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Samples
Devils Hole vein calcite: A Holocene vein calcite (DHC2-8) that precipitated 4.5–16.9 ka before present, was collected from the Devils Hole cave #2 in Nevada, USA (36.427138 N, 116.291172 W). It is postulated that DHC2-8 precipitated at extremely slow rates, i.e., 0.1–0.8 μm year−1, at a constant temperature of 33.7(±0.8) °C30. For this study, we prepared a ca 0.5 × 0.5 × 1.5 cm slab of calcite. First, we abraded the surface ca 0.5 mm of the slab with a slow-speed hand-held drill to remove impurities. Then, the slab was cleaned in de-ionised water in an ultrasonic bath for 5 min and dried in a vacuum oven at 30 °C before it was crushed and homogenised using an agate mortar and pestle. Material from the same vein was analysed for clumped isotopes in other studies5,32.
Cryogenic cave carbonate: A coarsely crystalline cryogenic cave calcite (MSK 2b) was obtained from Mitterschneidkar Eishöhle in the Austrian Alps (47.1165 N, 11.7407 E). The cave opens at 2258 m above sea level and contained perennial ice in the near-entrance part until 2007. Today the cave is ice-free, and the mean annual air temperature in the interior of the cave is 0.23 °C33. Coarsely crystalline cryogenic cave carbonates generally precipitate slowly and very close to 0 °C, otherwise powder-like fine-crystalline cryogenic cave carbonates form50. Cryogenic cave carbonates occur in several places in the inner part of the cave, and U–Th dating of these carbonates suggests the presence of perennial ice up to about 2600 years before present33. The sample crystals were crushed and homogenised using an agate mortar and pestle and were subsequently dried in a vacuum oven at 30 °C before isotope analysis. Additional information on the potential equilibrium nature of this sample is found in Supplementary Fig. 1.
Cave pool carbonate: A 3.5-cm thick subaqueous calcite sample (Obi 87-i) was collected in 2008 from a perennial pool (Silbersee) in Rasslsystem cave, which is part of the Obir Caves (46.5102 N, 14.5480 E), a series of karst caves in the Southern Alps of Austria, located at approximately 1100 m above sea level. The Obir Caves are hypogene in origin51, i.e., they were not connected to the surface and hence had a very stable microclimate until discovered during mining activities in the 1870s. The Silbersee pool, located in the inner part of Rasslsystem cave, has a surface area of 7 × 4 m and is on average ca 1 m deep. The pool water temperature between 1998 and 2002 was 5.4(±0.1) °C, closely corresponding to the long-term mean annual air temperature outside the cave at this elevation52. The sample analysed in this study is a 4 mm wide subsample retrieved from 2.7 cm above the base of Obi 87, and is estimated to have formed at about 1500 years before present, based on the U–Th dating of a lower layer in Obi 87 (a layer estimated to have formed about 3800 years before present at 1.5 cm above the base of Obi 87) and assuming a constant calcite growth rate of 5.3 μm year−1 (unpublished data, C Spötl). Although the water temperature about 1500 years before present is not precisely known, it likely corresponded to the mean annual air temperature outside the cave at that time in a similar way as the modern pool temperature does. Various temperature proxy data for the Alps suggest that the mean annual air temperature fluctuated by up to ±1.5 °C in the last two millennia before the industrial revolution53. Considering the ca 1.5 °C warming in the Alps during the past century, we estimate the water temperature of Silbersee pool from which Obi 87-i precipitated ca 1500 years ago to be 4.0(±1.5) °C. Experiments demonstrated that subaqueous pool carbonates can precipitate in oxygen isotope equilibrium with water54. Prior to isotope analyses, Obi 87-i was cleaned in de-ionised water in an ultrasonic bath, crushed and homogenised using an agate mortar and pestle, and dried in a vacuum oven at 30 °C.
Synthetic speleothem carbonate: A calcite sample (MHD1) was precipitated in a laboratory, under cave-like conditions55. Solutions super-saturated relative to calcite were pumped to flow down an inclined, sandblasted glass plate in a thin solution film (0.1 mm in thickness), precipitating CaCO3 along the flow path. The experiments were performed in an enclosed space, which allowed control of all surrounding conditions, such as pCO2, temperature, and relative humidity. Specifically, sample MHD1 was precipitated at 30.7(±0.3) °C, with an atmospheric pCO2 of 1007(±47) ppm and a relative humidity of 97.5(±1.2)%. The average δ13C and δ18O values of the atmospheric CO2 were −44.7(±0.8)‰ and −10.6(±0.6)‰ VPDB, respectively. The experimental solution was prepared by dissolving 5 mmol CaCO3 powder in high-purity water while bubbling tank CO2 through the water column. After the complete dissolution of CaCO3 powder, i.e., when there were no visible particles in the solution, the solution was stored for five days at the experimental temperature to obtain isotopic equilibrium among all dissolved inorganic carbon species. This resulted in an initial solution composition of pH = 6.34, [DIC] = 18.19 mM, δ13CHCO3− ≈ −31.9(±1.3)‰, and δ18OHCO3− ≈ −8.69(±0.11)‰ VPDB. After being exposed to lower pCO2 in the climate box, it took ca 18 s for the solution to reach chemical equilibrium with the atmospheric CO2, which increased the solution pH and led to super-saturation with respect to calcite. The calcite sample was scratched off the glass plate after the experiment was completed and corresponded to the first 5 cm of flow, i.e., the first 24 s of CaCO3 precipitation.
