GENERAL AND COMPARATIVE ENDOCRINOLOGY 32, 108-I 13 (1977)

The Effect of Iodide and Various lodocompounds on Initiation of Strobilation in Aurelial

MARK SILVERSTONE,* THOMAS R. TOSTESON,? AND CHARLES E. CUTRESS?

tThe Department of Marine Sciences, University of Puerto Rico, Mayaguez, Puerto Rico 00708; and

*The Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755

The effect of iodide and various iodocompounds on initiation of strobilation in Aurelia polyps was studied. Iodide was found to be active in initiating strobilation at concen- trations between 1O-4 and lo-’ M. The order of effectiveness of iodide, bromide and iodocompounds, tested at 10m6 M concentrations, was (highest first): DIT, MIT, I-, Ta, T,, Br-. The last had no effect. Results of experiments using different polyp concentrations indicated that strobilating polyps release a factor into the media that induces strobilation in other polyps. All agents shown to be active in initiating strobilation were tested in the presence of thiourea. At a concentration of 10e4 M, thiourea completely inhibited the effect of all of the agents except DIT, which was only partially inhibited. New buds were found to be capable of strobilating only when tested in the presence of older polyps. None of the agents tested induced strobilation in new buds tested in the absence of older polyps. The data indicate that none of the iodocompounds tested is itself responsible for initiating strobilation and that each must be altered to be effective. Because its effect persisted in the presence of thiourea, DIT may be similar in the putative actual compound.

Strobilation is the stage in the develop- ment of Scyphozoan jellyfish in which the sessile polyp metamorphoses to the free swimming medusa. The entire morphology and behavior of the organism changes in this adaptation to a new ecological niche. Prior to strobilation the polyp adheres to a hard substrate, asexually producing buds that detach from the parent polyp, adhere to the substrate and produce more buds in turn. This process continues until the en- vironmental conditions necessary for

’ Supported in part by a grant from the United States Commercial Fisheries and Development Act of 1964, the University of Puerto Rico, and the Fisheries Development Program, Department of Agriculture, Commonwealth of Puerto Rico, and Grant No. AM 18416 from National Institute of Arthritis, Metabo- lism and Digestive Diseases, National Institutes of Health, Bethesda, Md.

strobilation occur. After strobilation the remaining parts of the polyp grow, bud and strobilate again under appropriate condi- tions.

Percival (1923) pointed out the depen- dence of strobilation on temperature and noted that the optimum seawater tempera- ture varies between localities. It is known that polyps raised at ambient seawater temperature in Puerto Rico strobilate at summer temperature peaks of 29-30“ (Cut- ress, 1974). Chiba (1969) found that a high density of polyps in a culture dish hastens initiation of strobilation. These data are consistent with the hypothesis that certain polyps may release a chemical into the cul- ture medium that stimulates strobilation in other polyps. Similar findings have been reported for Chrysaora quinquecirrha (Loeb, 1968).

108 Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved. ISSN 00166480

IODINE IN STROBILATION 109

Recent work provides substantial evi- dence for a hormonal mechanism in strobi- lation of the Scyphozoan Aurelia aurita (Linni, 1758). Initiation of strobilation has been shown to be dependent on both iodide concentration and temperature, and inde- pendent of pH and salinity (Spangenberg, 1968). Iodide and thyroxine (TJ have been reported to be taken up by strobilating polyps. In addition to this there is evidence that strobilating polyps may synthesize iodinated compounds including T, (Spangenberg, 1972). However, other in- vestigators have reported that Aurelia polyps take up 1311 but organify little and that T4 synthesis was not detectable (Olman and Webb, 1974). Further studies on Cfrrysaoru indicate that iodide is involved in strobilation and that T4 may be synthe- sized in this species (Black and Webb, 1973). The object of this work was to assess the role of iodide in initiation of strobilation in Aurelia.

METHODS AND MATERIALS

The polyps used in these experiments were cul- tured from a sample of 30 polyps from stock cultures maintained in the Department of Marine Sciences, University of Puerto Rico. The stock cultures origi- nated from a group of polyps collected from the coast- al waters of Puerto Rico in June, 1972. Polyps were maintained in 6 in. finger bowls in a running seawater bath at ambient temperature. Every 2 days the polyps were fed newly hatched Artemia nauplii. The water in the culture dishes was changed after about 30 min of feeding. Under these conditions the polyps budded asexually. As the culture dishes became crowded with polyps new cultures were started.

In March, 1973 all but two of the culture dishes were placed in a water bath in which the temperature was maintained at 20 & 2”. The polyps that remained at ambient temperature strobilated in June, 1973 and these cultures were then discarded. Those maintained at 20” did not strobilate at that time nor at any time during the course of this study. At this temperature the polyps were maintained and cultured as before, though the rate of budding was greatly reduced. In the course of 5 months the original population of 30 polyps grew to at least lO,OOO-15,ooO individuals at the 20” temperature.

