observations concerning the metabolism of iodine by polyps of aurelia aurita

GtNtRAl AND COMP4R41lVI; ENDO<RiNOi il(~Y .%t, i32-i4() I lY?xl

Observations Concerning the Metabolism of Iodine by Polyps of Aurelia auritaf

MARK SILVERSTONE, VALERIE ANNE GALTON, AND SIDNEY H. INGBAR

The Department of Physiology, Durtmouth Medicul School, HunoLvr, N~MS Hampshirr 03775, and The Thorndike Laboratory oJ’Hurvard Medical School und the Department of‘ Medicine,

Beth lsrarl Hospit& Boston. Maxsuc~husc~tts 02215

Accepted June 17. 1977

In the Scyphozoan jellyfish, Aurelio auritcr, iodine is necessary for, and capable of initiating, the process of strobilation, by which the sessile polyp is transformed into the free-swimming medusa. Iodine metabolism in polyps of Aurelia auritcr was studied, therefore, with a view to elucidating its role in the initiation of strobilation. Polyps were incubated under varying conditions in artificial seawater containing inorganic’?. It was found that organification is the major means by which polyps accumulate iodine. Accumulation of ‘251 was greatly inhibited by methimazole, but not by perchlorate. and directly measured polyp/medium iodide concentration gradients were low ( v’. 3: I). Chromatographically im- mobile and only partially hydrolyzable “origin material” was the major product of iodine metabolism. Both MIT and DIT were formed and released into the incubation medium, with DIT in predominance. Another iodocompound that was chromatographically distinct from 3,5-T*, TS, and T, was also detected, but remains unidentified. No trace of ‘251-labeled T, was ever detected, even under conditions in which strobilation was induced. Coupled with evidence from previous studies, the findings suggest that T, is not the agent responsible for initiation of strobilation in Aureiiu.

Strobilation is the term applied to that stage in the development of Scyphozoan jellyfish in which the sessile polyp metamorphoses into the free-swimming medusa. It has been clearly demonstrated that scyphistomae of Aureliu uurita will not strobilate in the absence of iodine (Spangenberg, 1967; Silverstone rt al., 1977), but the function of iodine in respect to this process is not yet understood. Polyps apparently can synthesize mono- and diiodotyrosine (MIT and DIT), and it has been reported that traces of a compound chromatographically identical to the thyroid hormone thyroxine (Te) are also formed (Spangenberg, 1974). Although strobilation can be initiated by exposure to

f Supported in part by Research Grants AM 16172 and AM 18416 from the National Institute of Ar- thritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland.

inorganic iodide or to the precursors of TJ, MIT and DIT, compounds devoid of hor- monal activity in mammalian systems, the polyps respond slowly if at all to T, (Sil- verstone et al., 1977). To date, Scypho- zoans are the only invertebrates in which a function of iodide has been demonstrated.

In the present study the pathways and products of iodine metabolism in Aurcficr scyphistomae were examined with a view to elucidating the form in which iodine in- duces strobilation.

MATERlALS ANDY k#ETt-tODS The polyps used in these experiments were cul-

tured from a sample of the stock maintained in the Department of Marine Sciences, University of Puerto Rico. Polyps were maintained at 20” in Gin. finger bowls in iodide-free artificial seawater (ASW) (Instant Ocean. Aquarium Systems. Inc.) and were fed newly hatched Artemia nauplii twice a week. Studies OF iodine metabolism were conducted with suspensions of polyps in 55 Y .33-mm glass dishes to which carrier

0016~6480/78/0342-0132$01 .OOM, Copyright 0 1978 b> Academic PI-us, Inc. 411 rights of reproduction in any form rexwed

IODINE METABOLISM IN Aurelia 133

free rZ51 (Amersham-Searle) was added. In some cases incubation medium was measured. Aliquots of methimazole ( tOeaM), potassium perchlorate (IO-‘&f), homogenate and media were then hydrolyzed at 37” or potassium iodide (lO?U) was also added to the for 24 hr using either pancreatic protease or pronase incubation medium. (Sigma) (Gahon, 1972).