Stalagmite: A calcite sample (SPA121-02) was retrieved from a stalagmite in Spannagel Cave in the Austrian Alps (47.0803 N, 11.6717 E), an extensive cave system with the main entrance at 2523 m above sea level. SPA121-02 is a 4-mm-thin layer within SPA121, a stalagmite that records a long growth history from about 240 to 76 ka. SPA121-02 was formed at about 225 ka during Marine Isotope Stage (MIS) 7.4 when this high-alpine cave was buried beneath a warm-based glacier preventing the cave from freezing56. The growth of this stalagmite during MIS 7 likely occurred at constant temperatures around freezing point, i.e., 0(±2) °C. The relatively high δ13C values of SPA121-02 (about 7‰ VPDB, Supplementary Table 1) was attributed to the disequilibrium isotope effects during peak cold times56. A 3 × 6 × 4 mm large piece was cut out from the axial part of the stalagmite SPA121 using a diamond-coated band saw. The piece was then cleaned in an ultrasonic bath in de-ionised water, dried, and crushed and homogenised with an agate mortar and pestle before isotope analysis.
Cold-water coral: A scleractinian, azooxanthellate coral Desmophyllum pertusum (formerly known as Lophelia pertusa) (JR) was collected alive at Traenadjupet, Norwegian Sea (66.973333 N, 11.108833 E) at a water depth of 300 m during cruise POS325-356/1. The annual mean seawater temperature at the collection location is 7.2(±1.0) °C57. With a hand-held drill, a corallite was cut from the axis of the colony, and the septa were removed, i.e., only the theca walls were sampled. The sample was cleaned in an ultrasonic bath using de-ionised water and dried in a vacuum oven at 30 °C before being crushed and homogenised using an agate mortar and pestle.
Warm-water coral: A scleractinian, zooxanthellate coral Porites lutea (PC1_2005) was collected at the Rashdoo Atoll, Maldives (4.293776 N, 72.977115 E) at a water depth of ca 1 m. For isotope analysis, a ca 0.5 cm thick section was cut from the coral core. Based on sclerochronological analysis, this section corresponded to the growth in the year 2005 when the annual mean temperature at this location was 29.3(±1.0) °C58. The mean annual extension rate of the coral is ca 10 mm year−1. To remove material that may have been thermally altered when the section was initially cut from the colony, the surface 0.5 mm was scraped away. Then, the section was cleaned in an ultrasonic bath using de-ionised water and dried in a vacuum oven at 30 °C before being crushed and homogenised using an agate mortar and pestle.
Modern brachiopod shell: A terebratulid brachiopod Magellania venosa (Mv143-b) was collected from Punta Gruesa, Chile (42.409833 S, 72.424333 W) from 20 m below sea level. The annual mean temperature at the collection location is 11.4(±1.7) °C12. Magellania venosa is one of the fastest-growing modern brachiopods, with growth rates ranging from 3.8 mm year−1 (adult) to 17.3 mm year−1 (juvenile)59. For this study, we sampled a ca 2 × 3 cm area in the middle part of the ventral valve. First, we abraded the primary layer of the shell using a slow-speed hand-held drill and a diamond drill bit, cleaned the shell in an ultrasonic bath with de-ionised water, dried it in a vacuum oven at 30 °C, and finally homogenised the material using an agate mortar and pestle. The anterior part of the same specimen showed the largest offset from equilibrium in ∆47 values in a previous study12.
Cretaceous belemnite: A belemnite Belemnopsis sp. (66-4.65) was retrieved from DSDP Site 511 at the Falkland Plateau (51.004667 S, 46.971667 W). The investigated rostrum solidum shows excellent preservation based on cathodoluminescence, and trace element analyses44,60. Burial temperatures remained below 100 °C for this core, which makes the solid-state alteration of the clumped isotope composition of this sample unlikely61,62. The same sample in this study was analysed for its ∆47 values, together with other belemnites, to reconstruct Early Cretaceous southern high latitude palaeotemperatures44.
Mass spectrometry
We performed the CO2 clumped isotope analyses of sample carbonates on a Thermo Scientific 253 Plus gas source isotope ratio mass spectrometer during April 2019–March 2020, in three measurement sessions (April–August 2019, September–December 2019, and January–March 2020), following the method of Fiebig et al.25. Samples were measured in 5–10 replicates. Each replicate analysis consists of 13 acquisitions (10 cycles of reference and samples comparisons in each acquisition and 20 s integration time during each cycle). The raw clumped isotope values (indicated by subscript “raw” on the ∆ symbol) were calculated using the [Brand]/IUPAC isotopic parameters29,63.
