International Microgravity Laboratory 2/STS-65

PAYLOAD PROFILE: IML-2/STS-65

Mission Duration: 15 days

Date: July 8–23, 1994

Life Science Research Objectives
To study the early development of gravity-sensing organs in newts
To study the development and gravity-related behavior of jellyfish in space
To study the critical stages of plant embryogenesis in microgravity

Life Science Investigations
Developmental Biology (IML2-1)
Neurophysiology (IML2-2)
Plant Biology (IML2-3)

Organisms Studied
Cynopus pyrrhogaster (newt) adults and larvae
Aurelia aurita (jellyfish) polyps and ephyrae
Hemerocallis cv. Autumn Blaze (daylily) cells

Flight Hardware
Ambient Temperature Recorder (ATR-4)
Aquatic Animal Experiment Unit (AAEU)
Life Science Cell Culture Kit (CCK) (supplied by NASDA)
Plant Fixation Chamber (PFC) (supplied by NASDA)
Refrigerator/Incubator Module (R/IM)


Mission Overview

The second in the series of International Microgravity Laboratory payloads (IML-2) was launched on the Space Shuttle Columbia's STS-65 mission on July 8, 1994. After remaining in orbit around the Earth for 15 days, the Shuttle landed on July 23. The seven-member crew included a Japanese astronaut, who was the first Japanese woman in space.

Besides NASA, the European Space Agency (ESA) and the space agencies of Japan (NASDA), Canada (CSA), Germany (DLR), and France (CNES) sponsored experiments on the mission. Investigators from a total of 13 countries participated in research into the behavior of materials and life in microgravity.

The IML-2 payload consisted of more than 80 experiments in microgravity and life sciences, including five life science experiments developed by American researchers. Of these, Ames Research Center sponsored two experiments using newts and jellyfish. Kennedy Space Center (KSC) sponsored the PEMBSIS experiment, designed to study plant embryogenesis in microgravity.

Life Sciences Research Objectives

The objective of the newt experiment was to study the early development of gravity-sensing organs (Fig. 6). The utricle and saccule are specialized organs present in the inner ears of all vertebrate animals. They contain otoliths (or otoconia), calcium carbonate stones, which are deposited on a gelatinous membrane that lies over the sensory hair cells. The pull that gravity exerts on the otoliths is sensed by the hair cells, and information about the gravitational stimulus is transmitted to the brain via connecting nerve fibers. The experiment was designed to determine whether otolith production and development of otolith-associated receptor cells and nerve fibers may be altered in the microgravity environment of space.

The jellyfish experiment was designed to study behavior and development in space. Behavioral parameters studied included swimming, pulsing, and orientation. Study of developmental processes focused on gravity-sensing organs. The experiment also sought to determine the level of artificial gravity stimulus needed to counteract any negative effects of space flight.

The objective of the plant embryogenesis (PEMBSIS) experiment was to evaluate whether space flight affected the pattern and developmental progression of embryonic daylilies from one well-defined stage to another. It also examined whether cell division (mitosis) and chromosome behavior were modified by the space environment.

Life Sciences Payload

Organisms
Adults and larvae of the Japanese red-bellied newt species (Cynopus pyrrhogaster) were used in the newt experiment. This species was selected for study partly because the vestibular system of very young newts undergoes most of its development in a period of time equivalent to the planned mission duration. Furthermore, adult females can be induced to lay eggs by injecting them with a hormone. Their eggs develop in orbit and mature in the microgravity environment to provide scientists with a sample of embryos that have undergone early development in microgravity.

Moon jellyfish (Aurelia aurita) served as experiment subjects for the jellyfish experiment. Both the sedentary polyp stage and the free-swimming ephyra stage of the jellyfish were studied.

The PEMBSIS experiment studied embryogenically competent daylily (Hemerocallis cv. Autumn Blaze) cells.

Newt

Japanese Red-Bellied Newt (Cynopus pyrrhogaster)

Hardware
Newt adults and larvae were housed in cassette-type water tanks in the Aquarium Package within the Aquatic Animal Experiment Unit (AAEU), developed by NASDA, the Japanese space agency. The AAEU is a life support unit that can keep fish or other aquatic animals alive for at least 19 days in the Spacelab. It consists of a Main Unit, an Aquarium Package, and a Fish Package, each of which has an independent life support system. In IML-2, each cassette held an egg container with individual egg holes (6-mm diameter, approximately 12 mm deep).

A slow rotating centrifuge microscope and camera system, Nizemi, developed by DLR (formerly DARA), the German space agency, was used to examine and videotape the behavior of the jellyfish ephyrae and polyps at up to 15 varying levels of G and at a temperature of 28 °C (to facilitate swimming activity). The Nizemi provides observation of samples under variable acceleration levels between 10-3 and 1.5 G and a controllable temperature between 18 and 37 °C.

Jellyfish were housed in the European Space Agency's Biorack facility within Biorack Type I containers. For descriptions of the facility and containers, see IML-1.

A Refrigerator/Incubator Module (R/IM) held fixed jellyfish specimens. The R/IM is a temperature-controlled holding unit flown in the Shuttle middeck that maintains a cooled or heated environment. It is divided into two holding cavities and can contain up to six shelves accommodating experiment hardware. An Ambient Temperature Recorder (ATR-4) was placed inside the R/IM. For a general description of the ATR-4, see IML-1.

The PEMBSIS experiment used hardware provided by the National Space Development Agency (NASDA) of Japan. As part of the NASDA Life Science Cell Culture Kit, this experiment used six petri-dish-like Plant Fixation Chambers (PFCs). The PFCs were used to hold the cultured plant cells for the PEMBSIS experiment. These containers are completely sealed. The PFCs allow plant cells exposed to space flight to be fixed in orbit by insertion of a chemical fixative via syringe through a septum port.

