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Comparative study on amounts of trace elements in the solitary ascidians, Ciona intestinalis and Ciona robusta
Camp Eidwm. Phlsrol. Vol. 78A.No. 2,pp.2855288,1984
0300-9629:X4 $3.00+O.OO
,
Printed in Great B&in
COMPARATIVE STUDY ELEMENTS IN THE CIONA INTESTINALIS
1984 Pergamon Press Ltd
ON AMOUNTS OF TRACE SOLITARY ASCIDIANS, AND CIONA ROBUSTA
HITOSHI MICHIBATA Biological Institute, Faculty of Science, Toyama University,
Toyama 930. Japan
(Receired 30 August 1983) Abstract-l. The closely related ascidian species, C. intestinalis and C. rohusta have different lation patterns of metals. 2. The former had a higher amount of vanadium and lower amounts of iron and manganese tissues than the latter. 3. However, there was little difference in nitrogen and phosphate contents.
INTRODUCTION A new species of ascidian was first recognized and distinguished from C’ion~l intestinalis (L) by Hoshino and Tokioka (1967). According to their findings, the usually known cosmopolitan C. intestinalis has a very soft test, the surface of which is quite free of any foreign materials. Its mantle is more or less sprinkled with orange pigment spots that are especially dense near the tips of the siphons. The test of the other type, named C. robusta (Hoshino and Tokioka, 1967), however, is much thicker and harder and sometimes even harbours other organisms on its surface. Its mantle is scarcely pigmented. Moreover, the posterior end of the endostyle of C. intestinalis continues nearly straight to the retropharyngeal groove while that of C. robustu is curved toward the right dorsal side. The authors also indicated that the two species differ in the shape of the epicardium. Subsequently, Pisan6 and Rengel(1972) confirmed that the new species, C. robusta, was different from C. intestinalis due to the fact that in C. robustu self-fertilization took place in 60-100% of the cases, while in C. intestinalis, self-fertilization never occurred. On the other hand, it is well known that the ability of many species of ascidians to concentrate vanadium from sea water is one of the biochemical peculiarities which distinguish the ascidians from other classes of animals. Advancing this point of view a step further, Carlisle (1968) and Swinehart et al. (1974) proposed that the level and distribution of vanadium and other metals in ascidians were related to ascidian phylogeny. Biggs and Swinehart (1976) also pointed out that the concentration of vanadium in the tissues of ascidians was somewhat specific to the species. As it seemed interesting to find whether or not the amounts of trace elements contained in the tissues differ and whether these amounts are specific to species, in the present experiment, the vanadium, iron and manganese in some tissues of C. intestinalis and C. robustu were analysed mainly by neutron activation analysis. For the analysis of these trace elements, this may be the preferred technique since the sensi-
accumuin most
tivity of this method is much higher than that of atomic absorption spectrometry or spectrographic analysis.
MATERIALS AND METHODS Specimens of the solitary ascidian, Cionu intes/inu/is (L) were collected at Asamushi Marine Biological Station on the Bay of Mutsu, Japan. Specimens of C. rohusta were also collected at Asamushi and at Onagawa Fisheries Laboratory of Tohoku University. They were identified by Dr. T. Numakunai of Tohoku University. Not less than five specimens of each species were used in the present experiment. Each specimen was cleaned of extraneous materials. Blood was obtained via heart puncture, then plasma and corpuscles were separated by centrifuging the blood for 10 min at 3000 rpm. The test, mantle, stomach, gonad and branchial basket were rinsed three times with filtered sea-water. All tissues were carefully dried in procelain crucibles in a drying oven at 110 C to constant weight. Each sample, about 20 mg in dry weight. which was enough to analyse metals by means of the neutron activation analysis, was ashed in a muffle furnace at 5OO‘C for 24 hr. The ash was dissolved in 5.0 ml of 0. I N HNO, and put into a polyethylene capsule. For applying neutron activation analysis to measuring the amounts of vanadium and manganese, each sample was irradiated with thermal neutrons (thermal neutron flux: 5 x 10” n/cm’ set ‘. 2 min) in a TRIGA MARK II nuclear reactor at Rikkyo University. The radioactivity of j2V produced in the irradiated sample was measured with a 50-cm3 Ge(Li) gamma-ray spectrometer (Canberra Inc.) 2 min after the irradiation. Sixty min after the irradiation, the radioactivity of ‘“Mn was measured in the same manner as described above in order to avoid the interference of “Mg produced in the irradiated sample. The amounts of vanadium and manganese were determined by comparing the gamma-ray spectrograms with those of standard samples. After the radionuclides were allowed to decay to a negligible concentration. a part of each sample was submitted to analysis for iron by atomic absorption spectrometry (equipment: Hitachi GA-2 flameless atomic absorption spectrometer). The absorption line used was 3719.9nm. Besides the analysis of transition metals, a part of each dried sample was analysed for its nitrogen content bv the method of micro-Kjeldhal and for its phosphate content by Nakamura’s method (1950), which was a modification of the method of Allen (1940).