Data correction for the reference carbonates
In order to assign the long-term ∆47 (CDES90) and ∆48 (CDES90) values of the ETH 1, ETH 2, and ETH 3 carbonate reference materials finally used for sample correction (Table 1, see the next section), we followed the correction approach outlined by Fiebig et al.25. using equilibrated gases only (subscript “CDES90” on the ∆ symbol indicates that the ∆47 and ∆48 values of these carbonate reference materials are reported on the Carbon Dioxide Equilibrium Scale at a reaction temperature of 90 °C). A total of 36 aliquots of CO2 gases equilibrated at 25 °C and 54 aliquots equilibrated at 1000 °C were considered for the April–August 2019 period (Supplementary Data 1). Data correction for the reference carbonates consisted of two steps: correction for non-linearity followed by correction for scale compression25,64,65. These two steps are detailed below.
Correction for non-linearity: Within errors, the two sets of equilibrium gases, equilibrated either at 1000 °C or 25 °C, had identical slopes in ∆47 (raw) vs δ47 (Supplementary Fig. 3a) and ∆48 (raw) vs δ48 (Supplementary Fig. 3b) spaces, respectively, when the negative m/z 47.5 intensity is directly subtracted from measured m/z 47–49 intensities (scaling factor of −1, see below and in Fiebig et al.25). We, therefore, considered the slopes displayed by the merged data sets for the correction of non-linearity. In ∆47 (raw) vs δ47 space, the equilibrium gases showed a flat line such that non-linearity correction needs not be applied. In ∆48 (raw) vs δ48 space, the slope displayed by the merged data set was −0.0040(±0.0002).
Correction for scale compression: The intercepts for the 1000 °C and the 25 °C gases displayed in ∆47 (raw) vs δ47 and ∆48 (raw) vs δ48 spaces were compared to the corresponding theoretical values66 to constrain empirical transfer functions (Supplementary Data 1).
Finally, we combined the ∆47 (CDES90) and ∆48 (CDES90) values of ETH 1, ETH 2, and ETH 3 determined during the April–August 2019 period (Supplementary Data 1) with those reported in Fiebig et al.25 to calculate the long-term values listed in Table 1 (Supplementary Fig. 4). Shapiro-Wilks tests show that the combined ∆47 (CDES90) and ∆48 (CDES90) data set of the carbonate reference materials have a normal distribution with W-values  > 0.95 and p-values  > > 0.05.
Data correction for the carbonate samples
Unlike the method described in Fiebig et al.25, we did not make use of equilibrated gases to correct the samples but used our long-term ∆47 (CDES90) and ∆48 (CDES90) values obtained for ETH 1, ETH 2, and ETH 3 instead (Supplementary Data 2–4). This purely carbonate-based correction approach follows the principle of identical treatment of sample and reference materials and allows identification of subtle temporal drifts in the acid reaction environment and correction for them67,68,69. Correction of the sample data consisted of three steps: correction for non-linearity followed by correction for scale compression, and finally correction for variations in the reaction environment. These three steps are detailed below.
Correction for non-linearity: The negative background causing the non-linearities in ∆47 (raw) vs δ47, ∆48 (raw) vs δ48, and ∆49 (raw) vs δ49 spaces was corrected using Easotope70 by subtracting the intensities measured on the m/z 47.5 cup from the intensities measured on the m/z 47–49 cups, after multiplying the former by respective scaling factors. These scaling factors were determined empirically and enable one to calculate accurate negative backgrounds below m/z 47, m/z 48, and m/z 49 from the measured m/z 47.5 intensity. For the three periods of sample analyses, i.e., April–August 2019, September–December 2019, and January–March 2020, we determined the scaling factors in a way that no residual slopes remained between the respective measured values of the frequently analysed ETH 1 and ETH 2 standards in the corresponding ∆ vs δ spaces (Supplementary Figs. 5–7). The uniformity of the measured long-term ∆47 (CDES90) and ∆48 (CDES90) values of ETH 1 and ETH 2, also supported by experimental data71, allowed us to assume that they have identical ∆47 and ∆48 values (Table 1). Consequently, for the April–August 2019 period of sample analyses, scaling factors of −0.988, −0.906, and −0.648, respectively, were applied to correct m/z 47, m/z 48, and m/z 49 intensities based on the monitored m/z 47.5 intensity. For the September–December 2019 period, the corresponding scaling factors were −1.003, −0.938, and −0.581, respectively. For the January–March 2020 period, factors of −1.010, −0.92326, and −0.555, respectively, were applied.