Artificial seawater (Instant Ocean, Aquarium Sys- tems, Inc.) was employed in the incubations con-

ducted in these studies. The salinity of this medium, prepared with trace elements excluded, was 34 ? 1%0, at a pH of 8.0. Polyps were removed from the culture dishes and washed five times with this iodide- free artificial seawater prior to their use in experi- ments. The purpose of these washings was to remove all traces of iodide that the polyps may have carried over from the stock culture. Covalently bound iodide would not be washed out of the polyps in this pro- cess. Subsequent experimentation showed that these polyps, if they did contain iodide, did not contain enough to induce strobilation.

Effect of Iodide on Strobilation The effect of iodide concentration (KI) on initiation

of strobilation was studied. In each test 20 polyps were suspended in 10 ml of test solution (artificial seawater with iodide added) in a covered 48 x 33 mm stender dish (Wheaton) and placed in a constant tem- perature water bath. The percentage of polyps that were strobilating, as determined by the obvious ap- pearance of the first constriction, was noted at least twice daily during the 14 day test period. The polyps were not fed during this period. The following con- centrations of iodide were tested in this way at 29.5” (number of tests per concentration is given in par- entheses): 1O-4 M (4), low5 M (5), 10mB M (12), 5 X lo-’ M (4), IO-’ M (8), lo-* M (4), 1O-s M (4). In addition 12 tests were performed using iodide-free media and 9 with natural seawater. Tests were run concurrently at 20 and 29.5”.

Effect of Polyp Concentration on the Iodide Response Different numbers of polyps were tested in 10 ml of

media containing iodide at a concentration of 10m6 M. Concentrations of 5, 10, 20, 40, and 80 polyps were incubated as described above. Two tests were run for each polyp concentration.

E,ffects of T4, T3, MIT, DIT, on Strobilation Experiments were conducted to test the response

of the polyps to T1 and some of its analogues. Each of the following compounds was tested at a concentra- tion of 10-O M in iodide-free artificial seawater. Each test employed 20 polyps suspended in 10 ml of media (number of tests with each compound in parenthes- es): 3-iodo-L-tyrosine (MIT) (4), 3,S-diiodo-L-tyrosine (DIT) (41, 3,3’,5-triiodo-L-thyronine, Na salt (Ta) (8), L-thyroxine, Na salt (8). All products were purchased from Calbiochem Inc. except for DIT, which was provided by Nutritional B&chemicals Corp.

110 SILVERSTONE, TOSTESON AND CUTRESS

Inhibition of Strobilation The effect of thiourea. a compound known to in-

hibit T4 synthesis in the mammalian thyroid gland by blocking the organification of iodide, was tested on strobilation. Thiourea (Merck) was tested at concen- trations of 10m3, 10m4, and lo+ M with iodide (10m5, 10” M) and each of the agents in the previously de- scribed experiment at a concentration of 10m6 M. Each thiourea concentration was tested four times with each of the agents. Twenty polyps were em- ployed for each test, suspended in 10 ml of medium at 29.5”.

Eflect of Bromide on Strobilation In order to ascertain whether or not the effect seen

with iodide is also seen with other halogens, com- parative studies were made with bromide (KBr) at concentrations of low5 and lo-” M. These experi- ments were conducted in the same way as those de- scribed previously.

Effect of Polyp Age on Strobilation Six groups of polyps (10 polyps in 10 ml iodide-free

artificial seawater) were maintained at 20”. After at least 10 buds in each group had been produced, the buds were removed and placed in six new test dishes, 10 buds/dish. Each of the agents, iodide (10W4, 10e6 M) and Tl and its analogues ( 10U6 M) were tested for their effects on the new buds as compared to their parent polyps. These experiments were conducted in the same manner as described above.

RESULTS

Effect of Iodide on Strobilation

Figure 1 shows the percentage of polyps strobilating versus time for each concentra- tion of iodide tested. Data were pooled from all tests at each concentration and graphed as the mean percentage strobilat- ing at each time point. In Fig. 2 the average of the times at which half of the polyps in a test had begun strobilating are plotted for each iodide concentration. The rate of initi- ation of strobilation increases with iodide concentration. Tests in which the experi- mental medium was natural seawater gave a mean time for 50% strobilation of 169 + 11 (SE) hr. Using this value with the rela- tionship shown in Fig. 2, the concentration of iodide in seawater was estimated to be 6 x IO-’ M. This value closely approximates the value of 4.76 x lo-’ M iodide reported for seawater (Riley and Chester, 1972). Strobilation did not occur at all within the 14 day test period in any of the incubations in which iodide-free artificial seawater was the test medium or in those conducted at 20”. Similarly, no strobilation occurred in

100 -

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60 -

70 -

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TIME (HOURS 1

FIG. 1. The effect of iodide on initiation of strobilation. The rate of initiation of strobilation is shown for those concentrations of iodide that were tested and that induced strobilation within the 14 day test period. Each dot represents the percentage of polyps that were strobilating at the time point. The percentages are averages from all tests at each iodide concentration.