286
HITOSHI MICHIBATA Table
I. Metal
concentrations
m tiswcs
of the sohtary robusra
C.
ascldums.
Metal concentratmn (ng:mg c‘. inrrstinuli\
(’
irtrestmoli.s and
dry weight)
(‘. roi7u.w I .7 I 0.4
Metal
Tissue
Vanadium
TUIiC Mantle Stomach Gonad Branchial basket Biood cells Plasma
1.2 * 0.5 333.0 i 39.7 507.4 + 69.6 143.6 + 23.9 1037.5 7 109.2 1577.1 f 338.2 12.9 If- 0.4
IroIl
Tunic Mantle Stomach Gonad Branchial basket Blood cells Plasma
8i.h * 6 5 54.8 + 7.3 58.8 _+ 8.0 54.7 2 5.3 I73 8 k 24.3 7x.3 * 5.1 119.6 * 4.8
370.0 2 39.4 I-%?+ Il.4 2% 7 + 69.0 65 x + x.9 251.4127h 424.4 * 49.4 89 s i: 3.8
MatlgdtIW
Tunic Mantle Stomach Gonad Branchial basket Blood cells Plasma
4.8 * 7.4 * 20.2 t 13.2 + 17.3 * 107.6 * 15.4+
18X.8 :k 76.1 29.3 i_ 5.4 36.4 I x. I 77.0 & 14.9 69.‘) * 7.3 1..4*9.0 9.2 _+ 1.Y
Mean I standard II = 5.
1.5 0.9 3.1 1.1 0.8 13.x I9
33x.4 163.2 100.9 337.5 330.7 0.4
i: 23.x ?_ 14.6 + 7.5 ; 47 H 2 14.1 f: 0.2
error.
RESULTS
Metal concentrations detected in the various tissues are shown in Table 1. Metals in trace levels were found in all tissues examined. The vanadium content in each tissue was significantly higher in C. intestinalis than in C. robusta except in mantle. The highest value (1577.1 ng/mg dry weight) for vanadium was obtained from the blood cells of C. intestinufis. This value was about 5 times that in C. robusta blood cells. The leveis of iron found in most tissues of C. robusta were conversely higher than in those of C. i~testi~ul~s, The iron was present at the highest level of 424.4ng/mg dry weight in the blood cells of C. robusra. Relatively high concentrations of manganese occurred in the tissues of C. robusta but the levels in the blood cells and plasma were higher in C. intestinalis.
Nitrogen and phosphate were analysed as representatives of essential elements. In the contrast to the trace metals? there was little difference in the quantitative contents of these elements in the various tissues between the two species (Table 2).
