Dynamics of entomopathogenic nematode foraging and infectivity in microgravity
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
