Pergamon

Adv. Space Res. Vol. 14, No. 8, pp. (8)239--(8)247, 1994 Copyright © 1994 COSPAR

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DEVELOPMENT STUDIES OF A URELIA (JELLYFISH) EPHYRAE WHICH DEVELOPED DURING THE SLS-1 MISSION

D. B. Spangenberg,* T. Jernigan,** R. McCombs,* B. T. Lowe,* M. Sampson* andJ. Slusser*

* Eastern Virginia Medical Schoo~ Norfolk, VA 23501, U.S.A. ** NASA Astronaut Office, Johnson Space Center, Houston, TX 77058, U.S.A.

ABSTRACT

Aurelia polyps (scyphistomae) and ephyrae were exposed to microgravity for nine days aboard the space shuttle during the SLS-1 mission. During strobilation, polyps segment transversely and each segment develops into an ephyra. Polyps were induced to strobilate at 28°C, using iodine or thyroxine, at L(Launeh)-48h, L-24h, and L+Sh. Ephyrae developed in the groups tested in space and on Earth. The number of ephyrae formed per polyp was slightly higher in the L+8h groups as compared with those induced at L-24h and L-48h. On Earth, iodine is used by jellyfish to synthesize jellyfish-thyroxine (Jr T4) , needed for ephyra production. Since iodine-treated polyps strobilated and formed ephyrae in space, it appears that jellyfish can synthesize Jf-T 4 in space. Indeed, two groups of polyps not given inducer formed ephryae in space, presumably due to enhanced Jf-T 4 synthesis, utilization or accumulation. Some ephyrae that formed in space were also fixed in space on Mission Day (MD) 8; others were fixed post-flight. Examination of living ephyrae with the light microscope and fixed ones with the Scanning and Transmission Electron Microscopes revealed that those which developed in space were morphologically very similar to those which developed on Earth. Quantitation of arm numbers determined that there were no significant differences between space and Earth-developed ephyrae. Pulsing abnormalities, however, were found in greater numbers (18.3%) in space-developed ephyrae than in Earth-developed controls (2.9%). These abnormalities suggest abnormal development of the graviceptors, the neuromuscular system, or a defect in the integration between these systems in apparently mierogravity-sensitive animals.

INTRODUCTION

Metamorphosis of biological organisms from one form to another usually proceeds with precision on Earth, but little is known about the effects of microgravity on developing organisms. Indeed, very few biological organisms have completed their development while in space, due, most likely, to limited access to space and to short durations of many space flights.

Jellyfish were chosen for a study of their development in space partly because metamorphosis (strobilation) of warm water forms of Aurelia aurita occurs rapidly in the laboratory (within 5 days @ 28°C). Polyps (scyphistomae) divide transversely and each segment metamorphoses into an ephyra in sequential order (Fig. la & lb). The polyps and ephyrae are only two stages which occur within a complex life cycle (Fig 2). Polyps are easily grown in the laboratory where they bud asexually to form new polyps until they are induced to metamorphose with iodine/1/or thyroxine/2/. Within 24h following iodine treatment, the polyps synthesize jellyfish thyroxine (Jr-T4) which probably plays a role in the differentiation of new structures of the ephyrae /2/. Structures which form in the new ephyrae which are of particular interest are graviceptors (rhopalia) with statoliths and ocelli; the giant fiber nerve net; radial and coronal striated muscles; gastric filaments; arms with lappets; and nematocyst clusters. Also, ephyrae are able to pulse and swim, and they orient with respect to the gravity vector through a coordination between their graviceptors, nerve nets, and neuromuscular system.

(8)239

(8)240 D.B. Spangenberg etal.

Fig. 1. SEM of ephyra development

A. Developing ephyrae on strobila B. Newly-released ephyra C. SEM of arm of space-developed ephyra with graviceptor (G) and radial muscle (M) and D. SEM of arm of Earth-developed ephyra

Development of Aurelia During $1~-I

. r'l - --- -~" ~ ~ 1~ ,_

t

(s)241

Fig. 2. Life Cycle of Laboratory-Grown Aurelia aurita (not drawn to size).

