglycerides and free glycerol) via sequential enzymatic colour endpoint assays  using the manufacturer’s instructions and a dilution protocol  plus 4 nM L-glutamine (Sigma-Aldrich), 105 CPU (colony producing unit) E. coli  and tryptic soy broth on a 96-well microplate. We ran all samples in duplicate. We calculated background absorbance using a BioRad xMark microplate reader. After a 12-hour incubation at 37°C, we read absorbance following bacterial growth. We calculated bactericidal ability by subtracting background absorbance to account for any difference in plasma absorbance values, we averaged replicates for each control and sample and then divided the absorbance for each sample by the absorbance for the positive controls  [e.g. (1-(mean absorbance of sample/mean absorbance of positive controls))*100]. This provides the percent bacteria killed relative to the positive controls. We ran negative controls (containing only medium) to ensure that there was no background contamination. The mean CV across all plates was 4.37% and the CV was <5.98% on all plates.We used a bacterial killing assay involving the actions of opsonizing proteins, antimicrobial peptides and natural antibodies to assess the ability of blood plasma to eliminate a pathogenic bacterium, function . Followifunction , we diluWe measured baseline circulating concentrations of corticosterone , estradiol , progesterone  and testosterone  using a radioimmune assay. We performed assays following a protocol previously described by We assessed oxidative physiology by measuring the derivatives of reactive oxygen metabolite species (d-ROMs) and the effectiveness of antioxidant defences. Following methods described in Reproduction requires significant energy and nutritional resources, resulting in substantial changes to an animal’s overall physiology and behaviour. Thus, in trying to understand the impact of human activities such as tourism, reproductive status can have profound consequences. Oviparous animals such as iguanas deposit all of their energy into easily quantifiable units—eggs. Using high-resolution ultrasonography  we quantified total clutch volume (both number and size of follicles and eggs).10-transformed prior to inclusion in the statistical models. We used multivariate analysis of covariance (MANCOVA) to assess variation in physiology among iguanas, with separate models for each subspecies and separate models for each sex within each subspecies. Subspecies were included in separate models because of the known variation in the timing of breeding and unequal replication in sampling between subspecies. Further, the same suite of physiological products was not assessed for female and male iguanas, warranting different statistical models for each. There was moderate correlation among each of the dependent variables used in the models . We completed tests of multicollinearity and found the greatest variance inflation factor to be 2.602 .Progesterone, estradiol, testosterone and corticosterone values were not normally distributed; therefore, these values were logC. c. figginsi; May, June and September for C. c. inornata), tourism  and the interaction term for sample month*tourism as independent group variables. We included body condition as a covariate in all models. For models in which the sample month*tourism interaction was significant, we completed separate MANCOVA’s within each of the months. We also added ovarian follicle status (yes or no) as an independent variable for the month in which female iguanas were in reproductive condition.For female iguanas, we included the continuous dependent variables of progesterone (log-transformed), estradiol (log-transformed), triglyceride index, bacterial killing ability (log-transformed), corticosterone (log-transformed) and oxidative index in the MANCOVA models. We included sample month , triglyceride index, bacterial killing ability (log-transformed), corticosterone (log-transformed), and oxidative index in the MANCOVA models. These included sample month, tourism and the sample month*tourism interaction  as independent variables. We included body condition as a covariate in all models.P-value of <0.05 to be statistically significant. To compare groups within months, if the sample month*tourism interaction was significant, we used Bonferonni-adjusted pairwise differences to reduce the risk of inflated error. For each sample month, we included means and standard errors for each of the dependent variables in each possible combination of subspecies, site and sex.We considered a 24, 308 = 1.99, P = 0.004); therefore, we ran separate MANCOVA’s with the same dependent variables within each month . In May, females with follicles had a significantly higher triglyceride index  or no-tourism (P = 0.031) sites . Finally, estradiol differed significantly among sites  or moderate tourism (P = 0.003) than the site with high tourism. There was no significant difference in estradiol between the moderate-tourism and no-tourism sites (P = 0.999).There was a significant month*tourist interaction for the suite of physiological dependent variables , bactericidal ability (b), and oxidative index (c) for female <italic>C. c. inornata</italic> without (N) and with (Y) developing ovarian follicles in May