Initial experiments (Type I) were conducted to test the ability of polyps to accumulate radioiodine. Polyp suspensions contained 50 polyps/3 ml of ASW to which 2.5 &i of ‘%I had been added. In some suspensions, methimazole or perchlorate was added to the medium. The suspensions were incubated at 29”. At the end of each time period, the polyps in each dish were drawn up into 100-d capillary pipets (Yan- kee Micropet) together with a minimum amount of medium. Pipets were plugged with clay and centrifuged at 2OOOg (Sorvall glc centrifuge) for 30 min. This resulted in a tight polyp pellet. The volumes of the polyp pellet and supernatant were calculated from their individual lengths and the pipets were broken at the polyp-supematant interface. The radioactivity in each section was measured (Searle Automatic Gamma Counter, Model 1195). From these data, the total radioiodine accumulation by polyps as a function of the total radioiodine content of the system could be calculated. In addition, polyp/medium iodide concentration ratios could be calculated for vessels in which organification of resI was blocked by methimazole.

To determine the percentage of the total radioidine in the polyp homogenates and in the media that had been organified, organic and inorganic “‘1 in each were separated by short-term paper electrophoresis (1.5 hr using a constant current of 12 mA) in a glycine-acetic acid buffer, pH 8.6 (Galton and Ingbar, 1972). Carrier NaI was added as a marker. After electrophoresis, the paper strips were dried and stained with palladium chloride to visualize the inorganic iodide band; organically bound iodide remained at or near the origin. Both the “origin” and inorganic iodide bands were then cut out and the radioactivity in each was measured.

Subsequent experiments (Type II) were carried out to investigate further the organihcation reactions in the polyps and particularly to characterize the nature of the organic iodine compounds formed. Therefore, experiments were conducted using a greater polyp density and higher concentrations of rz51. Suspensions of 100 polyps in 3 ml of ASW containing 300 &i of rZ51 were employed. Methimazole and/or stable iodide was added to some of the suspensions.2 Suspensions containing polyps killed by boiling for IO min prior to incubation were also included. Incubation of the polyps was carried out for 2, 4, and 6 days at 4, 20, and 29”.3 Following the incubation period, the polyp suspensions were centrifuged at low speed, the supematants removed and saved, and the polyps washed by resuspending them in 3 ml of iodide-free ASW. The centrifugation and washing were repeated until the radioactivity in the supernatant was reduced to 0.1% of that in the original medium. The polyp pellets were resuspended and homogenized in 0.5 ml of Veronal buffer, pH 8.6, containing 3 mM KI and 1 mM methimazole to prevent further organification of iodide or deiodination of iodocompounds present. The total radioactivity in aliquots of homogenate and

Iodinated products in both hydrolyzed and nonhy- drolyzed homogenates and media were separated by ascending paper chromatography using butanol-2 N acetic acid (1: I) (BA) and butanol-dioxane-2 N NH40H (4:1:5) (BDA) as solvents. Samples of the preparations were applied directly to paper strips (Whatman No. I) and developed for 17 hr. Stable carriers, iodide (as NaI), MIT, DIT, Tlr 3,5,3’- triiodo-L-thyronine (T& and 3,5-diiodo-L-thyronine (3,5-T*) were applied to the strips for identification purposes. After development, the strips were dried. Carrier iodide was visualized with palladium chloride, and MIT, DIT, T,, T, and 3,5-T, with the diazotized sulfanilic acid method (Gahon and Ingbar, 196la). The strips were then cut into 0.5-cm segments and the radioactivity of each was measured. In order to confirm the identities of those compounds that cochromatographed with stable carriers, some strip chromotograms were placed against GAF non- screen X-ray him. After an adequate time (l-8 weeks), the carriers were visualized and their locations compared with those of the radioactive spots on the film. Appropriate sections were then cut from the strips, the radioactivity was measured, and the percentage of the total radioactivity in each of the sections was calculated.

In order to obtain convincing evidence of the iden- tity of the iodinated compounds, two-dimensional chromatography was also performed using BDA in both dimensions. The developed chromatograms were autoradiographed, and the carriers were visualized and then compared to the blackened areas of the film as already described.

’ Strobilation was observed to be occurring by 4 days in vessels containing lo-’ M NaI and incubated at 29”.

3 Polyps incubated at 4” survived only l-2 days and accumulated only small amounts of ‘?. No metabolism of lZsI was observed in boiled polyps.

Separation of iodocompounds was also achieved by high-voltage paper electrophoresis (Wain, 1973). Samples were applied to Whatman No. 1 filter paper sheets and supported vertically in coolant between the two electrode buffer troughs (formic acid:acetic acid:water, 1:4:45, pH 2.1). A voltage of 2 kV was applied for I hr and the paper was removed and dried. Chromatography was then performed in the second

dimension using BL>A solvent, and the spot*, \r~re identified b) autoradiography as described :rh!~\e

All of the experiment> reported herein WCI’C L.;LI-IW~ out several time\. Although the results present& :,1-e those of a single experiment. the rewlt\ 111’ rr”plic:tte experiments agreed clowi~.