DISCUSSION
Webb (1939) first pointed out the correlation of the presence of vanadium in ascidians with certain evolutionary traits of the class. He also suggested that the presence of vanadium was a primitive character which had been lost in the more specialized families. Many investigators, thereafter, analysed the metal content in ascidians. In general, several species in the order Aplousobranchia have a large vanadium content, among them the genera Eudistoma and Distaplia, and significant vanadium was found in the order Phlebobranchia. In contrast, some species in the order Stolidobranchia contain a small concentration of vanadium but the iron content is large (cf Swinehart et af., 1974; Biggs and Swinehart, 1976).
The present results have revealed that the closely related species, C. intestinalis and C. rohustn belonging to the order Phlebobranchia had different accumulation patterns of metals. Little difference between the two species was observed in the amounts of nitrogen and phosphate contained in each dried tissue (Table 2), while it should be noted that the amounts of trace metals were specific to species. Our unpublished observation that the embryo of C. intrstinah accumulates more vanadium and less iron and manganese than the embryo of C. ~~~2~~~~ also supports the above findings. This situation has a precedent. Carlisle (1958) reported that ~~lf~~u/u ~~~~~ttensis belonging to the order Stolidobranchia could be divided into two groups with regard to the metal accumulation pattern; in the population of the south coast of Devon about two-thirds of the Molugulu were found to have vanadium at a significant level but no niobium, whereas the remaining animals contained niobium at a detectable level but had only a trace of vanadium. Therefore, unless more species are submitted to analysis for their metal contents. we cannot conclude that the presence of several metals in ascidians reflects ascidian phylogeny if the trends are right. Taking another look at papers on trace elements. we become aware that there is considerable variation in the vanadium, iron and manganese contents of C intestinalis. The highest value of vanadium in blood cells was reported by Hielig et al. (1961) who found it present at 1.500ng/mg of the dry weight. Macara CJI al. (1979) found 2400 and 700 ng/mg of vandium in the body and blood cells, respectively. The whole animal also contained a very large amount of vanadium (1300 ng/mg of dry weight; Vinogradov, 1934). Ciereszko et al. (1963) determined a low value of vanadium (40 ng/mg of organic dry weight) in the siphon tips of C. intest~n~lis, and Goldberg rt crl. (1951) reported that vanadium was undetectable in the tunic, ovary, heart, siphon and branchial basket.
V, Fe and Mn in ascidian Table 2. Concentrations
_“.“.
--
287
of nitrogen and phosphate in tissues of the solitary ascidians, C. Concentration
Element
_._.-
tissues
Tissue __.
&g/m8 dry weight)
C. intestinalis
_____i_ ..,.... -
C. robusta
Nitrogen
Tunic Mantle Stomach Gonad Branchial basket Blood cells Plasma
6.1 rt 0.6 48.2 & 3.5 64.4 * 2.3 55.2 rt: 1.5 NA* 28.4 +- 2.2 2.9 It 1.2
II.1 + 1.5 49.0 f 0.7 63.2 + 2.2 60.4 + 1.9 NA* 30.6 t 1.5 2.6 * 0.8
Phosphate
Tunic Mantle Stomach Gonad Branch% basket Blood cells
l.OiO.1 18.8 + 0.6 43.8 rt 2.6 43.2 rt 3.2 32.9 I 2.4 24.7 It 3.4 12.1 i4.8
I.1 f 0.2 17.8 + 1.2 38.8 f I .3 55.2 & I .6 29.3 + 2.6 37.9 * 0.3 8.7 f 0.2
PlaSSXi
Mean + standard error. *NA = not analysed. n = 5.