Correction for scale compression: According to the principles outlined above, we used our long-term ∆47 (CDES90) and ∆48 (CDES90) values of ETH 1, ETH 2, and ETH 3 (Table 1) to project the non-linearity corrected, raw clumped isotope values of the carbonate samples to the CDES. We determined empirical transfer functions based on a comparison of our long-term mean ∆47 (CDES90) and ∆48 (CDES90) values of ETH 1, ETH 2, and ETH 3 (Table 1) with their corresponding, non-linearity corrected ∆47 (raw) and ∆48 (raw) averages over the three periods of sample analysis (Supplementary Figs. 5a–b, 6a–b, 7a–b). The application of these transfer functions to non-linearity corrected sample ∆47 (raw) and ∆48 (raw) values yields the ∆47 (CDES90,uc) and ∆48 (CDES90,uc) values of the samples (Supplementary Data 2–4).
Correction for subtle long-term variations in the acid reaction environment: When residuals between the accepted long-term and the measured ∆ (CDES90,uc) for all ETH 1, ETH 2, and ETH 3 replicate analyses are plotted against time, small but systematic temporal variations become detectable. For ∆47 (CDES90,uc), these residuals are on the order of ≤0.010‰ (Supplementary Figs. 8a, 9a, 10a), and for ∆48 (CDES90,uc) they are on the order of ≤0.030‰ (Supplementary Figs. 8b,  9b, 10b). We determined a residual vs measurement time function (Supplementary Data 2–4) and used it to further correct the ∆47 (CDES90,uc) and ∆48 (CDES90,uc) values in order to obtain the final clumped isotope compositions of the investigated carbonate samples (Table 2).
We used the non-linearity corrected ∆49 (raw) values of the carbonate-derived CO2 and the presumably uncontaminated equilibrated CO2 gases to check for potential contamination in the analyte. All ∆49 (raw) values of the carbonates fall within the range of the ∆49 (raw) values of the equilibrated gases, indicating no contamination of the investigated solids (Supplementary Figs. 11a–b, 12a–b, 13a–b). In addition, the lack of correlation between ∆48 (raw) and ∆49 (raw) of the measured analytes further argues that there is no contamination on ∆49 that would influence ∆48 (Supplementary Figs. 11c, 12c, 13c). All measured values can be found in Supplementary Data 1–4.
Acid fractionation factors
To be able to compare the experimentally measured clumped isotope compositions of a carbonate, i.e., the ∆47 (CDES90) and ∆48 (CDES90) values of the CO2 gas derived from the phosphoric acid digestion of that carbonate, with its theoretically predicted composition, i.e., the ∆63 and ∆64 values of the carbonate, we determined25 the clumped isotope fractionation factors associated with the 90 °C acid fractionation during our analysis. These are based on the long-term ∆47 (CDES90) and ∆48 (CDES90) values of ETH 1 and ETH 2 standards which were both potentially equilibrated at 600 °C68. The theoretically predicted calcite ∆63 and ∆64 values at 600 °C are 0.018‰ and 0.002‰28, respectively. These, combined with our experimentally measured ∆47 (CDES90) values of 0.212(±0.002)‰ and ∆48 (CDES90) of 0.140(±0.005)‰, yield acid fractionation factors of 0.194(±0.002)‰ for ∆63–∆47 and 0.138(±0.005)‰ for ∆64–∆48.
Numerical modelling
We used numerical models to simulate the evolution of the isotopic composition of the DIC during (1) CO2 absorption, i.e., the key process involved in coral calcification26, and (2) the laboratory carbonate precipitation of the synthetic speleothem27 (Supplementary Data 5).
(1) CO2 absorption simulations were constructed using the IsoDIC model to mimic the internal calcification environment of scleractinian corals26. Specifically, the modelled calcification environment consisted of an aqueous solution ([DIC] = 2 mM, δ13CDIC = 0, and pH = 8.8 for cold-water corals and pH = 8.5 for warm-water corals), which was exposed to a CO2-containing atmosphere (pCO2 = 1100 ppm72 and δ13CCO2 = −15‰73). The temperature of the modelled calcification environment corresponded to the mean growth temperatures of the cold- and warm-water corals, i.e., 7.2 °C and 28.9 °C, respectively. The catalytic enhancement of the inter-conversion between CO2 (aq) and HCO3− by carbonic anhydrase during coral calcification is simulated by increasing the rate constants of CO2 (aq) (de)hydration reactions26. The initial oxygen and clumped isotope compositions of both the DIC and air CO2 were assumed to be in isotopic equilibrium with the water (δ18OH2O = 0 VSMOW) at the above described temperatures.