RESULTS The accumulation of radioiodine by

polyps as a function of time, as seen in experiments of Type I, is shown in Fig. I. The percentage of added 125I accumulated by the polyps increased with time up to 4 days, reaching a maximum of approxi- mately 13% and declining thereafter. Total radioiodine accumulation was greatly inhibited by incubation with methimazole, but was not appreciably influenced by perchlorate (not shown). In accord with the latter finding were relatively low values for polyp/medium iodide concentration ratios, which never exceeded 3: 1.

The remainder of the data to be discussed was derived from Type II experiments. Or- ganic 125I present in polyps and medium combined increased up to 4 days and then declined, and its formation was again inhibited almost completely by methimazole. The fraction of total 1251 in the organic form was higher at 4 days of incubation than at 6 days, suggesting that deiodination of organic compounds was occurring, at least during that interval. Figure 2 depicts the

‘7 in polyps (% total ‘*‘I in culture)

‘_:ril-: 0 2 4 7

DAYS

FIG. I. Total accumutation of 9 by polyps incubated at 29”, in the presence and absence of methimazole (10-94). Results expressed as percentage of total ‘z31 in culture.

I34

‘*’ I orgonified

(% ?otol lz51 m culture)

25~

20 i

15-j

i

IO

5

media

PdYPS

I , I 1 2 4 6

DAYS

FIG. 2. Organic ‘9 in polyps and media during incubation of polyps at 29”. Results expressed as percentage of total 1251 in culture.

distribution of organic lz51 between polyps and medium in the same experiments. At 2 days, the proportions of organic iodine in polyps and medium were almost equal. At 4 days, a much greater fraction was present in the polyps, and between 4 and 6 days the organic 9 content in the polyps declined, while that in the medium increased.

Figure 3 depicts the effects of incubation temperature, and of either boiling of pqlyps or stable iodide, on the total organic lz51 content of the incubation system (polyps

‘*‘I orgaoified (% total ‘251 in culture 1 IO 20 _ 30

liE!itB 4O

29’ Boiled tissue

FIG. 3. Effect of temperature, boiling, and stable iodide on the formation of organic 9 by po!yps incubated for 4 days. Values indicate organic ‘=l present in polyps and media dombiired, expressed as percentage of total lzal in culture.

IODINE METABOLISM IN Aureliu 135

and medium combined), as measured at 4 days. Here, organic lz51 content was seen to be greater at 29 than at 20”, to be completely inhibited in boiled polyps, and to be little, if at all, affected by the presence of stable iodide at a concentration of lo-’ M.

Chromatographic analyses of 1251-labeled products present in polyps incubated with 1251 and 10W7 M [‘2’I]iodide at 29” for 4 days are shown in Fig. 4 (solvent BA) and Fig. 5 (solvent BDA). Several major peaks of radioactivity are evident, including a peak at the origin. The locations of the visualized carriers are indicated. Peaks of 1251 consis- tently cochromatographed with carrier iodide, MIT, and DIT. Another peak of lz51 with a greater Rf value than that of iodide was invariably observed in chromatograms developed in BDA. This material migrated to an area which overlapped both carrier 3,5-T2 and Ta, but did not coincide with either. Moreover, it clearly did not coincide with carrier Tq. Indeed, it is significant that radioactivity that cochromatographed with T, was never observed. The BDA system employed separated T, quite distinctly from 3,5-T* and TS, and hence from the unknown compound. These findings are emphasized in the two-dimensional chromatogram developed in BDA (Fig. 6)4 and in the chromatogram prepared by high-voltage electrophoresis followed by chromatography in BDA (Fig. 7). Radioactive spots corresponding in location, size, and shape to carrier MIT and DIT are evident (Fig. 7). Iodide and the unknown spot near the 35 T2-T, area are shown in Fig. 6. No radioactivity is visible in the regions corresponding to T4.

Chromatographic analyses were performed on homogenates of polyps and media incubated under all of the conditions employed and qualitative results were simi- lar to those cited above. As already indicated, this was true even under conditions

’ MIT and DIT were incompletely separated when BDA was the solvent system employed for both dimensions.