Many other data ranging from 10 ng/mg to hundreds of ng/mg of dry weight have been reported (Noddack and Noddack, 1939; Webb, 1939; Carlisle, 1968; Swinehart et al., 1974; Kustin et al., 1975; Botte et al., 1979). Concerning the content of iron, Bielig et al. (1961) reported that C. intestinalis contained 800 ng/mg dry weight of iron in the blood cells and 60 ng/mg in the plasma. Using an argon plasma emission spectrometer, Macara et al. (1979) found 350 and 40ng/mg dry weight of iron in the blood cells and plasma, respectively. However, a lower value of iron (50 ng/mg) was detected in the blood cells by Swinehart et al. (1974). The work of Papadopoulou and Kanias (1977) by neutron activation analysis revealed that the whole body, tunic and the rest of the body contained 880, 610 and 3OOOng~mg of iron, respectively. Botte CTaf. (1979) found 52 ng/mg of iron in the ovary tissue. In the case of manganese a similar diversity of analytical data has been reported. Noddack and Noddack (1939) reported that C. ~ntest~nal~s contained this element at a level of 120 ng/mg of dry weight. Carlisle (1968) found a level of about 95 ng/mg. Swinehart et al. (1974), however, reported that the manganese content in blood was less than 10 ng/mg. Botte et al. (1979) reported that manganese was undetectable in the ovary of this species by fiameless atomic absorption spectrometry. The cause of the diversity may be attributed to several possibilities. One of them may be advances in the analytical methods; the data obtained has become more accurate with the years. Generally speaking, the older data determined by emission spectrometry or calorimetry have higher contents of trace elements and less accuracy compared with more recent data determined by atomic absorption spectrometry, argon plasma emission spectrometry or thermal neutron activation analysis. It seems, therefore, the data extending over about 70 years are not comparable. The most possible cause is the confusion between these two species, C. inteJtinalis and C. robusta that have a certain similarity in their external appearance. They must have been indiscriminately analysed for metals. They had been, indeed, recognized as the
single species C. intenstinalis until Hoshino and Tokioka (1967) divided them into two different species. Therefore, the metal contents reported formerly may be divided roughly into two groups. One group has a higher content of vanadium and lower contents of iron and manganese, which probably corresponds to the species C. intest~nai~s. The other group has, on the contrary, a lower content of vanadium and higher contents of iron and manganese, which probably corresponds to the newly named C. robusta. On a distinction between two species, anyhow, reexamination should be expected not only from the mechanism of metal accumulation but also from various biological aspects. ~c~n~wie~~ernents-The author wishes to express his cordial thanks to Dr. T. Numakunai and the stafi; of Asamushi Marine Biological Station of Tohoku Universitv for nroviding many conveniences and to Professor Z.*Hoshino of Iwate University and Professor Emeritus T. Tokioka of Kyoto University for their valuable advice and encouragement throughout the course of the study. Thanks are also due to the staff of the Institute for Atomic Energy, Rikkyo University for their technical suggestions. This study was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan (56740303, 56104008).
REFERENCES
Allen R. J. L. (1940) The estimation of phosphorus. Biuthem. J. 34, 858-865. Bielig H. J., Pfleger K., Rummel W. and Seifen E. (1961) Aufnahme und Verteilung von Vanadin bei der Tunicate Ciona intestinalis I... Hoppe-Seyler *s Z. physiol. Chem. 326, 249-258. Biggs W. R. and Swinehart J. H. (1976) Vanadium in selected biological systems. In &feral Ions in Biological Sysfems, Vol. 6 (Edited by Sigel H.), pp. 141-196. Marcel Dekker, New York. Botte L., Scippa S. and de Vincentiis M. (1979) Content and ultrastructural localization of transitional metals in ascidian ovary. Dev. Growth Differ. 21, 483-491. Carlisle D. B. (1958) Niobium in ascidians. Narure, Lo&. 181, 932-933. Carlisle D. B. (1968) Vanadium and other metals in ascidians. Proc. R. Sot. R 171, 31-42.