A. Polyp which self-replicates by budding B. Strobilation stages: B and B1, early stages; B2, late stage showing ephyra formation. C. Post strobilation stages: C, end piece, C1-C3, free-swimming ephyrae D. Medusa E. Planula larvae

In the laboratory, polyps bud asexually indefinitely until they are induced to strobilate using iodine or thyroxine. Free-swimming ephyrae are released sequentially and end-pieces grow back to polyps when fed. Likewise, ephyrae grow into medusae when fed. Sexual reproduction in medusae and subsequent development of planula larvae into polyps is difficult to achieve in the laboratory-grown animals.

,,,~5 R 14:8-Q

(8)242 D.B. Spangenberg et al.

To determine whether ephyrae could develop from polyps in space, and whether such ephyrae could pulse normally, twenty-two groups of tiny jellyfish were sent into space as part of the payload of the NASA SLS-1 mission (Table 1). Some polyps were induced to strobilate (metamorphose) in space and others were induced~ 24h and 48h before launch. Thyroxine was used as inducer in some polyps whereas others were induced with iodine which polyps use to make Jf-T 4 (jellyfish thyroxine). Two control groups were in artificial sea water (ASW) with no inducer.

This paper reports the results of a detailed study of ephyrae returned to Earth after the SLS- 1 mission and their Earth-developed controls. Comparisons are made between numbers of ephyrae induced prior to or after launch and the numbers of arms formed in ephyrae which developed in iodine or thyroxine. Ultrastructural comparisons of the morphology of space-developed and Earth-developed ephyrae are also presented.

METHODS

Pre-fli~.ht activities.

Approximately 25,000 jellyfish polyps were combined in a large container of artificial sea water (ASW) and stirred to mix the animals to provide random distribution for the flight experiment. Twenty thousand polypsi were divided into groups of 200, rinsed six times in ASW and placed in pre-rinsed Kapok (polyester with polyethylene lining) bags for transport to Kennedy Space Center (KSC). At KSC, the animals were divided into groups of 100 and 66 and maintained in pyrex culture dishes at 19"C. Two days prior to loading t h e jellyfish for shuttle transport, the animals were rinsed five times in ASW. They were rinsed a sixth t ime immediately before packing them. During rinsing, the animals were screened and abnormal looking polyps i were removed. The five thousand polyps left at Eastern Virginia Medical School (EVMS) were treated in the same manner prior to packing the ground control of the experiment at the medical school.

Thyroxine in ASW was made just prior to packing the L-48h and L-24h samples, but thyroxine as well a s iodine solution, buffer and gluteraldehyde to be used in the Chemical Delivery Systems (CDS) was made three~ days prior to flight. The CDSs were developed by the principal investigator and the NASA Ames Research Center especially for the introduction of chemicals to the jellyfish in ASW while in space /3L The gluteraldehyde, cacodylate buffer, thyroxine and iodine solutions were placed in small baggies inside the syringe barrel of the CDSs. Iodine and thyroxine solutions were made up in ASW to achieve a final i concentration of 1 X 10"SM for induction of metamorphosis. The gluteraldehyde final concentration was 3 % and the final concentration of the buffer was 0.1M. The chemicals were maintained at 4°C until the experiment was packed at L-24h and then maintained at 22°C±2"C during the transport of the experiment to the launch pad. The organisms were exposed to these solutions for the duration of the experiment.

The organisms were rinsed six more times before being packed into Kapak bags prior to flight. Fourteen bagsl (Table 1) with one hundred polyps each (L-24h and L-48h) were placed in 150 ml of solutions with 1/3 volume of air and packed in a flight approved metal container called a zero box. This was Kit I exclusive of four groups which are presented elsewhere/4/. Six lithium fluoride (LiF) radiation dosimeters were added to s ~ of the bags prior to heat-sealing and these bags were positioned at each corner and in the middle of a zero box. Sixty-six animals in each of eight bags were placed in 100 ml of solutions in CDSs, with 1/3 volume of air and packed into another zero box (Kit II). A continuous temperature recorder (ATR) was packed wi~ the experiment. Controls which were similarly packed and housed at EVMS, closely followed the scheduled activities of the flight portion of the experiment.

In-fli~ht activities.

