Aurelia ephyrae develop eight graviceptors (rhopalia) during their metamorphosis from polyps, which are used for positional orientation with respect to gravity. In three experiments for each speed of 1/15, 1/8, 1/2, 1, and 24 rpm, groups of six polyps were rotated in the horizontal or vertical plane (control) using clinostats. Other controls were kept stationary in the two planes. Ten ephyrae from each group were collected after 5 to 6 days at 27 C in iodine and the number of statoliths per rhopalium were counted. Statistical analyses of statolith numbers revealed that horizontal clinostat rotation at 1/4 and 1/2 rpm caused the formation of significantly fewer statoliths per rhopalium than were found in controls. The finding that these slow rates of rotation reduces statolith numbers suggests that the developing ephyrae were disoriented with respect to gravity at these speeds, causing fewer statocytes to differentiate or to mineralize

Davis, S.

Ross-Clunis, H., III

Spangenberg, Dorothy B.

English

NASA Technical Reports Server

274 
EFFECTS OF CLINOSTAT ROTATION ON 
AURELIA STATOLITH SYNTHESIS 
D. Spangenberg, S.Davis and H. Ross-Cluni s , 111* 
Depa rtment of Pathology and *Department of 
Endoc rinology , Eastern Virginia Medical School, 
Norfolk, VA 23 501. 
Aure l ia ephyrae develop eight grav iceptors 
( rhopalia) during the ir metamorphosis from 
polyps, which a r e used for positional o rienta-
tion with respect to gravity. 
In three experiments for each speed of 1/15, 
1/ 8 , 1/4, 1/2, 1, and 24 rpm, groups of six 
poly ps were rotated i n the horizontal or verti-
cal plane (control) using clinostats . Othe r 
co ntrols were kept stationary in the two planes. 
Ten ephyrae from each group were collected after 
5- 6 days at 27°C in iodine and the number of 
s tatoliths per rhopal ium were counted. Statis-
tical anal ys es of statolith numbers r evealed 
tha t horizontal clinostat rotation at 1/4 and 
1/2 rpm caused the formation of significantly 
fewer statoli t hs per rhopalium than were found 
in contro ls. The finding that these slow rates 
of rotation reduces statolith numbers suggests 
tha t the developing ephy rae were disoriented 
with respec t to gravity at these speeds, caus -
ing fewer statocytes t o differentiate or t o 
mine ral ize . 
INTRODUCTION 
Using the Aurelia Metamorphosis Test System 
(Spangenbe rg, 1984), we have been investigating 
the effects of clinostat rotation in the hori-
zontal plane on the development of ephyrae and 
t he synthesis of their sta t oliths . 
Aurelia polyps are especially suited for 
gravi t y-rela ted research because they a re very 
small (2-4 mm), form ephyrae with g r avity 
senSing structures (rhopalia) in 5-6 days, and 
can be used for clinostat studies . During 
i odine-induced metamorphosis (Spangenberg, 
1967), ephyrae develop in sequential order from 
the oral to the aboral end of the polyps. Eight 
r hopalia with sacs of statoliths a t their distal 
ends form per ephyra . These statoliths are com-
posed of ca lcium sulfate dihydrate (Spangenberg 
and Beck, 1968) and only one statolith forms per 
statocyte. 
METHODS 
Two c l inostats, made according to the design 
of Tremor and Souza (1972), were used for thes e 
studies. Jellyfish polyps were impaled head 
Rcpril1Jtd, wilh permiJ'sion, from Physiologist. Copyrighl e 1985 ~rjCiln Physiological Society . 
Section ! - Reprint~ 
downwards on cactus spines embedded in paraffin 
in the conical bottoms of autoanalyzer capsul~~ 
(Figure I). The capsules were filled with 10 M 
iodine in artificial sea water (ASW) prepared 
according to Spangenberg (1967) . The caps of 
the capsules were filled with paraffin to elimi-
nate bubble formation in the capsules, and the 