<graphic mimetype="image" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="coac001f1"/></fig><fig id="f2">Supplementary Figure 2

Variation across months in progesterone (a) and estradiol (b) for female <italic>C. c. inornata</italic> without (open circles) and with (filled circles) developing ovarian follicles

<graphic mimetype="image" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="coac001f2"/></fig><fig id="f3">Supplementary Table 1

Descriptive tables

<graphic mimetype="image" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="coac001f3"/></fig>1, 22 = 9.30, P = 0.001), with greater progesterone in females with follicles or eggs  and high-tourism (P = 0.050) sites with no difference between sites with tourism (P = 0.900). Corticosterone  and sites with high tourism (P = 0.024), though there was no difference between the sites with tourism (P = 0.999). Finally, oxidative index , but there were no other pairwise differences (P < 0.089 in both cases).In June, the only significant difference among these physiological measures was for progesterone

<graphic mimetype="image" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="coac001f5"/></fig>2, 23 = 3.33, P = 0.048; P = 0.009). There were no other pairwise differences (P > 0.090 in both cases).In September, only corticosterone differed significantly based on tourism level

<graphic mimetype="image" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="coac001f9"/></fig><fig id="f10">Supplementary Table 4

Descriptive tables

<graphic mimetype="image" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="coac001f10"/></fig>2, 93 = 8.65, P < 0.001; P < 0.001 in both cases), but no difference between May and June (P = 0.498). Bactericidal ability also differed by site independent of month , with no other pairwise differences (P > 0.184 in both cases).Bactericidal ability differed by month independent of tourism

<graphic mimetype="image" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="coac001f11"/></fig><fig id="f12">Supplementary Table 6

Descriptive tables

<graphic mimetype="image" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="coac001f12"/></fig>12, 118 = 3.12, P = 0.001); therefore, we ran separate MANCOVA’s with the same dependent variables within each month (June and September). In June, there were no significant differences in any of the physiological measures between females with follicles and those without follicles . Triglyceride index differed among sites based on the level of tourism . There were significant differences among the levels of tourism for corticosterone  and high (P = 0.001) levels of tourism. There was no significant difference between the sites with tourism (P = 0.999). Oxidative index also differed based on the levels of tourism  and no tourism (P < 0.001). The difference between the site without tourism and the site with moderate tourism was not significant (P = 0.071).There was a significant month*tourist interaction for the suite of physiological dependent variables  and no-tourism (P < 0.001) sites . Corticosterone differed among sites  and the site with high tourist activity (P = 0.043). There was no significant difference between the high-tourism and no-tourism sites (P = 0.999). The oxidative index also differed among sites  and the site with no tourism (P = 0.004), with the latter two not significantly different from each other (P = 0.999).In September, there were significant differences among the levels of tourism for the same three variables, but the nature of the relationships was different. The triglyceride index differed among sites  and estradiol (b) for female <italic>C. c. figginsi</italic> with and without ovarian follicles in different months

