THE TRANSITION FROM TADPOLE TO FROG HAEMOGLOBIN DURING NATURAL AMPHIBIAN METAMORPHOSIS

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1 J. Cell Sci. 16, (i974) 143 Printed in Great Britain THE TRANSITION FROM TADPOLE TO FROG HAEMOGLOBIN DURING NATURAL AMPHIBIAN METAMORPHOSIS II. IMMUNOFLUORESCENCE STUDIES J. BENBASSAT Department of Medicine A, Hadassah University Hospital, Jerusalem, Israel SUMMARY Rabbits were immunized with frog or tadpole haemoglobin purified either by chromatography or by polyacrylamide gel electrophoresis. The obtained rabbit antisera were shown to be specific for frog or tadpole haemolysates by double diffusion, immunoprecipitation and immunonuorescence. Indirect immunofluorescent staining of peripheral blood smears of Rana catesbeiana tadpoles at the metamorphic climax revealed that 16 % of the red cells were stained with antibodies against frog haemolysates, while almost all of them (98 %) were stained with antibodies against tadpole haemolysates. These results are compatible with the possibility that some of the circulating red cells in metamorphosing tadpoles contain both tadpole and frog haemoglobins. INTRODUCTION During amphibian metamorphosis, the transition from tadpole to frog haemoglobin is associated with the appearance of a poorly haemoglobinized, morphologically immature microcyte in the circulation (Moss & Ingram, 1968; Vankin, Brandt & DeWitt, 1970), which incorporates actively precursors of protein and nucleic acid synthesis (Moss & Ingram, 1968; Benbassat, 1970; Thiel, 1970), and which contains frog haemoglobin (DeWitt, 1968). These findings indicate that the switch from tadpole to frog haemoglobin involves a replacement of the tadpole erythrocytes by a population of cells containing frog haemoglobin. The question, whether these 2 populations belong to different clones or to the same cell line is subject to controversy. Human embryonic and foetal haemoglobins (Kleihauer, Tang & Betke, 1967) as well as human foetal and adult haemoglobins (Betke & Kleihauer, 1958; Dan & Hagiwara, 1967; Gitlin, Sasaki & Vuopio, 1968) may coexist in the same cell. There is also evidence that frog and tadpole haemoglobins are present simultaneously in some of the erythrocytes of metamorphosing and adult anaemic Xenopus laevis (Jurd & Maclean, 1970; Maclean & Jurd, 1971). On the other hand, however, ithasbeen found that red cells of metamorphosing Rana catesbeiana tadpoles contain either tadpole or frog haemoglobins but not both (Rosenberg, 1970; Maniatis & Ingram, 19716). This paper reports the results of immunofluorescence studies of the content of individual erythrocytes of Rana catesbeiana tadpoles and frogs by means of specific rabbit antibodies against tadpole and frog haemolysates. It is shown that during