R 0 -

* 2.5- 5 s

- 1- I- MIT DIT Front

FIG. 4. Single-dimension chromatogmm (butanol- acetic acid) of homogenate prepared from polyps incubated with rz51 for 4 days at 29”. Locations of carriers are indicated. Each bar represents a 5-mm segment of chromatogram. The peaks are not an accurate quantita- tive reflection of the radioiodine present, as the origin and iodide peaks are off scale.

(29” plus 10u7 M iodide) in which strobilation occurred. Under some conditions, one or another peak was not seen, but addi- tional peaks were never observed. The distribution of total lz51 among the four major compounds generally present in polyp homogenates (origin material, MIT, DIT, and unknown X) is shown in Table 1. Only data for polyps incubated under conditions

BDA

FIG. 5. Single-dimension chromatogmm (butanol- dioxane-NH3) of homogenate prepared from polyps incubated with rz51 for 4 days at 29”. Locations of carriers are shown. Each bar represents counts per minute detected in 5-mm segment of chromatogram. The origin peak is off scale.

Hc,. 6. Autoradiograph of chromatogram of polyp homogenate developed in hutilnol-~~lozantl--.~~~,, it! bvrh

dimensions. Locations ofcarriers are shown by dotted lines. Radioacrivltl is observed in the area irt %I IT. Dl’! .!nd

of iodide. A third radioactive \pot did not coincide uith 7,i.T2- P,.. (‘I ‘!‘#

which permitted significant organification of 125I are included, and results obtained in both hydrolyzed and unhydrolyzed samples are given. The 4-day time period was again chosen since that is the time at which the system contained the greatest quantity of organic . lz51 It is evident that most of the radioactivity in the unhydrolyzed preparations was in the fraction that remained at the origin during chromatography; in fact only very small proportions of MIT, DIT. and X were present. The distribution of organified lz51 among the four major compounds present in the corresponding specimens of hydrolyzed medium is shown in Table 2. The distribution of lz5T in unhydrolyzed media (data not shown) was simi- lar to that shown for hydrolyzed media in Table 2, suggesting that the origin material in media was highly resistant to hydrolysis

and deiodination. Although compounds present in the media and the polyps were the same, MIT/DIT ratios were far greater in the polyps than in the media (Table\ I and 2).

The MlTiDIT ratio also changed with time (Fig. 8). In the polyp fracFion, this ratio increased up to 4 days and decreased thereafter. In samples of media, equal quantities of MIT and DIT were found after 4 days; however, after 6 days, relatively more DIT than MIT was present.

Attempts to hydrolyze the origin material with acid or alkali met with limited success. Although some MIT and DIT were released, the major product generated was inorganic iodide. In this respect, the origin material formed by polyps differs markedly from thyroglobulin, but resembles the radioiodinated origin material formed by


FIG. 7. Autoradiograph of chromatogram of polyp homogenate developed by high-voltage electrophoresis in the first dimension (horizontal) and paper chromatography in butanol-dioxane-NH, in the second (vertical). Locations of carriers are shown by dotted lines. Areas of radioactivity correspond to MIT and DIT. A third area does not coincide with 3,.5-T,, T,, or T,.

h 6 DAYS

FIG. 8. Effect of the duration of incubation of polyps at 29” on the 1251-labeled MIT/DIT ratio in polyps and media.

peripheral tissues during the deiodination of radioiodine-labeled T4 (Galton and Ingbar, 1961b).

DISCUSSION

Iodine is a,requisite factor in the hfe cycle of the Scyphozoan jellyfish, Aurelia aurita,

since it is essential for strobilation, the process by which the sessile polyp metamorphoses to the free-swimming medusa. Therefore, it seemed possible that studies of the products of iodine metabolism and their mechanisms of action in Au- relia might shed light on the actions of the thyroid hormones in higher forms. The present studies were undertaken to explore further the mechanisms of iodine metabolism in the polyps of Awe& and to reexam- ine the nature of the iodinated compounds that they form. The studies provide both some confirmation of previous observations and new information with regard to these questions.

A previous report has suggested that polyps are capable of taking up iodide, but that little is organified, suggesting that iodide itself may be responsible for the initiation of strobilation (Olman and Webb,

1974). The present results, however. strongly indicate that this is not the case: indeed, in our studies, iodine seemed to be metabolized mainly by organification, since the majority of accumulated iodine wa4 in the organic form and total radioiodine :tc. cumulation was greatly decreased by inclusion of methimazole in the incubalion medium. Iodide transport activity. in contrast, appeared to be relatively low. xince polyp/medium iodide concentration gradients, measured directly in the presence of methimazole, were low f - 3.0). and since inclusion of perchlorate in the incubation medium had little effect on total radioiodine accumulation. Moreover, the capacity of polyps to carry out organification of iodine appeared to be substantial, since enrich- ment of incubation media with 10’~’ &l stable iodide reduced only slightly the proportion of added “‘1 that was orgdnified.