<graphic mimetype="image" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="coac001f13"/></fig>1, 62 = 2.50, P = 0.064; 2, 62 = 4.57, P = 0.014; P = 0.03) and no-tourism (P < 0.001) sites .Bactericidal activity was not involved in any significant interaction and did not differ significantly by month ; therefore, we ran separate MANCOVA’s with the same dependent variables within each month . In May, triglyceride index differed among sites  and moderate tourism (P < 0.001) compared to the site with no tourism. There were significantly different corticosterone levels based on the level of tourism , but there were no other pairwise differences (P > 0.368 in both cases). Oxidative index differed among sites  and high tourist activity (P = 0.002). Further, oxidative index at the site with high tourist activity was also significantly greater than at the site with no tourist activity (P = 0.002). Bactericidal ability , but there were no other pairwise differences (P > 0.578 in both cases).There was a significant month*tourist interaction for the suite of physiological dependent variables  and moderate tourism (P < 0.001) compared to the site with no tourism. There was no significant difference in triglycerides between the sites with tourism (P = 0.586). However, there were significant differences in corticosterone based on the level of tourism  and high tourism (P = 0.002) compared to the site with no tourist activity. There was no significant difference between the sites with tourism (P = 0.999).Bactericidal ability , but there were no other pairwise differences (P > 0.132 in both cases). Iguanas at the site without tourism had significantly greater corticosterone than those at the moderate-tourism site (P < 0.001) and the high-tourism site (P < 0.001), but there was no difference between sites with tourism (P = 0.999).In June, triglyceride index differed among sites . There were significant differences in corticosterone based on the level of tourism  and no tourism (P = 0.005), but there was no difference between the moderate-tourism and no-tourism sites (P = 0.534). Oxidative index differed among sites  and no tourism (P = 0.050). There was no significant difference between the latter two sites (P = 0.802). Bactericidal ability  and the site with moderate tourism (P = 0.042), but there was no difference between the latter two sites (P = 0.999).In September, triglyceride index differed among sites  and September (P = 0.006), with no difference between May and September (P = 0.428). Testosterone levels did not differ among sites.Iguanas had significantly lower testosterone ; therefore, we ran separate MANCOVA’s with the same dependent variables within each month (June and September). In June, triglyceride index differed among sites  and no-tourism (P = 0.001) sites . There were no significant differences in corticosterone based on the level of tourism in June  and no tourism (P < 0.001). The difference between the site without tourism and the site with moderate tourism was not significant (P = 0.996). Bactericidal ability .There was a significant month*tourist interaction for the suite of physiological dependent variables  and no-tourism (P < 0.001) sites . Corticosterone differed among sites in September  and the site with moderate tourist activity (P < 0.001) having greater means than the site with high tourist activity. There was no significant difference between the moderate-tourism and no-tourism sites (P = 0.900). The oxidative index differed among the sites as well  or the site with no tourism (P = 0.006), with the latter two not significantly different from each other (P = 0.999). Bactericidal ability , but there were no other pairwise differences (P > 0.679 in both cases). Lastly, testosterone was not involved in any significant interaction, and levels in September were significantly greater than in June , the physiological metrics corresponded to tourism level in both subspecies and persist, to some extent, across seasons. These results suggest that anthropogenic disturbance is resulting in disparate physiologies in northern Bahamian rock iguanas, but more work is required to test the consistency of these changes over time.Overall, we found significant effects of tourism level and season on the physiology of both C. c. figginsi of both sexes at the high-tourism site had higher concentrations of energy metabolites (i.e. an index of triglycerides and free glycerol) than moderate- and no-tourism sites, and all fed populations (both moderate and high) had elevated energy metabolites in C. c. inornata (except during September when only high-tourist iguanas had elevated metabolites relative to other populations). These findings are not surprising given that supplemental feeding of many types of wildlife by tourists is a large part of the tourism industry in The Bahamas and elsewhere (The primary tourism effects manifested predominantly in two ways: (i) some physiological