288
HIT~SHI MICHIBATA
Ciereszko L. S., Ciereszko E. M., Harris E. R. and Lane C. A. (I 963) Vanadium content of some tunicates. Camp. Biochem. Phvsiol. 8, I 31- 140. Goldberg E. D., McBlair W. and Taylor K. M. (1951) The uutake of vanadium bv tunicates. Biol. Bull. 101, 84-94.’ Hoshino Z. and Tokioka T. (1967) An unusually robust Cionu from the northeastern coast of Honsyu island. Japan. Pub& Seio mar. bid. Lab. 15, 275-291. Kustin K., Ladd K. V. and McLeod G. C. (1975) Site and rate of vanadium assimilation in the tunicate Cionu intestinalis. J. gen. Physiol. 65, 315-328. Macara I. G,, McLeod G. C. and Kustin K. (1979) Tunichromes and metal ion accumulation in tunicate blood cells. Cotnp.B&hem. Physiol. 638, 299-302. Nakamura M. (1950) Calorimetric determination of phosphorus. Bull. ugric. them. Sot. Jupn 24, I-5.
Noddack I. and Noddack W. (1939) Die Hlufigkeiten det Schwermetalle in Meerestieren. ,4rk. Zool. 32.4, l--35. Papadopoulou C. and Kanias G. D. (1977) Tunicate species as marine pollution indicators. Mar. Polltrt. Rtdl. 8. 229-23 I. Pisani, A. and Rengel D. (1972) Comparative observation5 about self-fertilization in Cicmu inie.~iin~/i.~ and Cirmt ~ob~sza in Mar de1 Plats (Argentina). Acfa ~~~~~~~~~.F;Y~J. 3, 323-332. Swinehart J. II.. Biggs W. R.. Halko D. J. and Schroeder N. C. (1974) The vanadium and selected metal contents of some ascidians. Biol. Bull. 146, 302--312. Vinogradov A. P. (1934) Distribition of vanadium in organism. C.r. Acuctd. Sri. C~.R.S.S. 3, 454459. Webb D. A. (1939) Observations on the blood of certain ascidians. with special reference to the biochemistry of vanadium. J. elcp. Bioi. 16, 499.-523.
0300-9629:X4 $3.00+O.OO
,
Printed in Great B&in
COMPARATIVE STUDY ELEMENTS IN THE CIONA INTESTINALIS
1984 Pergamon Press Ltd
ON AMOUNTS OF TRACE SOLITARY ASCIDIANS, AND CIONA ROBUSTA
HITOSHI MICHIBATA Biological Institute, Faculty of Science, Toyama University,
Toyama 930. Japan
(Receired 30 August 1983) Abstract-l. The closely related ascidian species, C. intestinalis and C. rohusta have different lation patterns of metals. 2. The former had a higher amount of vanadium and lower amounts of iron and manganese tissues than the latter. 3. However, there was little difference in nitrogen and phosphate contents.
INTRODUCTION A new species of ascidian was first recognized and distinguished from C’ion~l intestinalis (L) by Hoshino and Tokioka (1967). According to their findings, the usually known cosmopolitan C. intestinalis has a very soft test, the surface of which is quite free of any foreign materials. Its mantle is more or less sprinkled with orange pigment spots that are especially dense near the tips of the siphons. The test of the other type, named C. robusta (Hoshino and Tokioka, 1967), however, is much thicker and harder and sometimes even harbours other organisms on its surface. Its mantle is scarcely pigmented. Moreover, the posterior end of the endostyle of C. intestinalis continues nearly straight to the retropharyngeal groove while that of C. robustu is curved toward the right dorsal side. The authors also indicated that the two species differ in the shape of the epicardium. Subsequently, Pisan6 and Rengel(1972) confirmed that the new species, C. robusta, was different from C. intestinalis due to the fact that in C. robustu self-fertilization took place in 60-100% of the cases, while in C. intestinalis, self-fertilization never occurred. On the other hand, it is well known that the ability of many species of ascidians to concentrate vanadium from sea water is one of the biochemical peculiarities which distinguish the ascidians from other classes of animals. Advancing this point of view a step further, Carlisle (1968) and Swinehart et al. (1974) proposed that the level and distribution of vanadium and other metals in ascidians were related to ascidian phylogeny. Biggs and Swinehart (1976) also pointed out that the concentration of vanadium in the tissues of ascidians was somewhat specific to the species. As it seemed interesting to find whether or not the amounts of trace elements contained in the tissues differ and whether these amounts are specific to species, in the present experiment, the vanadium, iron and manganese in some tissues of C. intestinalis and C. robustu were analysed mainly by neutron activation analysis. For the analysis of these trace elements, this may be the preferred technique since the sensi-
accumuin most
tivity of this method is much higher than that of atomic absorption spectrometry or spectrographic analysis.