2 144 J- Benbassat metamorphosis some of the circulating red cells are stained by both types of antisera. This rinding is consistent with the possibility that tadpole and frog haemoglobins are produced by erythroid cells belonging to the same red cell line. METHODS The source of the animals and the methods for blood collection, preparation of labelled haemolysates, polyacrylamide gel electrophoresis, reduction and alkylation of red cell proteins and electrophoresis in 8 M urea have been described previously (Benbassat, 1974). Preparation of the immunogen Red cells oirana catesbeiana adult frogs and premetamorphic tadpoles (total length mm) were washed 3 times with amphibian Ringer solution (Rugh, 1962). The visible buffy coat was removed after each centrifugation, and the washed cells were lysed in 10 volumes of M MgClj. The stroma, nuclei and the ribosomes were removed by centrifugation at g for 90 min at 5 C. About 2 ml of the ribosome-free supernatant were applied to a column of CM-cellulose (Serva) and washed through with o-oi M sodium phosphate buffer (ph 70) until the eluate was clear and without absorbance at 280 nm. Then the haemoglobin was eluted with 0-5 M Na 2 HPO 4 (ph 95), and used as immunogen. Immunization One ml of the eluted haemoglobin solution containing about 1 mg of the immunogen was mixed with complete Freund's adjuvant (Difco Laboratories, Detroit, Michigan), thoroughly emulsified and injected at different sites in the footpads and skin of adult New Zealand white rabbits. In some experiments the immunogen consisted of the major tadpole or frog haemoglobin fractions obtained by polyacrylamide gel electrophoresis (Benbassat, 1974). The haemoglobin bands were cutout, thoroughly homogenized as described by Maniatis& Ingram (1971a), and injected into the rabbits; 4 injections were given at 2- to 3-week intervals. One week after the last injection, the rabbits were bled by cardiac puncture. The sera were separated and kept frozen until used. Immunoprecipitation of labelled haemolysates 14 C-labelled haemolysates, prepared as detailed previously (Benbassat, 1974) from tadpole or frog erythrocytes, were incubated at 4 C for 48 h with antisera prepared as described in Immunization (above). After incubation the mixtures were spun for 10 min at 5000J* in a refrigerated centrifuge. The obtained precipitate was washed 3 times in cold Ringer's solution, dissolved in formic acid and mounted on planchets for determination of radioactivity. In other experiments, the immunoprecipitates were dissolved in deionized 8 M urea, reduced, alkylated and electrophoresed on polyacrylamide gels prepared in alkaline 8 M urea. All the immune precipitations were carried out at antibody excess as judged by standard immune precipitation curves (Fig. 6). Staining of tadpole and frog blood cells with fluorescent antibodies The indirect method of Weller & Coons (1954) was used. Air-dried smears were prepared from washed blood cells of early premetamorphic tadpoles, animals at the metamorphic climax, and adult frogs, and left at 20 C until used. The smears were covered with antisera against tadpole or frog haemoglobins and incubated for 30 min at 37 C with continuous shaking. Serum of non-immunized rabbits was used in control experiments. The coated smears were then washed 3 times with amphibian Ringer's solution and covered for 30 min with Fluorescein Conjugated Goat anti (Rabbit IgG) globulin (Microbiological Associates, Inc., Bethesda) diluted 1:60 in amphibian Ringer's solution. They were then washed again, dried and examined in u.v. illumination under glycerol buffered to ph 70 with sodium phosphate. Cell counts were performed from photographs of the smears.

3 RESULTS Synthesis of red cell protein during metamorphosis. II 145 Determination of the specificity of the antisera The characterization of the antisera was performed by double diffusion on agar, immunoprecipitation and characterization of the precipitated material by polyacrylamide gel electrophoresis in 8 M urea, and immunofluorescence. Double diffusion on agar. Agar-gel diffusion plates were used. The reagents consisted of undiluted antisera and of tadpole or frog haemoglobin solutions. The haemoglobin solutions consisted of either unfractionated haemolysates or purified major haemoglobin fractions after separation by polyacrylamide gel electrophoresis. Sera of rabbits immunized with tadpole haemolysates purified by chromatography produced one precipitin line when tested against haemolysates of prometamorphic tadpoles, animals at the metamorphic climax or against the purified major tadpole haemoglobin (T-J; the same antisera did not react when tested against frog or mouse haemolysates (Fig. 4). Sera of rabbits immunized with the purified major tadpole haemoglobin fraction (Tj), produced one precipitation line when tested against unfractionated tadpole haemolysates, purified major tadpole haemoglobin (T x ) and the purified minor tadpole haemoglobin fraction (T 2 ). The type of reaction obtained suggested an identity between the antigens (Fig. 5). Rabbit antisera against frog haemoglobin reacted with unpurified haemolysates of animals during the metamorphic climax and of adult frogs, as well as with the purified frog haemoglobin fractions (F x and F 3 ). One precipitation line was observed in all cases. The same antisera did not react when tested against tadpole or mouse haemoglobins (Figs. 6, 7). These results confirm the findings by Maniatis & Ingram (1971a) and by Jurd & Maclean (1970), that frog and tadpole haemoglobins are relatively strong immunogens for the rabbit. The absence of any detectable cross-reaction between these haemoglobins is consistent with the findings by Baglioni & Sparks (1963), Moss & Ingram (1968) and Aggarwal & Riggs (1969), that the transition from tadpole to frog haemoglobin involves the beginning of synthesis of a completely different set of polypeptide chains. The major tadpole haemoglobin (Tj) cross-reacted with the minor tadpole haemoglobin (T 2 ), while the major frog haemoglobin (F 3 ) cross-reacted with the minor one (Fj); this could indicate that these haemoglobin fractions share one or more common subunits. Haemolysates of animals at the metamorphic climax reacted with antisera against either tadpole or frog haemoglobin; this is in agreement with the finding that tadpoles at this stage of metamorphosis contain both types of haemoglobin (Fig. 8). Immunoprecipitation. The amount of antiserum needed to provide an excess of antibodies in the reaction mixtures was determined by standard immune precipitation curves. In these experiments a constant amount (3-4 /tg) of 14 C-labelled haemoglobin was titrated with increasing volumes (5-500 /A) of antiserum. The relative amount of antiserum which precipitated the maximal amount of radioactivity was used in further experiments (Fig. 1). In three different experiments, 76, 83 and 89% of the total radioactivity of IO C EL 16