metrics corresponded to tourism level in a step-wise fashion such that there were clear differences among high, moderate and no-tourism populations; or (ii) other metrics had an effect such that high- and no-tourism sites differed, but moderate sites were more similar to either high- or no-tourism sites. We also found some variation in physiological measures among sexes, subspecies and seasons. Specifically, Triaenodon obesus) (Amblyrhynchus cristatus) exposed to tourism in the absence of food provisioning show reduced immune function  , increasNegaprion acutidens) (C. c. figginsi during the nesting season (June), corticosterone was significantly lower at both high- and moderate-tourist sites than at the site with no tourists, but in September when all animals were post-reproductive, corticosterone levels in animals at the moderate-tourist site were elevated above the other sites. Similarly, for C. c. inornata populations, corticosterone was lower at high and moderate sites across seasons, except for early reproduction when females from the moderate-tourist site had elevated corticosterone relative to the other populations. The most likely explanation is that environmental variation between the northern and southern cay clusters is leading to differences in reproductive timing and thus physiology. Previous work has shown that nesting dates can vary by up to a week across these cays, even though they are in close proximity . Instead, during nesting season, C. c. figginsi males had higher corticosterone at high- and moderate-tourist relative to no-tourist sites, but during post-reproduction the corticosterone from high-tourist iguanas dropped significantly and corticosterone in male iguanas from no-tourist sites increased significantly .Easy and concentrated access to introduced food can lead to unnatural aggregation at feeding sites, which can lead to higher pathogen transmission (avian populations)  over the season, except during post-breeding when the oxidative index was only elevated in iguanas at the high-tourist site. Female C. c. inornata showed more seasonal fluctuations such that they had elevated oxidative indices at all tourist sites (high and moderate) in early reproduction, but only females from the moderate-tourist site had an elevated oxidative index during nesting and no differences among populations were present during post-reproduction. Interestingly, Galapagos marine iguanas also had higher d-ROMs at high-tourism sites and they were related to tourism intensity across sites in a dose-dependent manner. Given that marine iguanas, unlike rock iguanas, are not fed by tourists yet exhibited relatively similar patterns in both systems, this suggests that the relationship between tourism and oxidative stress could be independent from the feeding experienced by rock iguanas. However, absorptive states of digestion were associated with increased d-ROMs in corn snakes (Pantherophis guttatus) , suggesting that these iguanas may be experiencing a state of oxidative stress, such that their levels of antioxidants may not be sufficient to prevent harm from the reactive oxygen species being produced (C. c. figginsi apparently begins its nesting season 1–2 weeks earlier than C. c. inornata (C. c. inornata subspecies only), gravidity and nesting  and post-reproductive season . Therefore, it is not surprising that we found fluctuations in steroid hormones that correspond to the reproductive process, such as higher estradiol early in reproduction during vitellogenesis and higher progesterone during gravidity and right before and during nesting. Similar fluctuations are seen across species  and the reproductive cycle. Reproduction significantly alters iguana physiology  and then decrease during post breeding, including energy metabolites, bactericidal ability and the oxidative stress index. Past research has shown that both oxidative status and immunity shift with reproductive state and investment in a number of vertebrates, albeit in context-dependent ways , and potential inherent differences in natural history. For example, C. c. inornata is the northernmost member of its genus and sexual maturity in females requires 12 years . The slight variations in seasonal and tourist-based patterns between subspecies in body condition (Tables 1–3) and physiology are likely a reflection of both genetic .Additionally, the northern cays have historically been fed at high rates for longer, and thus we may be observing time-course differences in population density effects of tourism  -1752908 to S.S.F.], Utah State University Ecology Center [to A.C.W.], Shedd Aquarium [to C.R.K.], Dr Scholl Foundation [to C.R.K.], Paul M. Angell Family Foundation [to C.R.K], the Earlham College Biology Department [to J.B.I.] and the personal funds of most of the field assistants.Data related to published articles will be available on USU Digital Commons.suppl_data_coac001Click here for additional data file.