MATERIALS AND METHODS Specimens of the solitary ascidian, Cionu intes/inu/is (L) were collected at Asamushi Marine Biological Station on the Bay of Mutsu, Japan. Specimens of C. rohusta were also collected at Asamushi and at Onagawa Fisheries Laboratory of Tohoku University. They were identified by Dr. T. Numakunai of Tohoku University. Not less than five specimens of each species were used in the present experiment. Each specimen was cleaned of extraneous materials. Blood was obtained via heart puncture, then plasma and corpuscles were separated by centrifuging the blood for 10 min at 3000 rpm. The test, mantle, stomach, gonad and branchial basket were rinsed three times with filtered sea-water. All tissues were carefully dried in procelain crucibles in a drying oven at 110 C to constant weight. Each sample, about 20 mg in dry weight. which was enough to analyse metals by means of the neutron activation analysis, was ashed in a muffle furnace at 5OO‘C for 24 hr. The ash was dissolved in 5.0 ml of 0. I N HNO, and put into a polyethylene capsule. For applying neutron activation analysis to measuring the amounts of vanadium and manganese, each sample was irradiated with thermal neutrons (thermal neutron flux: 5 x 10” n/cm’ set ‘. 2 min) in a TRIGA MARK II nuclear reactor at Rikkyo University. The radioactivity of j2V produced in the irradiated sample was measured with a 50-cm3 Ge(Li) gamma-ray spectrometer (Canberra Inc.) 2 min after the irradiation. Sixty min after the irradiation, the radioactivity of ‘“Mn was measured in the same manner as described above in order to avoid the interference of “Mg produced in the irradiated sample. The amounts of vanadium and manganese were determined by comparing the gamma-ray spectrograms with those of standard samples. After the radionuclides were allowed to decay to a negligible concentration. a part of each sample was submitted to analysis for iron by atomic absorption spectrometry (equipment: Hitachi GA-2 flameless atomic absorption spectrometer). The absorption line used was 3719.9nm. Besides the analysis of transition metals, a part of each dried sample was analysed for its nitrogen content bv the method of micro-Kjeldhal and for its phosphate content by Nakamura’s method (1950), which was a modification of the method of Allen (1940).
286
HITOSHI MICHIBATA Table
I. Metal
concentrations
m tiswcs
of the sohtary robusra
C.
ascldums.
Metal concentratmn (ng:mg c‘. inrrstinuli\
(’
irtrestmoli.s and
dry weight)
(‘. roi7u.w I .7 I 0.4
Metal
Tissue
Vanadium
TUIiC Mantle Stomach Gonad Branchial basket Biood cells Plasma
1.2 * 0.5 333.0 i 39.7 507.4 + 69.6 143.6 + 23.9 1037.5 7 109.2 1577.1 f 338.2 12.9 If- 0.4
IroIl
Tunic Mantle Stomach Gonad Branchial basket Blood cells Plasma
8i.h * 6 5 54.8 + 7.3 58.8 _+ 8.0 54.7 2 5.3 I73 8 k 24.3 7x.3 * 5.1 119.6 * 4.8
370.0 2 39.4 I-%?+ Il.4 2% 7 + 69.0 65 x + x.9 251.4127h 424.4 * 49.4 89 s i: 3.8
MatlgdtIW
Tunic Mantle Stomach Gonad Branchial basket Blood cells Plasma
4.8 * 7.4 * 20.2 t 13.2 + 17.3 * 107.6 * 15.4+
18X.8 :k 76.1 29.3 i_ 5.4 36.4 I x. I 77.0 & 14.9 69.‘) * 7.3 1..4*9.0 9.2 _+ 1.Y
Mean I standard II = 5.