4 146 J. Benbassat it\ antiserum added Fig. 1. Immunoprecipitation of tadpole (solid line) and frog (dashed line) 14 C- labelled haemolysates by rabbit anti-tadpole hb and anti-frog hb antisera, respectively. The indicated amount of antiserum was incubated for 48 h at 4 C C with a constant amount of 14 C-labelled haemolysate containing 3 fig haemoglobin. After incubation the immunoprecipitate was washed, dissolved in formic acid and the radioactivity determined in a Nuclear Chicago Gas Flow Counter. Antigen Table 1. Immunoprecipitation of tadpole and frog labelled haemolysates TT globin, /*g TCAprecipitable cpm Anti-T* cpm precipitated by Anti-F* Control serum* Unfractionated (100%) 356(56%) 14 (3 %) 10(2%) tadpole haemolysatglobin (Tj) Unfractionated 4i 318(100%) frog haemolysate Purified major (100%) tadpole haemo- 82(60%) 21(6%) 6(4%) 240 (76 %) 7 (5 %) 24(7%) Purified major 2-O 112(100%) 5(4%) 79(7O%) 3 (3 %) frog haemoglobin (F 3 ) * Anti-T and anri-f refer to non-absorbed rabbit antisera against tadpole or frog haemolysates purified by chromatography on CM-cellulose. The control consisted of the serum of a non-immunized rabbit.

5 Synthesis of red cell protein during metamorphosis. II 147 unfractionated frog haemolysates could be precipitated by antisera against frog haemoglobin. The highest amounts of radioactivity, which could be precipitated by antisera against tadpole haemoglobin from unfractionated haemolysates were 56, 53 and 38%. Similar results were obtained when the purified major haemoglobin fractions were used as antigens in the reaction mixture. The amounts of tadpole haemoglobin precipitated by antibodies against frog haemoglobin and vice versa did not exceed those precipitated by sera of non-immunized rabbits. The results of a representative experiment are given in Table 1. In order to confirm that the antisera reacted with haemoglobin and not with any other red cell protein, which might have contaminated the immunogen, the immunoprecipitated material was characterized by polyacrylamide gel electrophoresis. For this purpose, immunoprecipitated haemolysates were dissolved in 8 M urea, in order to dissociate between the antibody and the labelled antigen. Possible S S bonds were reduced by the addition of mercaptoethanol and blocked by amidation. Then the dissociated protein subunits were resolved by electrophoresis on polyacrylamide gels prepared in alkaline 8 M urea. After electrophoresis the gels were sliced and the radioactivity was determined in each of the gel fractions. The radioactive profiles of the immunoprecipitated material were compared to those of the reduced and alkylated purified major haemoglobin components. As shown in Figs. 2 and 3, the purified major haemoglobin components of both tadpoles and frogs were resolved into one major and several smaller peaks of radioactivity (Figs. 2C and 3 c). The major peak consisted of both globin chains, which could not be separated electrophoretically under these conditions. The smaller peaks represented probably small amounts of contaminating red cell (haemoglobin or non-haemoglobin) protein. The pattern of resolution of the immunoprecipitated protein of frog haemolysates was almost identical with that of reduced and alkylated globin prepared from the purified major frog haemoglobin fraction (Fig. 2). The immunoprecipitated protein of tadpole haemolysates resolved into several peaks of radioactivity, one of which coelectrophoresed with the major tadpole reduced and alkylated globin (Fig. 3). The nature of the remaining peaks of immunoprecipitated radioactivity remains unclear: they could be globin subunits of the minor tadpole haemoglobins, or non-haemoglobin red cell proteins. Similar results were obtained whether the immunogen employed in the preparation of the antisera was purified by chromatography or by polyacrylamide gel electrophoresis. Immunofiuorescence. The antisera against frog or tadpole haemoglobins characterized in the two preceding sections, were used for immunofluorescent staining after cross-absorption with tadpole and frog lysed red cells, respectively. The degree of specificity of the antisera employed is shown in Figs and in Table 2. When peripheral blood smears of premetamorphic tadpoles were stained with antisera against tadpole haemoglobin, 98-7 % of the cells showed a brightfluorescence,which was evenly distributed throughout the cytoplasm; the nucleus was not stained (Fig. 9). Only occasional cells (less than 2 %) were stained when tadpole blood smears were treated with antisera against frog haemoglobin (Fig. 10), normal rabbit serum or antiserum against tadpole haemoglobin, which had been absorbed with tadpole haemoglobin.