Clarification of the products of iodine metabolism by polyps is of particular interest, since it may shed light on the nature of the iodinated material necessary to Initiate strobilation. In the present, as in most previous studies of Auwliu, the principal products of iodine metabolism by the polyps were MIT and DlT, but the relative proportions of each differed in relation to time of incubation and according to whether polyps or media were sampled. At an incubation temperature of W, MIT/DIT ratios in polyps exceeded unity at all time periods studied, increased with time up to 4 days. and then decreased. In contrast, in the medium, where MIT and DIT appeared mainly in the free form, MIT/DIT ratios decreased progressively with time. and ui- timateiy (at 6 days) were of the order of 0.2. It is unlikely that the declining MITiDIT ratio in the medium can be explained by preferential deiodination of MIT, since the organic lz51 content of the medium in,- creased progressively with time. The findings suggest that free iodotyrosines were lost or secreted from the polyps into the medium during the incubation. and that this was especially true 01’ DI’I‘,


TABLE 2 RADIOIODINATED COMPOUNDS IN THE MEDIUM OF POLYPS OF Aurelia

INCUBATED WITH INORGANIC 9 FOR 4 DAYS n

Incubation condition

Percentage of organitied r? lzSI in medium Organic lz51 in Organic ‘*V in in pronase hydrolysate (% total ‘=I medium (% total medium (% total in culture) lzsI in media) lz51 in culture) Origin MIT DIT X MIT/DIT

20” 88.3 12. 10.6 40.2 8.8 40.7 10.3 0.22 20° + 9 88.4 8. 7.1 38.1 7.0 48.4 6.6 0.14 29” 79.0 17. 13.4 32.7 27.8 28.2 1 I.3 0.99 29” + ‘*‘I 78.9 18. 13.2 41.0 4.0 23.1 32.0 0.17

a Percentages of different compounds present were obtained from chromatograms developed in either BDA or BAc solvent systems. Data in Tables I and 2 were obtained in the same experiment.

In addition to MIT and DIT, an iodinated compound was formed that has not been identified. Its mobility during paper chromatography and high-voltage electrophoresis resembled that of iodo- thyronines, but it was clearly separable from Tqr TB, and 3,5-T2. Other carrier iodo- thyronines were unavailable at the time these studies were performed. The compound remains unidentified.

Besides MIT and DIT and the unknown compound, one other class of iodinated material was formed, This was characterized by its immobility in the chromatographic systems employed. Such “origin material” comprised by far the major proportion of the organic 1251 present in unhydrolyzed polyps, and a substantial proportion in unhydrolyzed media. Origin material was resistant to hydrolysis by pronase, particularly that origin material found in the medium, which was almost entirely resistant. Origin material in polyps was only partly affected by pronase hydrolysis and liberated mainly iodide, with small proportions of MIT and DIT. Efforts to digest origin material with acid, alkali, and tryspin and chymotrypsin yielded either no hydrolysis or extensive deiodination. There- fore, the possibility cannot be excluded that the proportions of MIT, DIT, and the unknown compound that were actually formed differed greatly from those found.

Similarly, origin material may have contained iodinated compounds quite different from those actually detected.

A major finding of the present studies was the inability to detect the formation of Tg, despite previous reports to the contrary. In no instance, even when very large amounts of radioactivity were added, was even a trace of ?-labeled T4 detected in any of the analytic systems employed. In all of these systems, moreover, carrier T, could be clearly separated from the unknown compound described above. It is not clear, therefore, why T, purportedly has been found in Amelia in previous studies (Spangenberg, 1972, 1974), or in another Scyphozoan jellyfish, Crysaora quin- quecirrha (Black and Webb, 1973). It seems possible that T4 and the unknown compound that we have described were not separated from each other by the chromatographic systems used in those earlier studies in which formation of T, is said to have occurred. Another possibility is that the r311 employed in those studies may have contained material chromatographically distinct from iodide, as has been reported for some commercial preparations of this isotope (Ahn and Rosenberg, 1961). This could also explain the puzzling results of previous studies which indicate that some material was present that cochromatographed with Ts, but none that cochromato-

graphed with either MIT or DIT in polyps incubated with goitrogen (Spangenberg. 1974). In the present study, samples of the lZ51 employed were routinely tested for compounds chromatographically different from iodide, and any samples containing such materials were not used.