(2) To model the isotopic composition of the synthetic speleothem, simulations were constructed using the IsoCave model27, based on the conditions of the laboratory experiment55 (T = 30.7 °C, pCO2 = 1007 ppm, water film thickness of 100 μm, δ13CCO2 = −44.7‰, δ18OCO2 = −10.6‰ VPDB, δ13CCaCO3 = −6‰, δ18OH2O = −9‰ VSMOW, see above as well) and yielded an initial solution composition of pH = 6.3, [DIC] = 18.1 mM, [Ca2+] = 4.9 mM, δ13CHCO3− = −31.2‰, and δ18OHCO3− = −9.0‰ VPDB, which are close to the experimentally determined values (pH = 6.34, [DIC] = 18.2 mM, [Ca2+] = 5 mM, δ13CHCO3− ≈ −31.9(±1.3)‰, and δ18OHCO3− ≈ −8.69(±0.11)‰ VPDB, see above). More

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Rearing EPNs
Steinernema feltiae (SN strain) IJs were obtained from the International Entomopathogenic Nematode Collection (USDA-ARS, Byron Georgia USA). The nematodes were cultured in the laboratory using the White trap method41. Galleria mellonella (Vanderhorst Wholesale Inc. St. Marys, OH) were exposed to 100 IJs per larva. Infected G. mellonella larvae were incubated for 4 days at RT (20 ± 1 °C) and insect cadavers were transferred to White traps for IJ collection.
Why Steinernema feltiae was chosen
S. feltiae is a widely studied nematode and commercially used as a biocontrol agent in diverse systems. We selected S. feltiae for two reasons. (1) The dispersal behavior at the expected ISS temperature was the main factor in our decision. S. feltiae disperses without a quiescent period at temperatures from 15 °C to 30 °C. Some other EPNs such as S. carpocapsae IJs disperse normally at or above 25 °C, but at 20 °C and below, the nematodes have a quiescent period where IJs stay stand still for period of 40 min to 24h14. ISS ambient temperature is between 21 and 23 °C. We did not want to take chance with potential failure due to a temperature fluctuation so we chose S. feltiae, which is known to be active at the temperatures42 of the ISS. (2) S. feltiae is an intermediate forager thus incorporating behaviors of both foraging extremes43.
NASA safety certification, flight configuration, and safety experiments
The NASA safety certification was conducted by an implementation partner, NanoRacks, LLC (Houston, TX). The safety certification included filling out safety and experimental plan documents, submitting a bill of materials, and providing material safety sheets for the materials that would be flown to the ISS, their MSDS, and conducting safety and flight configuration experiments. To determine the risk of freezing tubes with moist sand on the ISS cold stowage, we sent three 15 ml conical tubes filled with 10% moist sand to NanoRacks for NASA safety testing. Briefly, play sand (Quikrete, Atlanta, GA) was washed three times in tap water and rinsed three times with MILLIQ water (MILLIPORE, Burlington, MA) using a gold pan (Garrett’s gravity trap, Garland, TX) then dried at 60 °C in the oven for 3 days or until all the moisture was evaporated. In a separate dish, dry sand was moistened with 10% MILLIQ water (w/v). Twelve ml moist sand was placed in 15 ml conical tubes that were rinsed three times with MILLIQ water and dried prior to use. We sent three tubes with moist sand to NanoRacks to be tested at NASA for safety. Next, we moved forward with flight configuration experiments for Specimens 2–6, prepared as shown in Fig. 5. Specimens 2–6 each with two replications were conducted once at 23 ± 2 °C and once at 15 ± 1 °C to determine whether nematodes can emerge and go through in the sand, infect the bait insects and reproduce within a month in a horizontal position. At the same time, we examined whether the tubes for Specimens 2–6 developed any discoloration or warp due to the byproducts produced during infection and decomposition of the cadavers for a month. Later, the flight configuration experiment was extended to six months in case return to Earth would be delayed due to lack of space in the resupply capsule. The lower temperature (15 °C) was tested as a precaution to determine whether nematodes still infect if the temperature fluctuated unexpectedly at ISS. We did one last safety test for Specimen 6 (insect only) to determine whether gasses released by decomposing insects could discolor or warp the tubes, causing a hazard on the ISS. For this experiment, two G. mellonella prepared in Fig. 5c were first placed in −80 °C overnight to kill insect larvae humanely and then the tubes incubated at 23 ± 2 °C. Two replications with a total four insect larvae were monitored monthly for over 6 months for discoloration and warping due to decomposing insect larvae. No warping or discoloration was observed, fulfilling safety requirements to move forward with the experiments.
Fig. 5: Experimental design of specimens for microgravity and their Earth control.

b Specimens 1–4. One end of the tube, designated as 1, had either two infected insect hosts for providing infective juvenile nematodes (Steinernema feltiae) or 2000 IJs in 0.5 ml of polyacrylamide gel. The other end of the tube, designated as 2, had either two healthy bait insects or infected insect. bS. feltiae IJs in gel. 4000 IJs/ml of polyacrylamide gel. c Two healthy Galleria mellonella larvae with wood shavings as shown.