1.5 0.9 3.1 1.1 0.8 13.x I9
33x.4 163.2 100.9 337.5 330.7 0.4
i: 23.x ?_ 14.6 + 7.5 ; 47 H 2 14.1 f: 0.2
error.
RESULTS
Metal concentrations detected in the various tissues are shown in Table 1. Metals in trace levels were found in all tissues examined. The vanadium content in each tissue was significantly higher in C. intestinalis than in C. robusta except in mantle. The highest value (1577.1 ng/mg dry weight) for vanadium was obtained from the blood cells of C. intestinufis. This value was about 5 times that in C. robusta blood cells. The leveis of iron found in most tissues of C. robusta were conversely higher than in those of C. i~testi~ul~s, The iron was present at the highest level of 424.4ng/mg dry weight in the blood cells of C. robusra. Relatively high concentrations of manganese occurred in the tissues of C. robusta but the levels in the blood cells and plasma were higher in C. intestinalis.
Nitrogen and phosphate were analysed as representatives of essential elements. In the contrast to the trace metals? there was little difference in the quantitative contents of these elements in the various tissues between the two species (Table 2).
DISCUSSION
Webb (1939) first pointed out the correlation of the presence of vanadium in ascidians with certain evolutionary traits of the class. He also suggested that the presence of vanadium was a primitive character which had been lost in the more specialized families. Many investigators, thereafter, analysed the metal content in ascidians. In general, several species in the order Aplousobranchia have a large vanadium content, among them the genera Eudistoma and Distaplia, and significant vanadium was found in the order Phlebobranchia. In contrast, some species in the order Stolidobranchia contain a small concentration of vanadium but the iron content is large (cf Swinehart et af., 1974; Biggs and Swinehart, 1976).
The present results have revealed that the closely related species, C. intestinalis and C. rohustn belonging to the order Phlebobranchia had different accumulation patterns of metals. Little difference between the two species was observed in the amounts of nitrogen and phosphate contained in each dried tissue (Table 2), while it should be noted that the amounts of trace metals were specific to species. Our unpublished observation that the embryo of C. intrstinah accumulates more vanadium and less iron and manganese than the embryo of C. ~~~2~~~~ also supports the above findings. This situation has a precedent. Carlisle (1958) reported that ~~lf~~u/u ~~~~~ttensis belonging to the order Stolidobranchia could be divided into two groups with regard to the metal accumulation pattern; in the population of the south coast of Devon about two-thirds of the Molugulu were found to have vanadium at a significant level but no niobium, whereas the remaining animals contained niobium at a detectable level but had only a trace of vanadium. Therefore, unless more species are submitted to analysis for their metal contents. we cannot conclude that the presence of several metals in ascidians reflects ascidian phylogeny if the trends are right. Taking another look at papers on trace elements. we become aware that there is considerable variation in the vanadium, iron and manganese contents of C intestinalis. The highest value of vanadium in blood cells was reported by Hielig et al. (1961) who found it present at 1.500ng/mg of the dry weight. Macara CJI al. (1979) found 2400 and 700 ng/mg of vandium in the body and blood cells, respectively. The whole animal also contained a very large amount of vanadium (1300 ng/mg of dry weight; Vinogradov, 1934). Ciereszko et al. (1963) determined a low value of vanadium (40 ng/mg of organic dry weight) in the siphon tips of C. intest~n~lis, and Goldberg rt crl. (1951) reported that vanadium was undetectable in the tunic, ovary, heart, siphon and branchial basket.
V, Fe and Mn in ascidian Table 2. Concentrations
_“.“.
--
287
of nitrogen and phosphate in tissues of the solitary ascidians, C. Concentration
Element
_._.-
tissues
Tissue __.