IODINE IN STROBILATION 111

T

L

04 6 5rl61 lo-‘ 16’ 16’

IODIDE CONCENTRATION (moles/L)

FIG. 2. Increasing time for 50% strobilation initi- ation with decreasing iodide concentration. Each dot represents the average of the times at which half the polyps in a test had begun strobilating for each iodide concentration (bars represent standard error).

those tests in which the iodide concentra- tion was less than lo-’ M.

Effect of Polyp Concentration on the Iodide Response

Figure 3 shows the inverse linear rela- tionship between polyp co.ncentration and the mean 50% strobilation times in the test cultures with an iodide concentration of 10m6 M. The time required to induce strobi- lation decreases when there are more polyps in the group.

01 0 I 5 IO 20 40 #O

““YlEl OF POWPI

FIG. 3. The effect of polyp concentration on the iodide response. Each dot represents the average time for half the polyps in a test to begin strobilating at an iodide concentration of 10es M (r = 0.98; P < 0.001).

Effect of Zodide, T4, T3, MIT and DZT on Strobilation

The effects of these compounds, tested at a concentration of 10m6 M, are listed in Table 1 as the mean times required for 50% of the polyps in each test to begin strobila- tion. DIT and MIT appear to be the most effective of the agents, followed by iodide, T3 and the least effective, Tq. Except for T4 all of the agents induced strobilation in all polyps within the 14 day test period. Only 40% of the T4 treated polyps strobilated. With polyps exposed to MIT and DIT strobilation began sooner than with those tested with 1O-6 M iodide and later than with those tested with 10e5 M iodide.

TABLE 1 TIMES FOR 50% STROBILATION INITIATION WITH EACH OF THE INDUCERS

AND DIFFERENT CONCENTRATIONS OF THIOUREA”

Time (hr) for 50% strobilation initiation; thiourea 04):

Inducer (Ml

1, 10-S DIT, lO-6 MIT, 1O-B I, 10-B T,, IO-& T,, 1O-6

None

95 t 2.07 112 k 2.81 119 k 3.15 128 k 5.20 164 2 9.96 336 (40%)’

10-Z 10-4 10-S

Ob 0 - 0 233 k 28.04 110.5 2 4.1 0 0 122 ? 7.3 0 0 193 -t 9.43 0 0 193 k 9.36 0 0 336 (4%)’

D Each value represents the mean time at which half the polyps had begun strobilation (shown with standard error).

b 0 = no strobilation within the test period. c Did not reach 50% during the test period.

112 SILVERSTONE, TOSTESON AND CUTRESS

Inhibition of Strobilation

These data are summarized in Table 1. In these experiments the effects of iodide, Tq, T3, MIT and DIT in the presence of thiourea were tested. Thiourea, when tested at a concentration of 10T3 M, com- pletely inhibited the effects of all of the agents, i.e., no strobilation occurred within the 14 day test period. No inhibition was observed when the agents were tested with thiourea at a concentration of low5 M. However, in the presence of thiourea at a concentration of 10e4 M the stimulatory ef- fects of all of the agents were completely inhibited, except for that of DIT.

Effect of Bromide on Strobilation

Bromide failed to induce strobilation at any of the concentrations tested. It did not appear to affect the polyps in any way.

Effect of Polyp Age on Strobilation Induction

The response of new buds to iodide ( 10e4 M), T, and its analogues ( 10e6 M), with or without the presence of older polyps was observed. During the incubation period the older polyps produced buds. These buds strobilated at the same time as the parent polyps. The times required for 50% of the polyps in these tests to begin strobilation are listed in Table 2. The new buds that were tested separately from the parent polyps failed to strobilate in most cases in the presence of the agents tested.

DISCUSSION The objective of this work was to assess

the role of iodide in initiation of strobilation in Aurelia. The data reported here confirm previous reports that iodide is necessary for this process to occur. The results of the tests in which natural seawater was the ex- perimental medium and those in which dif- ferent concentrations of iodide were tested made it possible to calculate the iodide concentration in natural seawater. That this

TABLE 2 TIMES FOR SC% STROBILATION INITIATION FOR

POLYPS AND NEW BUDS WITH

EACH OF THE INDUCERS’

Time (hr) for 50% strobilation initiation

Inducer (M) Polyps Buds

I, 10-d 72.6 2 1.50 240 (30%)c MIT, lo+ 119 k 3.15 240 (IO%)= DIT, 1O-6 112.5 k 2.8 240 (lO%)c TI, lo-& 164 k 9.96 ob T,, 1O-B 240 (20%)’ 0

’ Each value represents the mean time at which half the polyps had begun strobilation (shown with standard error).