The present study does not provide direct evidence as to the nature of the iodinated compound that is presumed to initiate strobilation. It does, however, provide some indirect evidence to this point. It is known that iodide itself is ineffective. since addition of iodide fails to initiate strohila- tion in the presence of goitrogenic agents, such as methimazole (Silverstone or ~1.. 19771, and we have shown that methimazole is indeed active in inhibiting organification of iodine in this system. The data also suggest that T, is not the initiating factor, since no synthesis of T, was detect- able under conditions in which strobilation occurred (29” and added stable I). This con- clusion is consonant with previous studies that have shown T, to be relatively inactive in initiating strobilation when added directly to the incubation medium (Sil- verstone et ul., 1977). The data suggest that the concentration of the initiating factor is critical for strobilation. Clearly, polyps could synthesize all of the iodinated compounds under any of the conditions employed. However, the absolute amounts of each compound formed were greatest under the conditions that permitted strobilation (29”. IO--’ M I).

shown that DI’P is the predominant iodin-

ated amino acid released from the polyp:, into the medium. This finding might aiso suggest that DIT is the initialing agent, since previous studies indicate that polyp\ undergoing strobilation release into the medium compound(s) that are capable of initiating strobilation in other polyps in I he absence of added iodide (Spangenherg. 1971).

REFERENCES Ahn, C. S., and Rosenberg, I. N. (1961). Non-iodide

radioactivity in commercial solutions of sodium radio-iodide !I 13’). Endoc~rindogy 68, 60-61,

Black, R. E., and Webb, K. L. (1973). Metabolism of ‘“‘I in relation to strobilation of Ch~suoru quirr-

quecirrhrr (Scyphozoa). Camp. Biochrm. Physiol. 45A, 1023-1029.

Galton, V. A. (1972). Some effects of altitude on thyroid function. Endocrinobgy 91, I393-1403.

Galton, V. A.. and Ingbar. S. H. (1961a). The influence of reserpine. herotonin and metabolites of tryp- tophane on the degradation of thyroxine and it5 derivatives. Endocrinc&~gy 68, 435-449.

Galton, V. A.. and ingbar, S. H. (196(b). rhe mecha- nism of protein iodination~ during the metabolism of thyroid hormones by peripheral tissues. Entk~ crinolog.v 69, 30-M.

Galton, V. ‘4.. and lngbar. S. H. (1972). Decrease in the thyroidal internal iodide pool induced by i-nitro-1 -tyrosine (MNT), an inhibitor of iodotyrosine deiodination. Endocrinology 91, 1476-1480.

Olman. S.. and Webb, K. ( 1974). Metabolism of Is’1 in relation to slrobiiation of Audio ~uritu I.. (Scyphozoa). /. E.rp. Mrrr. Biol. Ecol, 16, 1 I?- 122.

It is possible that the unknown compound detected is related to strobilation. but this possibility cannot be tested until its identification is achieved. Earlier studie\ have strongly suggested that DIT is closely related to the initiating compound, since, in equimolar concentrations, it induced strobilation more rapidly than did iodide, MIT, T:%, or T,. In addition, DIT was the only compound of those tested that was active in the presence of goitrogen (Silverstone et ~1.. 1977). In the present studies we have

Spangenberg, II. B. (1967). Iodine induction of metamorphosis in Artrc/ic~ moitcr. .I. E.t-/l. Zoml. 165, 441-449.

Spangenberg, D. B. (1977). Thyroxine induced 3trobi- lation in Aweliu crurita. .I. E-I-[I. Zord. 178, 183- 194.

Spangenberg, D. B. (1974). Thyroxine in early strobilation in Aurelitc crurifa. Amer. Zool. 14, 825-83 I.

Silverstone, M.. Tosteson, T. R., and Cutress, C. E. (1977). The effect of iodide and various iodocom. pounds on initiation of strobilation in Auwliri.

Gen. Camp. Endncrinol. 32, 108-I t3. Wain. W. H. (1973). The biosynthesis of thyroxine:

Incorpov&tian of [U-ICI tyrosine into thyroglobrt- lin by mouse thyroid glands in viva and in vitro. J. Endocrirwl. 56, 17% 185

observations concerning the metabolism of iodine by polyps of aurelia aurita

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