&g/m8 dry weight)
C. intestinalis
_____i_ ..,.... -
C. robusta
Nitrogen
Tunic Mantle Stomach Gonad Branchial basket Blood cells Plasma
6.1 rt 0.6 48.2 & 3.5 64.4 * 2.3 55.2 rt: 1.5 NA* 28.4 +- 2.2 2.9 It 1.2
II.1 + 1.5 49.0 f 0.7 63.2 + 2.2 60.4 + 1.9 NA* 30.6 t 1.5 2.6 * 0.8
Phosphate
Tunic Mantle Stomach Gonad Branch% basket Blood cells
l.OiO.1 18.8 + 0.6 43.8 rt 2.6 43.2 rt 3.2 32.9 I 2.4 24.7 It 3.4 12.1 i4.8
I.1 f 0.2 17.8 + 1.2 38.8 f I .3 55.2 & I .6 29.3 + 2.6 37.9 * 0.3 8.7 f 0.2
PlaSSXi
Mean + standard error. *NA = not analysed. n = 5.
Many other data ranging from 10 ng/mg to hundreds of ng/mg of dry weight have been reported (Noddack and Noddack, 1939; Webb, 1939; Carlisle, 1968; Swinehart et al., 1974; Kustin et al., 1975; Botte et al., 1979). Concerning the content of iron, Bielig et al. (1961) reported that C. intestinalis contained 800 ng/mg dry weight of iron in the blood cells and 60 ng/mg in the plasma. Using an argon plasma emission spectrometer, Macara et al. (1979) found 350 and 40ng/mg dry weight of iron in the blood cells and plasma, respectively. However, a lower value of iron (50 ng/mg) was detected in the blood cells by Swinehart et al. (1974). The work of Papadopoulou and Kanias (1977) by neutron activation analysis revealed that the whole body, tunic and the rest of the body contained 880, 610 and 3OOOng~mg of iron, respectively. Botte CTaf. (1979) found 52 ng/mg of iron in the ovary tissue. In the case of manganese a similar diversity of analytical data has been reported. Noddack and Noddack (1939) reported that C. ~ntest~nal~s contained this element at a level of 120 ng/mg of dry weight. Carlisle (1968) found a level of about 95 ng/mg. Swinehart et al. (1974), however, reported that the manganese content in blood was less than 10 ng/mg. Botte et al. (1979) reported that manganese was undetectable in the ovary of this species by fiameless atomic absorption spectrometry. The cause of the diversity may be attributed to several possibilities. One of them may be advances in the analytical methods; the data obtained has become more accurate with the years. Generally speaking, the older data determined by emission spectrometry or calorimetry have higher contents of trace elements and less accuracy compared with more recent data determined by atomic absorption spectrometry, argon plasma emission spectrometry or thermal neutron activation analysis. It seems, therefore, the data extending over about 70 years are not comparable. The most possible cause is the confusion between these two species, C. inteJtinalis and C. robusta that have a certain similarity in their external appearance. They must have been indiscriminately analysed for metals. They had been, indeed, recognized as the
single species C. intenstinalis until Hoshino and Tokioka (1967) divided them into two different species. Therefore, the metal contents reported formerly may be divided roughly into two groups. One group has a higher content of vanadium and lower contents of iron and manganese, which probably corresponds to the species C. intest~nai~s. The other group has, on the contrary, a lower content of vanadium and higher contents of iron and manganese, which probably corresponds to the newly named C. robusta. On a distinction between two species, anyhow, reexamination should be expected not only from the mechanism of metal accumulation but also from various biological aspects. ~c~n~wie~~ernents-The author wishes to express his cordial thanks to Dr. T. Numakunai and the stafi; of Asamushi Marine Biological Station of Tohoku Universitv for nroviding many conveniences and to Professor Z.*Hoshino of Iwate University and Professor Emeritus T. Tokioka of Kyoto University for their valuable advice and encouragement throughout the course of the study. Thanks are also due to the staff of the Institute for Atomic Energy, Rikkyo University for their technical suggestions. This study was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan (56740303, 56104008).
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