- Email: [email protected]
In vivo oxygenation of hemoglobin in early embryos of Daphnia magna
Comp. Eiochem. Physiol.Vol. 1074 No. 1, pp. 127-131, 1994 Printed in Great Britain
In vivo oxygenation of Daphnia magna Michiyori Kobayashi Department
0
of hemoglobin
0300-9629/94 $6.00 + 0.00 1993 Pergamon Press Ltd
in early embryos
and Yukio Takahashi
of Biology, Faculty of Science, Niigata University, Niigata 950-21, Japan
The concentration of hemoglobin (Hb) in early embryos of Daphnia magna is higher than that in hemolymph of adult Daphnh magna.The oxygen tension in ambient water at which Hb in early embryos was 50% oxygenated was 30 torr in eggs of &poor animals and 17 torr in those of Hb-rich animals. These agreed with the data of in uiuo oxygenation of the Hb in the hemolymph of their respective parents. Hb in early embryos of I-&rich animals required a longer time for deoxygenation than that in early embryos of Hb-poor animals. A longer time was required for early embryos Hb than for adult animals Hb to be deoxygenated in an anoxic environment. Key words: Hemoglobin;
Daphnia magna.
Comp. Biochem. Physiol. 107A, 127-131,
1994.
Introduction The
concentration
of hemoglobin (Hb) in influenced by the concentration of oxygen dissolved in water and undergoes marked variations with the oxygen concentration in a living environment (Fox and Phear, 1953; Hoshi and Kobayashi, 1972; Calvalho, 1984). Hb-poor animals living in water of high oxygen concentration are colourless and Hb-rich animals living in low oxygen concentration are bright red. Daphnia magna usually reproduces by parthenogenesis. It is known that Hb also exists in parthenogenetic eggs and that its concentration varies depending on the concentration of Hb in the parental hemolymph on the concentration of Hb in the parental hemolymph (Teissier, 1932; Fox et al., 1949; Kobayashi and Nezu, 1985). No difference is found between the molecular weight of parthenogenetic egg Hb and that of parental Hb. Purified Hb from parthenogenetic eggs has a similar oxygen affinity to its respective parental Hb (Kobayashi Daphnia magna is acutely
Correspondence to: M. Kobayashi, Department of Biology,
Faculty of Science, Niigata University, Niigata 950-21, Japan. Received 18 February 1993; accepted 26 March 1993.
et al., 1990). Parental Hb is considered to have transferred into the parthenogenetic eggs (Dresel, 1948). The physiological functions of Hb in adult Daphnia magna have been investigated in relation to the amount of oxygen consumption (Kobayashi and Ho&i, 1984) swimming activity in a low oxygen environment (Kobayashi and Gonoi, 1985), oxygen saturation in uivo (Hoshi and Yahagi, 1975; Kobayashi and Tanaka, 1991), etc. for both Hb-poor and Hb-rich animals. Little attention has been directed to the significance of egg Hb of Hb-poor animals. For Hb to demonstrate its function in viva, Hb should not stay saturated with oxygen but should release the oxygen. Absorption spectral changes are induced by the release of oxygen from Hb. Measurement of the absorption spectrum of Hb in living early embryos is possible by means of microspectrophotometry. In this paper, the relationship between the oxygen saturation of Hb in early embryos and the oxygen concentration in environmental water was investigated and compared with the corresponding data for parental Hb in order to characterize the physiological functions of early embryos Hb. 127
128
Michiyori Kobayashi and Yukio Takahashi
Materials and Methods We used female Daphnia magna and their early embryos. Rice bran and Chlorella were provided as food. Hb-rich animals were reared in a low oxygen environment and Hb-poor animals in an air-saturated environment. Of the eight stages of embryonic development of Daphnia magna according to Fox (1948), stages 1 and 2 were used as early embryos. Adults measuring 2.5 mm in body length were used. In determining the Hb content, crushed early embryos or hemolymph was collected on a microslide (0.05 mm in light path) to read the absorbance of light. In vivo oxygenation of Hb To examine the absorption spectrum of Hb in a living body, an adult animal was immobilized by fixing its carapace to the tip of a tungsten wire with instant-drying reagent (Aronalpha, Konishi Co., Osaka), and the animal inserted into the absorption cell. Both sides of the cell were then sealed with glass cover slips by means of Araldite adhesive (Konishi Co.). Embryos were removed from the maternal brood pouch and placed in a 0.3 mm-square mesh of a net in an absorption cell. Water of a known oxygen concentration was allowed to flow into the cell by means of a peristaltic pump at a flow rate of about 2 ml/min. The absorption spectrum was read using a microspectrophotometer (Zeiss UMSP 80). The absorption spectra were measured at the dorsal region of the adult animal and at the center of the early embryo. A spot of monochromatic light of diameter 0.08 mm was illuminated. The hemolymph of a single animal was placed on a microslide (0.05 mm light path), and its absorption spectrum was measured at 400-450 nm with a microspectrophotometer. The Hb concentration was calculated using an oxyHb extinction coefficient of 126.6 mM-’ cm-’ at 414 nm (Hoshi and Kobayashi 1971). The Hb concentration in hemolymph from Hb-rich and Hb-poor animals were 0.92 and 0.25 gHb/lOOmm hemolymph, respectively. The oxygen partial pressure in the ambient water was monitored with an oxygen electrode (Yellow Springs Instrument Co., Ohio). Experiments were carried out at 25°C.