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Preparing IJs for the microgravity experiments
S. feltiae IJs were removed from the White trap 4 days after the beginning of emergence and rinsed three times in deionized water to remove residual cadaver-derived metabolites and pheromones (Supplementary Table 1) as described by Kaplan et al.14. Rinsed IJs were resuspended to infect the Galleria mellonella larvae for Specimens 1, 2, and 3 and used to prepare IJs in polyacrylamide gel (75 ml of water 1 g of a polyacrylamide gel (Soil Moist, JRM Chemical, Cleveland OH) with a density of 4000 IJs/ml for Specimens 4 and 5 as shown in Fig. 5a for both microgravity experiments and their corresponding Earth controls.
Six concurrent experiments in microgravity for emergence, infection and reproduction and space flight
We designed multiple concurrent experiments that were conducted at the International Space Station U.S. National Laboratory (ISS) with controls conducted on Earth. The experiments were designated as Specimens 1–6 (Fig. 5). The concurrent experiments were conducted to capture the maximum amount of information about the EPN infection process and life cycle in microgravity and make sure that usable data was returned even if microgravity interrupted part of the EPN life cycle. The space limitations of conducting an experiment on the ISS also had to be considered. Microgravity causes changes in many variables (not just gravity), which may impact a multistep infection requiring the cooperation of two organisms (nematode and its mutualistic bacteria). Furthermore, some of the facilities on the ISS, such as cold stowage (−80 °C), have limited capacity and high demand. A timeline of the experiments (Specimens) is presented in Fig. 2 and Supplementary Table 1.
We used a modified sand column assay44,45 shown in Fig. 5. The experimental units consisted of 15 ml conical tubes (Fig. 5) filled with S. feltiae infected G. mellonella larvae or 2000 S. feltiae IJs in 0.5 ml of polyacrylamide gel were placed in one end of the tube and the healthy larvae were placed on the other end of the tube (CELLTREAT scientific products, San Diego, CA). Based on preliminary evidence, the set-up allows a 3-day time delay for the nematodes to move through the sand and infect the bait larvae. However, we did not know what delay there would be under microgravity when setting up the experiment.
Specimen 1 was prepared as shown in Fig. 5a to determine whether nematodes reproduce, form the IJ stage, emerge, disperse, travel 10 cm in moist sandy soil, find a bait insect and infect/invade the host in microgravity (Fig. 1a–d). If the infection failed, this specimen was also designed to determine whether it was due to insect host-immune response. Specimen 1 was placed in cold stowage (−80 °C) 7 days after the launch at ISS, which was after the anticipated time of IJ invasion. A corresponding Earth control was prepared for both IJ invasion and insect host immunity response. Since we did not know how freezing or a month in −80 °C storage would affect hemocyte counts, we prepared additional Earth controls to be analyzed on the day the Earth Specimen 1 controls were placed in −80 °C (Supplementary Table 1). Three replicates were prepared for each treatment; microgravity, corresponding Earth control and additional Earth control for hemocyte counts. A total of 18 insects in nine individual tubes were analyzed for IJ invasion, to determine the IJ developmental stage and hemocyte counts for both Space and Earth specimens. The hemocyte counts were done for the additional Earth control (no freezing) on the day the other set was placed in the −80 °C freezer. Mean hemocyte count for the additional Earth control (no freezing) was 2.8 ± 1.7 from three replicates.
Specimen 2 was prepared in the same manner as Specimen 1 except that the tubes were not frozen to allow IJs to develop and emerge from the bait insect. Unfortunately, they died upon return to Earth. Three replicates were prepared for each treatment; microgravity and Earth control with a total of six tubes analyzed.
Specimen 3 was prepared the same as Specimen 1 except that it did not include a bait insect and the location of the infected insect was on the opposite side (Fig. 5). This specimen was designed to determine whether nematodes can continue their development and reproduction during the flight and emerge in microgravity. Since we did not know whether IJs could emerged from consumed cadavers under microgravity, we prepared Specimen 3. Sand was included to make sure that the emerged nematodes have a place to live until the experiment was returned to Earth and to make a good comparison to Specimen 1. Furthermore, Specimen 3 was kept at ambient temperature to test IJs dispersal after returning to Earth. Again, three replicate tubes for each treatment (microgravity and Earth control) were prepared with a total of six replicate tubes.
Specimen 4 was prepared the same as specimen 1 except that it had 2000 IJs in 0.5 ml of polyacrylamide gel instead of an infected insect (Fig. 5a). This was a backup experiment just in case Specimens 1 and 2 failed to emerge in microgravity to infect bait insects. The column contained sand in the middle (causing a time delay) so the infection event would occur in space not on Earth. The experiment had to be delivered to NASA 36 h before the rocket launch. The sand provided enough time delay (2–3 days) that IJs would only find the bait insects when they reached the ISS or when the Dragon Capsule was in space. This experiment had an Earth control like the other experiments and one additional Earth control was included to determine whether the bait insects were infected before the rocket launch. Three replicates were prepared for each treatment; microgravity and two sets of Earth controls with a total of nine tubes analyzed.