To evaluate the biomechanical performances of the sacroiliac screw fixation of the first sacral vertebra with different lengths of screws using the Finite-Element Method.First, pelvic CT images were generated from a healthy volunteer, and multislice sagittal views were produced to determine the axis for the first sacral vertebra geometric osseous fixation pathway (GOFP). Subsequently, according to the geometric size and mechanical parameters of the iliosacral screw, the screw models with the same diameter of 7.3 mm and different lengths of 80 mm, 90 mm, 100 mm, 110 mm, 120 mm, 130 mm and 140 mm were built. Then the seven screws were assembled with the pelvic model. The maximum von Mises stress and the shape variables were evaluated for the pelvis and the screws.Results are shown for the pelvic and GOFP screw, respectively. The simulation results show that the maximum von Mises stress in the cortex of the pelvic ring of the pelvis with the 130-mm length screw is the lowest among the pelvic models with different screws. Moreover, the peak displacement of the pelvis with the 130-mm length screw is the smallest. These results indicate that under the standing condition, a 130-mm length screw can decrease the stress concentration and result in a more effective transfer of stress within the reconstructed pelvis. In addition, the displacement of the screw with a 130-mm length is the smallest among all the considered screws. The peak von Mises stresses in the 130-mm length screw and the cortex are still within a low and elastic range below the yielding strengths of the materials.Through the finite element analysis, the GOFP can be used as a safe and effective way for iliosacral screw fixation. The optimal length of the screw may be 130 mm length. Percutaneous iliosacral (IS) screw fixation is a common surgical intervention method for longitudinal sacral fractures that is used to stabilize pelvic and acetabular fractures. However, due to the complicated structure in the pelvis and the deep location, this treatment requires high accuracy in screw implantation –3. S1 scIn this study, we demonstrated a computer-aided method for determining an optimal OFP for percutaneous IS screw fixation. The biomechanical performances of the sacroiliac screw fixation of the first sacral vertebra with different lengths of screws were evaluated using the Finite-Element Method (FEM).Ethical approval was obtained from the Human Research Ethics Committee, The Affiliated Hospital of Inner Mongolia Medical University, Hohhot, China.A healthy male adult volunteer  was enrolled in this study. No tumors and severe bony deformities were diagnosed prior to the study. The pelvis was scanned using a spiral CT scanner  at 120 kV with a slice thickness of 0.625 mm and a matrix of 512 × 512 pixels. The generated images were converted into DICOM format and were further processed using medical imaging software  to obtain the STL format files for the 3D reconstruction of the pelvises. Visualization software  was subsequently used to produce multi-slice sagittal views (thickness of 1.0 mm) of the 3D reconstruction images. First, we defined the geometric boundary of the safe zone on each sagittal view for the first sacral vertebra. Thus, the inscribed ellipse of the boundary was obtained from each view. Subsequently, the X, Y, and Z coordinates of the center in each inscribed ellipse  were calculated. Finally, the least-squares methods  were useTo perform the simulations, we defined the properties of the materials , such asAfter performing the mesh control of each part, to ensure the perfect contact between the different structures, application regions of the loads onto the Z, X, and Y axes were set along of sagittal, coronal, and axial planes, respectively . A 500 NAmong them, the screw displacement of 120 mm length was the largest, and the screw displacement of 130-mm length was the smallest, while the displacement increased when the length of the screw increased to 140 mm. The von Mises stress distribution in the pelvis showed that the maximum stress in the cortex of the pelvic ring with the screw length of 110 mm was the highest among all pelvic models. When the screw length was 130 mm, the stress decreased to a low level, and the stress increased when the screw length is 140 mm., The results indicate that the cortex of the pelvis with the screws of 110 mm and 120 mm length was prone to stress concentration and fracture, while the 130-mm length screw can reduce the stress concentration in the cortex and result in effective transferring of stress within the pelvis Figs. .Fig. 2ThFor accurate IS screw placement, more studies were done on the structure and boundary of bony in the safety area, especially the analysis of projection or image based on CT , 20, 21.Cruz’s research shows that there was a significant size difference in the analyzed sacral vertebra, differing on a wider size in men than in women. However, there was no significant statistical difference between vertebral size and age. Age does not influence the width of the surgical corridor. The surgeon has a safe corridor considered narrower when inserting screws in a female pelvis than when in a male one .To ensure the safety of the premise of the iliac body, it is recommended to use a long-threaded screw. Sagi  comparedThe screw path was defined as McLaren’s methods . The patWhether the optimal anatomical pathway obtained by our algorithm is also the optimal biomechanical solution is still not fully understood and should be verified in the subsequent studies. Furthermore, the optimal pathway provides a maximum safe range, rather than the surgery screw pathways. As long as the screw stayed within the pathways, its safety and reliability should be guaranteed.Fradet’s study had shown that sacroiliac screws provided forces to failure 2.75 times higher than sacral fixation screws . On the The lower bone density  led to a decrease of the IS (20–35%) and force to failure (5–20%) values, which is consistent with published experimental results showing decrease of IS by 40% and force to failure by 30% for a 30% density reduction , 32. By Some limitations should be considered when evaluating the simulation results. In this study, a pelvic model based on a healthy volunteer was used to build the finite-element model for evaluating the mechanical performance of iliosacral screws in the fixation of the first sacral vertebra. The effect of osteoporosis on the evaluation will be considered in future work. Also, to make the simulation results more convincing, the grid convergence analysis, and axial pullout and flexion/extension toggle forces on the screws representing intra and post-operative loads for complete biomechanical analysis should be calculated, these studies will be carried out in the next work.