cap-capsule junction was covered with pressure 
sensitive tape to prevent leakage of the solu-
tion or evaporation . The capsules were tightly 
held in a 9 inch long glass tube with plastic 
jOiners and the tube was attached to the shaft 
of the clinostat. 
Cor k -_+--c 
"o·m· .. Rh oo""m Days . 
p 
Splfl 8 
Polyp 
VE R TIC AL RO T ATIO N 
HOR I ZON T Al RO T A liON 
Figure 1. Orientation of the polyps and 
developing ephyrae during clinostat rotation . 
Three tests were done for each cl inostat 
speed using groups of 6 polyps (one per capsule) 
and speeds of 1/15 , 1/8, 1/4, 1/2, 1, and 24 rpm 
were used. For each test, polyps were (1) 
rotated in a horizontal plane; or (2) rotated in 
the vertical plane; or (3) kep t stationary in 
the horizontal position and placed near to (1); 
or kept sta tionary in the vertical position and 
placed near t o (2) . After 5-6 days at 27°C , the 
polyps fo rmed ephyrae in all of the groups, and 
the ephyrae we r e r emoved from the spines in the 
capsules and squashed in a wet film . The excess 
ASW was removed to flatten the animals, causing 
the statoliths to spread, so that the number of 
statoliths per rhopalium per ephyra could be 
counted and r ecorded (Figure 2) . Statistical 
analyses were done on the number of statoliths 
formed per rhopalium using an ANOVA and the 
Duncan's New Multiple Range Test . 
Figure 2. Statoliths spread in ephyra squash 
preparation. 
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RESULTS 
Comparison of the numbers of statoliths 
formed by ephyrae which had developed during 
clinostat rotation in the horizontal plane with 
controls were made using an ANOVA which revealed 
significant differences between the groups at 
the p(.OI level and the Duncan's New Multiple 
Range Test. The latter test showed that those 
ephyrae which developed during rotation in the 
horizontal plane or 1/4 or 1/2 rpm had signifi-
can tly fewer statoliths than controls which were 
rotated at these rates in the vertical plane or 
were kept stationary (Table 1). Ephyrae rotated 
in the horizontal plane at the other speeds of 
rotation did not have significantly different 
statolith numbers than controls . 
Table 1. Numbers of statoliths in ephyrae which 
developed during clinostat rotation. (120 ephy-
rae in 3 tests per rotation speed). 
Rotation 
Speed 
(rpm) 
1/15 
1/8 
1/4 
1/2 
24 
Duncan's New Multiple Range Test 
Clinostat Clinostat Control Control 
(Horizont. (Vertical (Hori<:ont. (Vertical 
Plane) Plane) Plane) Plane) 
25.7 25.6 26 . 3 23.8 
22.0 19.9 19.2 17. 0 
*20. 2 25.1 25. 0 27.3 
*22.7 25. 4 25.5 24.5 
22 . 0 16.9 20.9 17 . 4 
32.7 2 7.6 30.5 29. 6 
*Mean numbers of statoliths per rhopalium of 
ephyrae rotated in the horizontal plane which 
are significantly different from controls . p=. OI 
DISCUSSION 
A variety of developmental and maturational 
effects have been found in plants and anima ls 
following clinostat rotation in the horizontal 
plane (Miquel, 1984; Brown, 1984; and Walgemuth 
and Grills, 1984). Clinostat effects have been 
found using both slow and fast clinostat rota-
tion. The reduced statolith fo rmation in the 
jellyfish ephyrae rotated at 1/4 and 1/2 rpm 
is a response to slow clinostat rotation. Other 
biological systems responding to slow clinostat 
rotation in the horizontal plane include: the 
formation of more developmental anomalies in R. 
pipiens and~. laevis following rotation of --
their egg at 1/4 rpm (Tremor and Souza , 1972); 
disturbed tobacco protoplast regeneration by 
rotation at 4 rpm (Iversen and Baggerud, 1984); 
and the delay of flower formation, seed produc-
tion, and seed maturation which was found by 
Hoshizaka (1984) following rotation of Arabi -