The rocket launch was delayed 1 day from December 4 to 5. The IJs had 2 days, 21 h and 20 min to go through 10 cm sand to reach the bait insect as opposed to 1 day 21 h and 20 min as planned launch day December 4. This was very concerning. At the earliest time (1 and ½ h) after the rocket launch, we analyzed the additional Earth control (three replicates) for insect invasion in two steps. First, we placed the IJs in a petri dish and added water to rinse the IJs off the surface of the insects. Each replicate was analyzed on a separate petri dish. The bait insects were alive and in all three replicates had IJs on them. Next, we washed all the insects to remove all the IJs from their surface and looked inside the insects to determine whether any of the IJs were inside the insects. No IJs were found inside the bait insects suggesting that none of the insects were infected while they were on Earth.
Specimen 5 contained S. feltiae IJs in polyacrylamide gel with a density of 4000 IJ/ml (Fig. 5b). A total volume of 10 ml of IJ acrylamide suspension was placed in each of three 15 ml conical tubes. This specimen was prepared to determine how 33 days of space flight would affect IJ adjustment (movement) and infectivity after returning to Earth. Upon return to Earth at the earliest time (3 days after returning), IJ movement was compared to an Earth control and 10 days later, IJ infectivity was tested. This was also a backup experiment in case nematodes died due to the effect of sand because IJs were known to safely travel on Earth in polyacrylamide gel43. Like the other experiments, three replicate tubes (120,000 IJs) for each treatment (microgravity and Earth control) were prepared with a total of six replicate tubes (240,000 IJs).
Specimen 6 had two G. mellonella last instar larvae in 15 ml conical tubes in wood shaving (Fig. 5c). This was to determine if the infection failed in Specimen 1 due negative effects on the insect host. At the end of the space flight, insects in both microgravity and Earth control were dead at different developmental stages. Several insect stages were observed in the various tubes (larvae, pupae and adult), all of which died. A total of 12 insect larvae in six replicate tubes were analyzed.
All Specimens, except for Specimen 1, were kept at ambient temperature on the ISS, during the flight back to Earth, and for 3 days while being shipped to our laboratory for further analysis on nematode IJ fitness and infectivity.
Handing over the experiments to NanoRacks for delivery to NASA for launch
Specimens 1–4 and 6 in Fig. 5 were assembled at Kennedy Space Center (KSC) Space Station Processing Facility (SSPF) on December 2 (Supplementary Table 1). Specimen 5 in Fig. 5b was prepared on Nov 29 at the Shapiro lab in Byron, GA, and kept at 5 °C until December 2. After all of the specimens were prepared (Supplementary Fig. 1), Specimen 1 replications were placed in a sealed bag and Specimens 2–6 were placed in a separate sealed bag. They are weighed and placed in Nanotracks’ NanoLab in a Horizontal form (Supplementary Fig. 1). The Nanolab lid was sealed with Velcro. After that, the Nanolab was delivered to NASA and placed in the Dragon Capsule on SpaceX Falcon9 rocket for CRS-19 on the same day (Supplementary Table 1). The experiments in the Nanolab were placed in a horizontal position and kept 20 °C for 2 days, 21 h and 20 min in the Dragon capsule until the launch on December 5, 2019. Experiments were kept in a horizontal position during the launch, in microgravity in Space, and at ISS at ambient temperature until returned to Earth.
Earth controls: Specimen 1 for Earth control was kept in a sealed plastic bag and Specimens 2–6 were placed in a separate sealed plastic bag at ambient temperature at the KSC SSPF until launch on December 5. Specimen 1 Earth controls were taken to the Shapiro Lab by car and kept at RT until December 11. Specimens 2–6 were shipped by FedEx at ambient temperature using cool packs for maintenance of the temperature to the Pheronym R&D laboratory, Davis, CA
In orbit and return to Earth
Specimen 1 (the three 15 ml conical tubes) was transferred to cold storage on December 12 at 346/14:30 (8:30am CST) by European Space Agency Astronaut Luca Parmitano (https://blogs.nasa.gov/stationreport/2019/12/12/). Specimens 2–6 were kept in the NanoLab on the Dragon Capsule which returned to Earth on January 7, 2020 and was retrieved from the Pacific Ocean by Space X. NanoRacks received the samples on Jan 9, 2020, in their facility in Houston, TX and shipped Specimen 1 (kept frozen) in dry ice overnight by FedEx to the Shapiro Lab in Byron, GA. The Shapiro Lab in Byron GA placed the samples in −80 °C until analysis. On the same day (Jan 9), NanoRacks shipped Specimens 2–6 at RT overnight by FedEx to Pheronym, Davis, CA. On January 10, 2020 at 10 AM, Specimens 2–6 were received by Pheronym Lab, and were inspected immediately for dead, alive sluggish or active nematodes, pictures were taken, and videos recorded before removing the Teflon seals. Pictures were taken using a Nikon D60 (Nikon, Tokyo, Japan). Videos were taken with a hand-held USB Celestron microscope (Celestron, Torrance, CA) and/or iPhone 6 S (Apple, Cupertino, CA).