Liver sinusoidal endothelial cells (LSECs) facilitate the efficient transport of macromolecules and solutes between the blood and hepatocytes. The efficiency of this transport is realized via transcellular nanopores, called fenestrations. The mean fenestration size is 140 ± 20 nm, with the range from 50 nm to 350 nm being mostly below the limits of diffraction of visible light. The cellular mechanisms controlling fenestrations are still poorly understood. In this study, we tested a hypothesis that both Rho kinase (ROCK) and myosin light chain (MLC) kinase (MLCK)-dependent phosphorylation of MLC regulates fenestrations. We verified the hypothesis using a combination of several molecular inhibitors and by applying two high-resolution microscopy modalities: structured illumination microscopy (SIM) and scanning electron microscopy (SEM). We demonstrated precise, dose-dependent, and reversible regulation of the mean fenestration diameter within a wide range from 120 nm to 220 nm and the fine-tuning of the porosity in a range from ~0% up to 12% using the ROCK pathway. Moreover, our findings indicate that MLCK is involved in the formation of new fenestrations—after inhibiting MLCK, closed fenestrations cannot be reopened with other agents. We, therefore, conclude that the Rho-ROCK pathway is responsible for the control of the fenestration diameter, while the inhibition of MLCK prevents the formation of new fenestrations. The vascular endothelium constitutes a functional barrier between the circulating blood and peripheral tissues. Endothelial cells are highly diverse, serving different functions depending on their location in the organism. Located in the liver capillaries, liver sinusoidal endothelial cells (LSEC) provide highly effective filtration functions, allowing for controlled bidirectional transport of macromolecules and solutes. The absence of a basement membrane and the presence of sub-micrometre-sized fenestrations lacking diaphragms give rise to the extreme hyperpermeability of LSEC , making It is widely accepted that fenestrated morphology is associated with healthy LSEC. Loss of fenestrations (defenestration) is a hallmark of liver pathology , or aginData on isolated LSEC provide in-depth insights into the structure, dynamics, and regulation of fenestrations. During the last few decades, several crucial discoveries were made about cellular regulation of fenestrations . FenestrThe existing four hypotheses describing the regulation of fenestrations in LSEC (summarised in ) indicatIn general, the hyperpermeability of the endothelium was shown to be controlled by the non-muscle myosin II . The conIn the present study, we fill the gap in the knowledge about the regulation of fenestrations by phosphorylation of MLC. We show that in LSEC, both phosphorylation and dephosphorylation of MLC results in an altered fenestration number and diameter. We analyse the effects elicited by various inhibitors on LSEC, describing parameters such as porosity (the fraction of cell area covered by fenestrations), fenestration size (distribution of fenestration diameter), the distribution and shape of long fibres and actin mesh, and the level and localisation of monophosphorylated (pMLC) and diphosphorylated (ppMLC) myosin regulatory light chain. To better understand the correlation between pMLC and actin cytoskeleton, we used blebbistatin to prevent the contraction of activated myosin. Moreover, to connect the role of MLC with actin cytoskeleton, we used an actin depolymerising agent, cytochalasin B, alongside the inhibitors. To quantify the morphology of LSEC, we used modern high throughput scanning electron microscopy (SEM), a well-established microscopic technique in LSEC morphology research. We also provide data acquired with structured illumination microscopy (SIM), a method with resolution better than those imposed by the diffraction limit of light, that would allow us to colocalize pMLC and ppMLC to sieve plates and fenestrations. The selected methodology and data analysis methods were carefully selected based on our recent reports ,29. We sIn order to unravel the role of MLC phosphorylation in the regulation of fenestrations, we firstly labelled mono- and diphosphorylated MLC (pMLC and ppMLC) in LSEC and studied the cells with SIM .The freshly isolated LSEC in the primary culture display heterogenous morphology. SIM provides the super-resolution necessary for the selection of well-spread and well-fenestrated LSEC. Moreover, it enables the co-localization of the antibody with the cytoskeleton, fenestrations, and sieve plates. We showed that pMLC/ppMLC colocalize with the actin filaments surrounding sieve plates. We did not detect any antibody signal within the sieve plates, neither in the control nor in LSEC treated with inhibitors. In non-fenestrated areas of LSEC, p/ppMLC was uniformly distributed and colocalized with actin filaments. Moreover, ppMLC was additionally observed in the nuclear region of all treated