The jellyfish were kept in the mid-deck locker (22"C_2°C) until they were transferred to the 28°C incubator at L+8h. Controls on Earth were kept at EVMS at 22°C--.20C to simulate the mid-deck locker temperature

Development of Aurelia During SLS-I

TABLE 1 Space Flown Jellyfish in Kits 1 and 2

(8)243

100 Polyps in 149 ml ASW induced on Earth to metamorphose with:

@ L - 24h

T_~@ L - 24h

I a @ L - 48h

T 4 @ L - 48h

I 2 @ L -24h

T 4 @ L - 24h

@ L - 48h

T 4 @ L -48h

"12 @ L - 24h

*T @ L - 24h

*I @ L - 48h

*T 4 @ L - 48h

66 Polyps in 100 ml ASW induced in space to metamorphose with:

I ~ @ L + 8 h I I ! @ L + 8 h ] * * I z @ L + 8 h

T @ L + 8 h T @ L + 8 h * * T @ L + 8 h

66 Polyps in 100 ml ASW induced on Earth to metamorphose with:

*Fixed on ground after flight **Fixed in space

and were transferred to 28°C--. 2"C very shortly after the flight animals were transferred. At this time the L+8h groups were induced with iodine or thyroxine by pushing in the plunger of the syringes of the CDSs and releasing the inducers into the bags with the jellyfish. On MD (Mission Day) 8, four of the L+8h groups were fixed with glutaraldehyde and cacodylate buffer which were injected at the same time into the bags containing the jellyfish in ASW. The fixed specimans were maintained at ambient temperature in the Space Lab because refrigeration was not available.

Post-flight activities.

Testing. Within three hours post-flight, the jellyfish experiment was unpacked at Dryden Field (California, USA) and the ephyra counting, coding, and testing begun using the Aurelia Metamorphosis Test System/5/. Ephyrae were placed in a test tube with ASW and observed for pulsing/swimming ability. Ephyrae with pulsing abnormalities were videotaped. The ephyrae were then placed in wet films under a dissecting microscope in order to count their structures, including the number of arms (rhopalia'and statolith data is presented in another paper/4/). Photomicrographs were made of both normal and abnormal ephyrae. Data analysis was performed using an analysis of variance and Tukey's HSD Test/6/.

Fixation. Fixation of some of the the L+8h groups (Table 1) was continued on Earth, with an additional exposure to fresh gluteraldehyde fixative for two hours. L-24h and L-48h groups of ephyrae were fixed for four hours and all groups were rinsed four times for 10 minutes and stored in sodium cacodylate buffer at 40C. Within three days, the samples were transported to EVMS and were post-fixed in 1% Osmium for one hour for TEM studies. Some of these ephyrae were embedded immediately in Polybed 812 and others were stored @ 4"t2 in buffer for future studies. For Scanning Electron Microscope (SEM) studies, ephyrae were critical point dried and coated with gold/palladium prior to examination under a Phillips 515 SEM. For further details of these procedures refer to Spangenberg /7/ and Spangenberg and Kuenning/8/. The TEM studies were done using a Phillips 301 Transmission Electron Microscope.

RESULTS AND DISCUSSION

Numbers of Ephyrae Formed in Space and in Controls.

The number of ephyrae formed in space was compared with the number formed on Earth in all groups of animals.

(8)244 D.B. Spangenberg et al.

TABLE 2 Comparison of the Number of Ephyrae Formed per Polyp in Groups Induced by Iodine or Thyroxine in Space and on Earth

Time Induced

L + 8h

Iodine Induced

3.26

L - 48h

Thyroxine Induced

2.97

3.30

L + 8h 3.69 3.82 Earth

L - 24h 2.93 3.09 Space

L - 24h 2.64 3.44 Earth

L - 48h 2.72 2.64 Space

3.52

Location

Space

Earth

Comparison of total number of ephyrae produced in space and on Earth in the L+8h, L-24h, and L-48h groups revealed an average of 2.74 ephyrae/polyp formed in space and 2.95 ephyrae/polyp formed on Earth. Ephyrae which formed in space from Kits I and II totaled 5289 as compared to the 5692 formed by the Ear th based controls. One of the most notable results was that in the non-induced ASW groups in space, 1.09 ephyrae/polyp formed and 0.00 ephyrae/polyp formed on Earth.