6 148 J. Benbassat When frog peripheral blood cells were stained with antisera against frog haemoglobin, 99-2 % of the cell showed a bright fluorescence, which was most prominent around the nucleus; the remaining parts of the cytoplasm and the nucleus appeared unevenly stained (Fig. 11). Less than 3% of the cells were stained when frog blood k E Q A ) c / 10 li ft Fraction no. Fig. 2 I Fraction no. Fig. 3 i i i Figs. 2, 3. Electrophoresis of I4 C-labelled reduced and alkylated globin in alkaline 8 M urea. Immunoprecipitation, reduction and alkylation of the immune precipitates, electrophoresis and fractionation were carried out as previously described (Benbassat, ) Fig. 2. A, untreated frog haemolysates; B, immune precipitates of frog haemolysates treated with antibodies against frog haemoglobin; c, purified major frog haemoglobin (F 3 ). Origin to the left, migration towards the anode. Fig. 3. A, untreated tadpole haemolysates; B, immune precipitates of tadpole haemolysates treated with antibodies against tadpole haemoglobin; c, purified major tadpole haemoglobin (T^). Origin to the left, migration towards the anode. smears were treated with antisera against tadpole haemoglobin (Fig. 12) or with control sera. Imtnunofluorescent staining of peripheral blood cells of animals at the metamorphic climax The results of the immunofluorescent staining of the peripheral blood cells of animals 40 40

7 Synthesis of red cell protein during metamorphosis. II 149 at the metamorphic climax are given in Table 2 and Figs. 13 and 14. Of these cells, 98'1 % exhibited a bright fluorescence after treatment with antisera against tadpole haemoglobin. The pattern offluorescenceof the cells was identical with that of tadpole red cells stained with antisera against tadpole haemoglobin (Fig. 13). In contrast to the homogeneity of the smears stained with antisera against tadpole haemoglobin, smears treated with antibodies against frog haemoglobin exhibited a mosaic-like pattern of 16% positively stained cells (Fig. 14). Table 2. Staining of frog tadpole peripheral blood smears with fluorescent antibodies Total no. of % cells showing Stage Antiserum used* cells counted fluorescence Premetamorphosis Anti-tadpole Hb Premetamorphosis Anti-frog Hb Metamorphic climax Anti-tadpole Hb Metamorphic climax Anti-frog Hb Adult Anti-tadpole Hb Adult Anti-frog Hb The antisera were prepared by immunizing rabbits with frog or tadpole haemolysates purified by chromatography on CM-cellulose. DISCUSSION The degree of specificity of the antisera employed was determined by double diffusion on agar, immunoprecipitation of labelled haemolysates and fluorescent staining of peripheral blood smears. The amounts of labelled tadpole and frog haemoglobin, precipitated by antisera against frog and tadpole haemoglobin, respectively, did not exceed those precipitated by control sera of non-immunized rabbits. Less than 3 % of the tadpole or frog blood cells were stained with antisera against frog or tadpole haemoglobin, respectively, while more than 98 % of the cells in tadpole and frog peripheral blood smears were stained with antibodies against the haemoglobin of the same developmental stage. Therefore, the finding that 16% of the peripheral blood cells of metamorphosing animals were stained with antisera against frog haemoglobin, while almost all of them were stained with antisera against tadpole haemolysates, suggests the coexistence of both types of haemoglobin in some of the erythrocytes. These results conform with the evidence presented by Shukuya (1966) and by Jurd & Maclean (1970) that some of the erythroid cells of metamorphosing tadpoles contain both tadpole and frog haemoglobins. The presented observations are consistent also with the coexistence of human embryonic and foetal haemoglobins (Kleihauer et al. 1967) and of human foetal and adult haemoglobins (Dan & Hagiwara, 1967) within the same erythrocyte. They are at variance, however, with the conclusions of Rosenberg (1970) and Maniatis & Ingram (19716), that during metamorphosis the erythroid cells carry either frog or tadpole haemoglobin but not both. The reason for this inconsistency is uncertain. One possibility could be, that the