and control samples. The use of SIM has an additional advantage—it allows not only the assessment of differences in phosphorylation levels but also provides a precise intracellular distribution of pMLC/ppMLC. In particular, after CytB treatment, we observed that the distribution of the antibody is no longer uniform, but rather presented in grouped dots, which corresponds to actin dots . In contTo test the hypothesis that the phosphorylation of MLC is involved in the regulation of fenestrations and to decouple the mechanism regulated via either ROCK or MLCK pathways, we divided our experiments into two groups: first, involving inhibitors affecting ROCK/MLCP and second, involving inhibitors affecting MLCK in a calcium-dependent or independent manner. Next, we presented the effects induced by the non-muscle myosin II ATPase inhibitor, blebbistatin.n = 3) and shifted the distribution of fenestration diameters towards smaller values . Moreover, the diameter of the fenestrations was significantly smaller. The peak was shifted, by >25 nm, to 118 ± 45 nm. We observed broadening of the distribution of the fenestrations’ sizes towards smaller values. The detection of fenestrations in the range of 50–90 nm was reported to be overburdened with the error arising from the quality of SEM images [The proportion of fenestrations > 200 nm in the overall fenestration distribution decreases from 17% to 12% and 8% for the control, Y27632, and Y27632+CytB-treated cells, respectively. The mean fenestration size calculated from the maximum of the peak was 145 ± 52 nm for the control and 145 ± 47 nm for Y27632-treated cells. To enhance the effect of the inhibitor, after 30 min of treatment with 10 µM Y27632, 21 µM CytB was added for another 30 min. The combined effects of the two agents resulted in the additional increase of the LSEC porosity of 1.35–1.75-fold (3.4–5.1 percentage points) of the control  caused almost a complete loss of fenestrations, without any visible effect on the level of actin polymerization . After 2n = 3), but its effect was not as prominent as ML-7, and never reached a complete loss of fenestrations. We observed insignificant trends towards the reduction of fenestration diameter . In contrast to ML-7, the combined effect of 20 µM KN93 and CytB (30 min + 30 min) was the same as with CytB alone. Moreover, we did not observe changes in the level of actin polymerisation after KN93 treatment  to investigate sole inhibition of MLCK in a calcium-dependent and independent manner, without influencing the ROCK pathway. We showed that the inhibition of MLCK induces significant dephosphorylation of ppMLC and a reduction in pMLC after one hour. We observed a dose-dependent and reversible increase in the fenestration diameter, including an increase in the proportion of large (>200 nm) fenestrations. However, by increasing the dose of ML-7, the porosity decreased down to zero. It indicates that phosphorylated (activated) MLCK is involved in the process of fenestration formation. Previous reports showed that fenestration lifespan is ~20 min, but some fenestrations last for more than 2 h . MoreoveIn particular, we showed that after 10 µM of Y27632 treatment, a decrease in pMLC and ppMLC occurred, with a simultaneous increase in the porosity. Actin filaments became less prominent than in the control cultures and more actin mesh was present. Existing actin filaments often became rounded and within closed actin circles, sieve plates were observed. Our results are in agreement with the results presented by Venkatraman and Tucker-Kellogg . In thatThe effects of CalA and Y27632 on pMLC/ppMLC confirmed the desired influence of MLC phosphorylation; however, we could not clearly establish the effect of pMLC or ppMLC on fenestrations. Both ML-7 and Y27632 reduced the ppMLC, but only Y27632 had a significant effect on pMLC, indicating that pMLC plays an important role in the regulation of fenestration diameter. However, the regulation is probably more complex. CytB heavily increased pMLC and at the same time, had no effect on fenestration diameter, other than increasing the number of fenestrations. The results are in agreement with previous reports ,46. In aWe want to emphasize here that the fenestrated morphology is not a direct indicator of the condition of LSEC. By using inhibitors presented above, we could uncouple the porosity from the cell condition. We observed a decrease in viability and mitochondrial and endocytic activity after CytB  with a sThe observed weak effect of blebbistatin on fenestrations in LSEC was unexpected. The uncoupling of the regulation via phosphorylation of MLC should induce effects similar to ML-7 or Y27632. Indeed, it was similar to Y27632, as the rounding of actin fibres is observed; however, only an insignificant trend in the reduction of fenestration diameter was observed after the treatment. Blebbistatin inhibits only one isoform of myosin-II. The observed lack of effect on fenestration number