dopsis following clinostat rotation at 1/4 rpm . 
A feature common to clinostat studies is the 
finding (as in the jellyfish statolith research) 
that rotation of organisms at some clinostat 
speeds elicit biological responses whereas no 
response occurs following rotation at other 
speeds. Lyon (1979) while referring to his ex-
periments with Coleus and tomato plants reported 
that the difference in rotational time require-
ments for organs With different patterns of 
growth and hormonal controls illustrates the 
impossibility of setting a fixed rule for opti-
mal rate of rotation of a clinostat. Iverson 
and Baggerud (1984) were unable to decide from 
their experiments which type of clinostat (fast 
or slow) provides the best simulated micro-
gravity environment. These authors concluded 
that the response of their organisms indicated 
them to be a sensitive tool for studies of 
gravity effects which should be further tested 
in a real microgravity environment. Gruener 
(1984) also concluded that "it is impossible at 
present to assess the fidelity with which clino-
stat rotation simulates zero hypogravity encoun-
tered in space". Brown (1984) compared bio-
logical responses of plants rotated on the 
clinostat with those exposed to the microgravity 
environment of space. He found that circumnuta-
tion in plants occurred more vigorously in space 
than on the earth-based clinostat. 
The mechanisms through which clinostat-rota-
tion in the horizontal plane reduces statolith 
numbers in developing ePhYrae is not known. In 
other organisms, such rotation has been reported 
to: cause mixing of intracellular constituents 
in frog eggs (Tremor and Souza, 1972); reduce 
movement of an auxin in Coleus and tomato plants 
(1970); significantly alter the functionai 
interactions between the elements of a proto-
typic synapse in cultured cells of X. laevis 
(Gruener, 1984); cause a decrease i~ the content 
of starch grains in chloroplasts in protoplasts 
of tobacco using the slow but not the fast 
clinostat and to produce minor differences 
between protein patterns of rotated protoplasts 
and controls (Iversen and Baggerud, 1984). 
Statolith synthesis in rhopalia of Aurelia 
ephyrae appears to be sensitive to disorienta-
tion of the organisms with respect to gravity . 
It is, therefore, possible that statolith syn-
thesis will also be affected by exposure of 
developing ephyrae to the microgravity of space. 
Indeed, changes occurring in rhopalia following 
development in space could provide information 
which can lead to the identification of mechan-
isms through which microgravity affects stato-
lit~ synthes i s and t hrough which gr avi t y in f lu-
ence s normal develo pment of s t a toliths and 
rho palia on ea rth. 
AC KNOW LE Da-I ENTS 
The authors are grateful to Dr. C. Ward for 
the loan of a clinostat and to Dr. T. Hoshizaka 
for supplying several clinostat motors . This 
research was funded by NASA, grant #NAG 2- 343 . 
REFERENCES 
1. Brown, A.H. 1985. NASA Conf.Publ. 2336:37-39. 
2. Gruener, R. 1985. NASA Conf.Publ. 2336 : 35-36. 
3. Hoshizaka,T. 1985. NASA Conf.Publ.2336:35-36. 
4. Iversen, T. and C.Baggerud. 1984. The PhYSi-
ologist. 27:S127-S130. 
5. Lyon, C. 1970. Plant Physiol. 46:355-358. 
6. Spangenberg, D. 1984. Mar. Environ. Res. 
14:281-303. 
7. Spangenberg, D. and C. Beck. 1968. Trans . Am. 
Neurosc. Soc. 87:329-335. 
8. Spangenberg,D. 1967. J.Exp.Zool. 165:441-450. 
9. Tremor, J. and K. Souza. 1972. Space Life 
Sci . 3: 1 79-1 91. 
10.Walgemuth, D. and G. Grills. 1984. The Phys-
iologist 27:S99-S100. 
275 


Effects of Clinostat Rotation on Aurelia Statolith Synthesis

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