8 150 J.Benbassat antisera against tadpole and frog haemoglobins employed by Maniatis & Ingram (1971 a) may have had a higher degree of monospecificity than those used in the present investigation. As shown in Fig. 3, treatment of tadpole haemolysates with antisera against tadpole haemoglobin resulted in the precipitation of several protein subunits in addition to the main tadpole globin; this additional immunoprecipitate could have consisted either of minor tadpole globin fractions or of non-haemoglobin red cell proteins. It is possible, therefore, that the antisera against tadpole haemoglobin employed in the present study were not monospecific. Thus, the antigen involved in the immunofluorescent staining of the red cells with antisera against tadpole haemoglobin, could have been a non-haemoglobin protein, present in the red cells of premetamorphic and metamorphosing tadpoles, but not in mature frog erythrocytes. Alternatively, the discrepancy in results could be explained by differences in the sensitivity of the methods employed for immunofluorescent staining. The indirect 'sandwich' method for immunofluorescent staining employed in this paper is more sensitive than the direct 'double label' method used by Maniatis & Ingram (1971). Conceivably, small amounts of tadpole haemoglobin in a cell containing principally frog haemoglobin may have escaped detection by direct immunofluorescent staining, particularly when applied to cells already pretreated with fluorescent antibodies against frog haemoglobin. I am grateful to Dr I. M. London for advice and encouragement and to Dr S. Baum for help in the staining with fluorescent antibodies. This investigation was supported by a Public Health Service International Research Fellowship (FOTW5-1224) while the author was a postdoctoral fellow in the Department of Medicine, Albert Einstein College of Medicine, New York, and by a grant from the Joint Research Fund of the Hebrew University and Hadassah. REFERENCES AGGARWAL, S. J. & RIGGS, A. (1969). The hemoglobins of the bullfrog, Rana catesbeiana. I. Purification, amino acid composition and oxygen equilibria.^, biol. Chem. 244, BAGLIONI, C. & SPARKS, C. E. (1963). A study of hemoglobin differentiation in Rana catesbeiana. Devi Biol. 8, BENBASSAT, J. (1970). Erythroid cell development during natural amphibian metamorphosis. Devi Biol. 21, BENBASSAT, J. (1974). The transition from tadpole to frog haemoglobin during natural amphibian metamorphosis. I. Protein synthesis by peripheral blood cells in vitro. J. Cell Sci. 15, BETKE, K. & KLEIHAUER, E. (1958). Fetaler und blebender Blutfarbstoff in Erythrozyten und Erythroblasten. Blut 4, 241. DAN, M. & HAGIWARA, A. (1967). Detection of two types of hemoglobin (HbA and HbF) in single erythrocytes by fluorescent antibody technique. Expl Cell Res. 46, DEWITT, W. (1968). Microcytes response to thyroxine administration. J. molec. Biol. 32, GITLIN, D., SASAKI, T. & VUOPIO, P. (1968). Immunochemical quantitation of proteins in single cells. Blood 32, 796. JURD, R. D. & MACLEAN, N. (1970). An immuno-fluorescent study of the haemoglobins in metamorphosing Xenopus laevis. J. Embryol. exp. Morph. 23, KLEIHAUER, E., TANG, T. E. & BETKE, K. (1967). Die intrazellnare Verteilung von embryonalem Hamoglobin in roten Blutzellen menschlicher Embryonen. Ada haemat. 38, 264. MACLEAN, N. & JURD, R. D. (1971). The haemoglobins of healthy and anaemic Xenopus laevis. J. CeU Sci. 9,