and size may indicate that other myosin isoforms are involved in the regulation of fenestrations. A good candidate is myosin VIIa, which was reported to be highly expressed in rat LSEC . Having Several inhibitors, dyes, and chemicals were used. They are collated in TM . Parenchymal cells were removed by differential centrifugation, and LSEC were separated using immunomagnetic beads conjugated with antibodies against CD146 . After isolation, cells were seeded according to the experiment type. LSECs were seeded on Ø13 mm glass coverslips for SIM, in standard 48-well plates for endocytosis assays, or in 16-well plates with glass bottoms  for SEM. All surfaces were coated with 0.2 µg/mL of human fibronectin for 10 min prior to seeding. LSECs were incubated for 3 h at 37 °C with 5% CO2 before adding inhibitors.The experiments followed protocols approved by the local Animal Care and User Committees. All experiments were performed in 3–5 bioreplications with the total use of research animals of about 20 mice. LSECs were isolated using the modified protocol described in . BrieflyTo investigate the role of the actomyosin (actin-myosin complex) cytoskeleton in changes in the fenestration number and size, we proposed the sequential use of several widely used inhibitors . The schCytochalasin B (CytB) is used to depolymerise filamentous actin, and increases the number of fenestrations [trations . CytB istrations ,50, withtrations ,46. We a(−)-Blebbistatin blocks myosin-II ATPase activity. It was reported that the addition of 20 µM blebbistatin decreased fibre tension by ~80% and the effect was independent of MLC phosphorylation [rylation . Blebbisrylation . We applML-7 hydrochloride is a reversible and ATP-competitive MLCK inhibitor. By blocking phosphorylation of MLCK, it blocks both calcium-calmodulin-dependent and independent MLCK-induced phosphorylation of MLC. The phosphorylation of MLC can still be facilitated by ROCK. We tested 1, 5, 10, and 20 µM (1 h incubation), observing a dose-dependent effect on fenestrations. However, the highest concentration reduced cell viability after 2 h of treatment .Y27632 dihydrochloride is a ROCK inhibitor that causes relaxation of myosin tension in two ways: by preventing the direct phosphorylation of MLC via ROCK and by preventing ROCK-dependent phosphorylation/inactivation of MLCP. MLCK still phosphorylates MLC, but MLCP is constantly active, causing an elevated rate of the dephosphorylation of MLC. Y27632 dihydrochloride was used at a concentration of 10 µM (1 h incubation).Calyculin A  is a potent inhibitor of protein phosphatase 1 and 2A. It inhibits MLCP and other phosphatases. Phosphorylation of MLC via ROCK and MLCK occurs normally, but MLCP does not reverse it. We tested 1, 10, and 100 nM for 30 min, as longer treatments were detrimental to cell viability.SEM was used to analyse the detailed changes in LSEC morphology as it provides high spatial resolution. SEM images were used for quantitative image analysis. The method of sample preparation was presented in detail previously . Here, 1SIM is a super-resolution technique that allows optical visualization of LSEC, with their well-fenestrated morphology. The images were acquired using a DeltaVision OMX V4 Blaze imaging system  equipped with a 60X 1.42NA oil-immersion objective  and three sCMOS cameras. We used 488, 568, and 642 nm lasers for excitation. Image deconvolution and 3DSIM reconstructions were completed using the manufacturer-supplied softWoRx program . Images projections were analysed. It is necessary to check cell morphology together with specific staining to exclude dead cells. After isolation, up to 5% of primary cells die after seeding, despite remaining well-attached to the surface. LDH experiments  indicateSEM data were analysed according to the semi-automatic, threshold-based method described previously . AdditioIn addition to the standard way of presenting the fenestration distribution, we also discussed the ratio of fenestrations larger than 200 nm to all fenestrations. Because the area of fenestration is the quadratic function of their radius, the impact of large fenestrations on the porosity and filtration function of LSEC is huge. The porosity of the control groups varied between animals from 4–7%. In order to present the effect of the inhibitor relative to the control for all animals, we presented the data as the net increase/decrease in percentage points relative to the corresponding control.t-test.Data were analysed and visualized using Origin Pro software . SEM and SIM images were analysed using Fiji . The staThe aim of this study was to verify the hypothesis that MLC phosphorylation regulates fenestrations in LSEC. The size and number of fenestrations are strictly connected with the liver function. Small fenestrations in rabbits and too few fenestrations in chickens were associated with long chylomicron remnant retention in the blood stream and high susceptibility to atherosclerosis ,55. More