Jellyfish were sent into space at different time periods following their induction with iodine or thyroxine so that the animals would be in different stages of development when they were introduced to the microgravity environment. Earlier studies/2/revealed that the Jf-T 4 hormone is synthesized within twenty four hours after administration of iodine. The iodine-induced L-24h and L-48h polyps had, therefore, apparently begun synthesis of hormone prior to launch. Those polyps induced in space to form ephyrae, however, were required to synthesize the hormone in space. Polyps induced with thyroxine pre-launch would have ingested thyroxine and begun metamorphosis events prior to launch whereas those induced in space had to take up the thyroxine while in space. Of the space-developed groups of ephyrae, the L+8h animals induced in space gave rise t o more ephyrae/polyp than the L-24h or L-48h animals which were induced on Earth and developed in space. Since the L+8h animals were exposed to inducer for 24h to 48h less than the other groups, the inference is that the space-induced epyrae may have developed at a faster rate to produce more ephyrae, or that t he Earth-induced groups made fewer ephyrae because of some pre-flight activities, including launch, that may have affected their developmental rate. In all but one group (L-24h), however, the Earth-induced, Earth- developed polyps made more ephyrae than the space-exposed organisms.

The development of ephyrae in two groups sent into space without inducer was of considerable significance. Earth control animals maintained in the same batch of ASW and exposed to a comparable temperature range, did not form any ephryae. In addition, two more controls in the same batch of ASW tested postflight did not form ephyrae. It is known that the J f -T 4 hormone is secreted into the medium/1/, and also that the jellyfish obtain a small amount of iodine from their nutrition. The jellyfish were fed on the same schedule as EVMS controls prior to flight. The formation of ephyrae in replicates in ASW in space suggests that hormone production, utilization, or accumulation in the medium (with possible subsequent feed-back on the organisms) was different in space animals compared to their ground-based controls. If the induction response was caused by a localized build-up of hormone in the ASW in space, then the implication is that secretory products from~ cells or aquatic animals may accumulate differently in space and create unexpected biological responses.

Number of Arms/Ephyrae.

The number of arms formed in ephyrae can vary between four and sixteen, although most animals have eight.

Development of Aurelia During SLS-I (8)245

Arm numbers, therefore, are considered an indicator of normal development. The mean number of arms formed in ephyrae which developed in space and their ground-based controls is given in Table 3.

TABLE 3 Comparison of the Number of Arms Formed in Space-flown Ephyrae and Their Earth Controls Induced by Iodine or Thyroxine

Time Induced

L + 8 h

Arms/Ephyra ] No. Ephyra

73 8.19

L + 8h 8.64 56 Earth

L - 24h/ 8.47 79 Space L - 48h

8.72 80 Earth L - 24h/ L - 48h

Location

Space

Ephyrae induced in space by iodine had significantly fewer arm numbers/ephyra than their controls p=0.02 using the Tukey's HSD test/6/. Ephyrae induced with thyroxine in-flight or iodine or thyroxine pre-flight formed higher numbers of arms which were not significantly different from each other. Since eight is the number considered most normal, the iodine-induced in-flight animals apparently had more normal ephyrae with respect to arm number than the other groups, including the ground-based controls.

Radiation Dosimetry

Radiation dosimeters revealed very low X-irradiation exposure of the jellyfish in space. Readings from the flight group ranged from 91 to 407 mrad. The control was slightly lower ranging from 82 to 381 mrad. Aurelia polyps can produce normal ephyrae after exposure to 5000 rad /9/ given at the time of T4-induced metamorphosis.

Morpholo~ of Space-developed and Earth-developed Ephyrae.

Lim'at Microscopy and SEM. Examination of several hundred ephyrae from space and their ground-based controls, with the dissecting microscope, revealed no morphological differences in the space-developed ephyrae as compared with the controls. Distribution of arms, gastric filaments, and nematocyst dusters was normal as compared with controls. (Rhopalia and statolith numbers are presented elsewhere/4/.) In addition, twenty ephyrae were examined and photographed with the SEM. Ciliation on the surfaces of the ephyrae was normal in both the space-developed and ground-based controls.

TEM. Examination of the various structures of the ephyrae at the cellular level is still in progress. To date, examination of graviceptors (rhopalia) and radial muscle of seven randomly selected ephyrae (4 space-developed and 3 controls) at the TEM level, reveals a close similarity between the groups. The space developed animals had well-developed rhopalia with touch-plates, as did the controls. Rhopalia of space-developed animals were of the same shape and size range as controls and ciliation was normal on their surface. Likewise the radial muscles developed well in the space-developed ephyrae as they did in Earth-controls (Fig. lc & ld).