9 Synthesis of red cell protein during metamorphosis. II 151 MANIATIS, G. M. & INGRAM, V. M. (1971a). Erythropoiesis during amphibian metamorphosis. II. Immunochemical study of larval and adult hemoglobins of Rana catesbeiana. J. Cell Biol MANIATIS, G. M. & INGRAM, V. M. (19716). Erythropoiesis during amphibian metamorphosis. III. Immunochemical detection of tadpole and frog hemoglobins in single erythrocytes. J. Cell Biol. 49, Moss, B. & INGRAM, V. M. (1968). Hemoglobin synthesis during amphibian metamorphosis. I. Chemical studies on the hemoglobins from larval and adult stages of R. catesbeiana. J. molec. Biol. 32, ROSENBERG, M. (1970). Electrophoretic analysis of hemoglobin and isozymes in individual vertebrate cells. Proc. natn. Acad. Set. U.S.A. 67, RUGH, R. (1962). Experimental Embryology. Minneapolis: Burgess. SHUKUVA, R. (1966). As cited by Frieden, E. in Metamorphosis (ed. W. Etkin & L. I. Gilbert). New York: Appleton-Cenrury-Crofte. THIEL, E. (1970). Red blood cell replacement during the transition from tadpole to frog hemoglobin in Rana catesbeiana. Comp. Biochem. Physiol. 33, VANKIN, G. L., BRANDT, E. M. & DEWITT, W. (1970). Fine structure of erythroid cells during thyroxjn-induced metamorphosis of bullfrog tadpoles. Am. Zool. 10, 321. WELLER, T. H. & COONS, A. H. (1954). Fluorescent antibody studies with agents of Varicella and Herpes Zoster propagated in vitro. Proc. Soc. exp. Biol. Med. 86, {Received 15 February 1974)

10 is* J. Benbassat Figs Double diffusion in 1 % agarose. The reagents consisted of undiluted rabbit antisera (central wells) and haemoglobin solutions in concentrations varying between O-O2-O-3 mg/ml (peripheral wells). Central wells: Figs. 4 and 5, rabbit anti-tadpole hb serum; Figs. 6 and 7, rabbit antifrog hb serum; the antisera in Figs. 4 and 6 were prepared by immunization of rabbits with haemolysates purified by chromatography on CM-cellulose; the immunogen used for the preparation of the antisera in central wells Figs. 5 and 7 consisted of the purified major hb fraction. Peripheral wells: T, unfractionated haemolysate of a prometamorphic tadpole; Tj, purified major tadpole hb fraction; T 2, purified minor tadpole hb fraction (see Fig. 8); E, haemolysate of an animal at emergence of the front legs (beginning of metamorphic climax); C, haemolysate of an animal at the end of the metamorphic climax; F, haemolysate of an adult frog; F lt purified minor frog hb fraction; F 3, purified major frog hb fraction (see Fig. 8); M, mouse hb.

11 Synthesis of red cell protein during metamorphosis. II 8 A B E C F Fig. 8. Polyacrylamide gel electrophoresis (discontinuous Tris-glycine/Tris-HCl alkaline system) of haemolysates of R. catesbeiana tadpoles and frogs. Unstained gels. A, early premetamorphic tadpole (total length, 35mm, estimated age 2-3 weeks); B, prometamorphic tadpole (total length, 85 mm, hind legs 2 mm, estimated age, at least 1 year); E, tadpole at the beginning of the metamorphic climax (total length, 98 mm, hind legs 45 mm, 2 days after emergence of front legs); C, animal at the end of metamorphic climax (tail almost completely reabsorbed, 12 days after emergence of front legs); F, adult frog.

12 154 J. Benbassat Figs R. catesbeiana tadpoles and frogs. Peripheral blood smears treated with fluorescent antibodies, x 750. Fig. 9. Tadpole blood cells stained with antibodies against tadpole haemoglobin. Fig. 10. Tadpole blood cells stained with antibodies against frog haemoglobin. The fluorescent round body is probably either a white cell or an artifact.

13 Synthesis of red cell protein during metamorphosis. II Fig. II. Frog blood cells stained with antibodies against frog haemoglobin. Fig. 12. Frog blood cells stained with antibodies against tadpole haemoglobin.

14 156 J. Benbassat Figs. 13, 14. R. catesbeiana metamorphosing tadpoles. Peripheral blood smears treated with fluorescent antibodies, x 750. Fig. 13. Stained with antisera against tadpole haemoglobin. Fig. 14. Stained with antisera against frog haemoglobin.