Immune response assessment of Specimen 1
The primary immune defense in insects against multicellular parasites including EPNs is encapsulation. Immune cells (hemocytes) bind to the nematode and one another to form a multicellular envelope; we therefore assessed immune response based on hemocyte activity and encapsulation46. G. mellonella larvae were dissected longitudinally. The presence of hemocytes adhering to the nematode body was confirmed at 400–600X. The number of nematodes showing hemocyte activity was compared between treatments.
Assessing the IJs after returning to Earth
When Specimen 2, 3, 4, and 5 were received, S. feltiae IJs were observed visually to assess whether they were alive, sluggish, or dead before removing the seals. Microgravity and corresponding Earth controls were inspected at the same time. There were several IJs visible prior to opening the tubes. Subsequently, we unsealed the tubes for Specimen 2, 3, and 4, removed half of the sand to a 10 cm petri dish and harvested the IJs by washing the sand three times with water. The IJs were then rinsed with MILLIQ water to test for dispersal behavior at Pheronym and sent to the Shapiro-Ilan’s lab for assessing symbiotic bacteria load. In case we missed any live IJs in Specimens 2, 3, and 4, we baited the other half of the sand with two G. mellonella larvae and incubated them at 22 ± 1 °C for three days before inspecting for dead or live insects. After that the samples were shipped to the Shapiro-Ilan’s lab for a second inspection and analysis of the bait insects for IJ presence.
Specimen 5 was in polyacrylamide gel which was transparent, making visually scoring easy dead or alive and taking videos (Supplementary Video 1 and 2). After inspection, three ml of IJs with a density of 4000 IJs/ml were shipped in gel at ambient temperature overnight by FedEx to the Shapiro-Ilan lab to conduct infectivity experiments.
Dispersal assays of Specimens 2 and 3
Dispersal assays were conducted as described by Kaplan et al.14,15 with a few differences. Petri dishes (6 cm) containing 0.9% agar with a gel strength: ≥900 g/cm2 (Caisson Agar, Type I, Smithfield, UT) were used for the assays. Briefly, IJs from Specimens 2 and 3 were tested for dispersal immediately by rinsing three times with MILLIQ water after the harvest from sand. The assays were conducted in the afternoon between 2 and 3 PM. Approximately ~26–55 IJs (due to a limited number of IJs) in 10 µL of water were placed in the center of an agar plate with 6 cm diameter Petri dishes and counted. The assays were run for 30 min during which IJs were free to move on agar plates. After 30 min, IJs inside and outside the 1.3 cm ring were counted. IJs remaining inside the 1.3 cm ring were considered non-dispersed, and those outside were considered to have dispersed. Percentage dispersal was calculated as the number dispersed relative to the total number of IJs on the plate. Three replications for Specimens 2 (proxy Earth control) and Specimen 3 (treatment, microgravity), a total of six plates were analyzed.
Infectivity after 33-day space flight, Specimen 5
Infectivity was assessed based on procedures describe by Mbata et al.46. IJs were extracted from gel by dilution and centrifuged at 582g to concentrate them. A total of 960 IJs per replication were pipetted onto filter paper (Whatman No. 1) in a 0.35 mm Petri dish. A single greater wax moth, Galleria mellonella (L.) larva was added to each Petri dish. Insects were incubated in the dark for at 25 °C for 72 h and then dissected under a stereomicroscope to determine the number of invading IJs. There were five insects per replicate of Specimen 5 (total six replicates microgravity and Earth controls) and insect only (no IJs) control. A total of 35 insects were analyzed.
Comparison of symbiotic bacterial load per nematode in Specimens 2 and 3
Methods to compare symbiotic nematode bacteria load among treatments were based on those described by Kaya and Stock1,5. IJs were surface sterilized with 0.5% NaClO and then washed three times with sterile distilled water (centrifuging at 582g for 2 min between each wash). The final pellet was suspended in 0.5 ml. Single IJs were homogenized for 60 s with a sterile motor-driven polypropylene pestle and then transferred onto 60 mm nutrient agar plates. The plates were incubated at 25 °C. The number of bacterial colonies per IJ was assessed at 3 and 7 days.
Statistical analysis
For comparisons of infectivity, dispersal behavior, symbiotic bacteria load, and immune response the treatments effects (from space) were compared to Earth controls using Student’s t tests (SAS version 9.4, SAS 2002). Based on the inspection of residual plots, numerical data were log transformed prior to analysis47 (SAS, 2002).
Ethics statement
We use the invertebrate model system Steinernema feltiae and Galleria mellonella for this study, in accordance, the study was exempt from ethics committee approval.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article. More