* 0 = no strobilation within the test period. c Did not reach 50% within the test period.

value is very close to the reported value for iodide concentration in seawater suggests that the effect seen with the artificial seawa- ter media occurs at concentrations that may be termed “physiological” and that the phenomenon is a meaningful one. The lack of activity on the part of bromide suggests that the initiation of strobilation has a spe- cific requirement for iodide.

The results presented here do not appear to be consistent with the hypothesis that T4 is the agent responsible for initiation of strobilation in this system. The results of the experiments in which the polyps were exposed to iodide, T4, T3, MIT, and DIT indicate that T4 was far less active than any of the other agents tested. This lack of ac- tivity does not appear to be due to permea- bility factors. All of the agents tested are of the same order of magnitude in molecular weight. Thus, molecular size would not seem to be an important factor in differ- entiating their effects on strobilation, Both variations in polyp concentration (Fig. 3), and previous reports concerning sub- stances released into the medium by strobilating polyps (Spangenberg, 1974) in- dicate, by virtue of the rapid onset of strobi- lation in polyps exposed to this factor in the

IODINE IN STROBILATION 113

medium, that the active agent rapidly en- ters the polyps. Autoradiographic studies indicate that T4 does in fact readily enter the organism (Spangenberg, 1974).

The results of the experiments with thiourea are also inconsistent with the hypothesis that T4 is the active agent (Table 1). The singular response to DIT at the thiourea concentration of 10m4 M is highly significant. These data suggest that DIT may be similar to the agent that directly induces strobilation. Since thiourea inhibits thyroid hormone synthesis by inhibiting the organification of iodide, the data suggest that DIT is the fully iodinated form of the active agent. That DIT is partially inhibited may be due to either an inhibition by thiourea of some necessary modification of DIT or a general toxic effect of thiourea that accounts for the inhibition.

The result of the experiments on the re- sponse of new buds to the test compounds provides further information on the nature of the role of iodide in this system (Table 2). That new buds readily strobilate when in the presence of older polyps that are strobilating, but not in their absence, suggests that new buds develop the capac- ity to synthesize the active agent and that none of the agents tested is actually the final product.

It has been reported that Aurelia polyps synthesize a compound that co- chromatographs with T4 (Spangenberg, 1974). preliminary data in this laboratory have detected an iodocompound in polyp homogenates that closely, though not exactly, co-chromatographs with T4. These

results are to be published soon. Thus, it appears that elucidation of the role of iodide in initiation of strobilation must await further studies on the response of polyps to other iodinated compounds and further analysis of compounds produced by Polyps*

REFERENCES Black, R., and Webb, K. (1973). Metabolism of Is11 in

relation to strobilation of Chrysaora quin- quecirrha (Scyphozoa). Cornp. Biochem. Physiol. 45A, 1023-1029.

Chiba, Y. (1969). Ecological factors affecting the strobilization of Aurelia aurita. Bull. Marine Biol. Station Asamushi 3, 173-178.

Cutress, B. (1974). Unpublished data. Loeb, M. (1968). Studies of the life history of the sea

nettle, Chrysaora quinquecirrha. In “Quarterly Progress Report of Project JF-31-1, A Study of the Biology of Sea Nettles to Develop Potential Methods for Control of their Abundance.” United States Department of the Interior, Fish and Wildlife Services, Bureau of Commercial Fisheries. Public Law 89-720, Jellyfish Act. Ref- erence No. 68-76b.

Olman, J., and Webb, K. (1974). Metabolism of 13tI in relation to strobilation of Aurelia aurita L. (Scyphozoa). J. Exp. Marine Biol. Ecol. 16, 113-122.

Percival, E. (1923). On the strobilation of Aurelia au- rita. Quart. J. Microsc. Sci. 67, 85-100.

Riley, J. P., and Chester, R. (1971). “Introduction to marine chemistry,” pp. 465. Academic Press, New York.

Spangenberg, D. (1968). Iodine induction of metamorphosis in Aurelia aurita. J. Exp. Zool. 165, 441-449.

Spangenberg, D. (1972). Thyroxine induced metamorphosis in Aurelia. J. Exp. Zool. 178, 183-194.

Spangenberg, D. (1974). Thyroxine in early strobila- tion in Aurelia aurita. Amer. Zool. 14(2), 825- 832.