Pulsinu Abnormalities.

Although space-developed ephyrae examined immediately post-flight were morphologically very similar to ground-based controls, close examination of their pulsing behavior revealed certain abnormalities in 18.1%

(8)246 D.B. Spangenberg et al.

of them as compared with only 2.9% abnormalities in the ground-based controls. found are listed in Table 4.

The types of abnormalities

TABLE 4 Pulsing Abnormalities in Ephyrae Which Developed in Space or on Earth

Space

Earth

Incompl. pulse

12

0

Spasms

11

Arms out of synch.

4

After- twitch

6

Jerky pulse

Uncoord.

13

4

"wave"

0

Only seven out of the 236 ephyrae which developed on Earth and that were examined had a total of nine pulsing abnormalities. Of the 218 ephyrae examined among the space-developed ephyrae, forty animals showed fifty-three abnormalities. Ephyrae with incomplete pulses, spasms, aftertwitches, uncoordinated pulsing, and '~aves" were found only in the space-developed groups. One control was found with a jerky pulsing which was not seen in the space-developed ephyrae. In an earlier study/5/, pulsing abnormalities were found in ephyrae that had developed in petroleum-related chemicals. Aniline, biphenyl and cresol caused a reduced strength and frequency of pulsing, while benzene, petroleum oil, and pyrene caused uncoordinated pulsing.

Development of Other Animals in Space

While not all organisms flown on space missions in the past developed well in space, some did. Fundu/us (fish) were flown on the joint Apollo-Soyuz mission (9 days) and on the USSR unmanned Cosmos 782 for 19.5 days. They reported that development proceeded normally, if not more rapidly than normal. Guppies flown for 5 days on board Cosmos 1514 were returned to Earth on approximately day 12 of pregnancy. The pregnant fish tolerated space flight conditions well. The female allowed to bear young gave birth to 25 normal young and two anomalous and underdeveloped embryos/10/. Six out of 35 Japanese quail chicks hatched unaided following their incubation on Mir /11/. These six chicks showed every evidence of normal development, reacted to visual and auditory stimuli, displayed motor activity, vocalized, and ate. According to the authors/ll/, this study confirmed the possibility of normal embryonic development in space. Miquel and Souza/12/recently reviewed the effects of space exposure on developing animals. They report that Drosophila flown on the shuttle D-1 mission did not show any drastic influence on the developmental process. However, there was a decreased proportion of hatching of embryos in space, a decreased oogenesis rate in females and a shortening of male life. The effects of microgravity on jellyfish likewise did not show any drastic effects on the developmental processes. However, the abnormal pulsing in some of the organisms suggest subtle effects which may reflect abnormality in the development of the graviceptors, the nerve nets, or the neuro-muscular system, which will require further studies at the cellular level.

ACKNOWLEDGMENTS

This research was funded by NASA grants NAG2-343 and NAGW-1784 to the senior author. The authors are grateful to the following people for their assistance with the Jellyfish-in-Space E, xperiment:EVMS Jellyfish Research Team: Dr Jim Shaeffer, Dr Ron Spangenberg, Mark Hughes, Guy Shelton, Debbie Leete, Alison Barnes, Mike McCombs, Anna Shore and Mike Prokopchak; NASA ARC: Dr Gary Jahns, Dr. C. Winget, Shelli Jones, Bonnie Dalton, Gini McCollough, Daniel Cheng, and Tom Nelson; NASA HQ: Dr Thora Halstead and Dr Ron White.

Devdopment of Aurelia During SLS-I (8)247

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10. J.R. Keefe, Vertebrate development in space. In: NASA DevelopmentalBiology Workshop, eds. K.A. Souza and T.W. Halstead, NASA Technical Memorandum 86756, Alexandria, VA, (1984)

11. G.I. Meleshko, Y. Shepeloev, T.S. Guryeva, K. Boda and W. Sabo Embryonic development of birds in weightlessness. Kosmicheskaya Biologia i Aviakosmicheskaya Meditsina 25(1):37-39 (1991) Reported in USSR Space Life Sciences Digest 32:24 Washington DC).

12. J. Miquel and K. A. Souza, Gravity Effects on Reproduction, Development, and Aging. In: Advances in Space Biology and Medicine, Vol. 1, ed. S. L. Bonting, JAI Press, Greenwich, Conn. (1991).