Jellyfish


Jellyfish (Aurelia aurita)

Operations

Preflight
PEMBSIS cell cultures were prepared about a week before launch. Twelve chambers were filled with a semi-solid medium. Six were transported to KSC and kept in an unlit incubator at 222 °C until they were loaded into the Shuttle. The other six were used as ground controls.

Approximately 36 hours before launch, 148 prefertilized newt eggs were loaded into the three cassettes of the AAEU. Four adult newts were also loaded into the cassettes; two cassettes each contained one newt apiece, while a third contained two. Fresh, aerated water at 24 °C circulated continuously through the unit. A similar unit was maintained at KSC as a ground-control.

Twenty-four hours before launch, four groups of six jellyfish polyps each were given iodine in artificial sea water (ASW) to induce strobilization of polyps into the ephyrae form.

Shortly before flight, the jellyfish samples were loaded into a total of 10 Nizemi cuvettes containing ASW and placed in Type I containers. For the behavior study, a group of normal ephyrae and a group of ephyrae without statoliths were placed in the Biorack 22 °C incubator. The third group of ephyrae was placed in the Biorack 1-G centrifuge. Two groups of polyps were used for the development study. One group was placed in the incubator and the other was placed in the 1-G centrifuge. A similar set of equipment was maintained at the KSC ground-control facility.

Figure 6

Figure 6. Together, the semicircular canals and the otolith organs make up the vestibular apparatus of the inner ear, which provides information to the brain about balance and motion in 3-D space. The gravity-dependent otolith organs, lined with hair cell receptors and otoconia, detect linear acceleration of the head. When the head moves, the otoconia lag behind, bending the hair cell receptors and changing the directional signal to the brain.

Inflight
On flight days 6, 8, and 11, the crew carried out video observations of newt eggs to document the rate of development. The crew also made observations of the adult newts at specified times. On both the fifth and ninth days of flight, an adult newt was found dead, causing the loss of some eggs because of contamination. The remaining two adult newts survived the flight and were recovered live upon landing.

One cuvette from each group of jellyfish ephyrae and polyps were videotaped on the rotating microscope/centrifuge at intervals throughout the mission to determine the G-threshold for the swimming behavior of the ephyrae. On flight day five, both the flight and ground-control groups of ephyrae with statoliths that had been hatched on Earth were fixed. On flight day 13, two of the four groups of polyps that had been strobilation-induced were fixed. The remaining ephyrae and polyps were returned to Earth for postflight analysis.

To provide a comparison between flight-fixed and ground-fixed groups in the PEMBSIS experiment, the crew fixed some cultures shortly before landing. The fixative was a three-percent glutaraldehyde (balance water) solution. Each chamber was fixed with a 20-ml injection of fixative.

Postflight
The flight cassettes containing the newts were retrieved approximately six hours after landing. Some of the larvae were fixed and preserved for later analysis, while some were tested to estimate how space flight affected the gain of the otolith-ocular reflex and measure the otolith volumes and areas of associated sensory epithelia.

Living jellyfish were counted, coded, and photographed beginning at five hours postflight. The pulse rate, numbers of arms, rhopalia, and statoliths were counted in each of the ephyrae. Those with abnormal pulsing were videotaped after landing and again approximately 24 hours later. Some of both the flight and control jellyfish were allowed to form clones, which were then examined for arm number and other structural differences.

After the PEMBSIS cell culture chambers were recovered from the Shuttle, specimens of living cells and somatic embryos were photographed, counted, and chemically fixed within nine hours of landing, before their first division cycle on Earth was complete. Chromosomes were measured and compared within and among cultures.

Results

Newt Study
According to morphogical analysis, both flight and ground controls developed at the same rates. Analysis of three-dimensional reconstructions showed that flight-reared larvae had a larger mean endolymphatic sac (ES) and duct volume and a larger average volume of otoconia within the sac when compared to similarly staged ground controls. Furthermore, the appearance of otoconia in the ES was greatly accelerated in the larvae reared in microgravity.

Jellyfish Study
Ephyrae that developed in microgravity had significantly more abnormal arm numbers as compared with 1-G flight and ground controls. As compared to controls, significantly fewer ephyrae that developed in space swam when tested postflight. Polyps budding in space produced more buds and were developmentally ahead of ground controls. Although development through budding and through metamorphosis proceeded well in space, some jellyfish are apparently more sensitive to microgravity than others, as evidenced by their abnormal arm development.

Daylily Cell Study
Cytological changes and chromosomal aberrations were seen in both flight-fixed and ground-fixed flight cells. A substantial number of binucleate cells, cells possessing two nuclei, were also found in the flight samples. The ground-control samples were all uninucleate.

Additional Reading

NASA. STS-65 Press Kit, July 1994. Contained in NASA Space Shuttle Launches Web site: http://www.ksc.nasa.gov/shuttle/missions/missions.html.

Snyder, R.S., comp. Second International Microgravity Laboratory (IML-2) Final Report. NASA RP-1405, July 1997.

Wiederhold, M.L., W.Y. Gao, J.L. Harrison, and R. Hejl. Development of Gravity-Sensing Organs in Altered Gravity. Gravitational and Space Biology Bulletin, vol. 10(2), June 1997, pp. 91–96.

Wiederhold, M.L., H.A. Pedrozo, J.L. Harrison, R. Hejl, and W. Gao. Development of Gravity-Sensing Organs in Altered Gravity Conditions: Opposite Conclusions from an Amphibian and a Molluscan Preparation. Journal of Gravitational Physiology, vol. 4(2), July 1997, pp. P51–P54.