0.3
0.2 !I
% 0.0
0.6
0.4
400
450
550
500
wavelength,
500
nm
Fig. 1. Absorption spectra of Hb in early embryo (a) and parental hemolymph (b) of Hb-poor and those (c, d) for Hb-rich Daphnia magna.
lymph and early embryos. The absorbance should directly reflect the Hb concentration, since the absorption spectrum was read in a microslide of light path 0.05 mm. The figure shows that early embryo from both Hb-poor and Hb-rich animals contain several times as much concentration of Hb as their parental hemolymph. Absorption spectrum of Hb in a living body
Figure 2 shows the absorption spectra of early embryo from Hb-rich animal which was subjected to water of different oxygen concen-
Results and Discussion 510
Concentrations of Hb in early embryos and in parental hemolymph
Figure 1 compares the Hb concentrations from the absorption spectra of parental hemo-
530
170
550
Wavelength,
590
mn
Fig. 2. In uiuo recorded absorption spectra in early embryo removed from Hb-rich Daphnia mama. (a) deoxygenated Hb, (b-e) partially deoxygenated Hb, (f) oxygenated Hb.
In oivo oxygenation of Daphnia Hb
129
Oxygen saturation of Hb in vivo
dp d E
o 100
s
50
0 0
30
oxygen
60
90
pressure,
120
150
torr
Fig. 3. Effect of the ambient oxygen pressure on in uiuo oxygen saturation of Hb in Daphnia magna. Open circles:adult Hb-rich animals, closed circles: adult Hbpoor animals, open squares:early embryos from Hb-rich animals, closed squares:early embryos from Hb-poor animals.
trations. The absorbance was measured at the alpha and beta bands. The oxygen saturation of Hb in early embryo from Hb-rich animals was obtained from the change in absorbance at 562 nm where the absorbance varied greatly (Fig. 2), and that in embryo from Hb-poor animal from the change in absorbance at 420 nm.
L 0
The relationship between the oxygen tension and oxygen saturation of Hb in early embryos is shown in Fig. 3. Deoxygenation of Hb in early embryo from Hb-poor animal (open squares) started at an oxygen tension below 120 torr, and the oxygen tension at which Hb was 50% deoxygenated being approximately 38 torr. Deoxygenation of Hb in early embryo from Hb-rich animals (closed squares) started at a lower oxygen tension (approximately 70 torr). The oxygen tension at which Hb was 50% deoxygenated in early embryo was also lower (approximately 17 torr). These data were similar to those obtained from adult Hbpoor animals (closed circles) and Hb-rich adult animals (open circles) that were collected from the same populations. This agreed with the finding that purified Hb from early embryos and parental Hb has the same affinity for oxygen. Deoxygenation of Hb in early embryos
The time required for early embryo Hb to be deoxygenated by consumption of oxygen by the early embryo itself was measured in a small air-tight vessel filled with anoxic water (Fig. 4A,B). Deoxygenation of Hb was completed in a relatively short time, but Hb in embryo from Hb-rich animals required a little more time for deoxygenation. However, completion of deoxygenation in relatively short times, seems to be attributable to the diminution of dissolved oxygen due to
I
200
400
600
Time,
600
1000
1200
second
Fig. 4. Time courses for a change in oxygen saturation of Hb for Hb-poor embryos (A) and that for Hb-rich embryos (B) of Daphnia magna in anoxic water.
Michiyori Kobayashi and Yukio Takahashi
130
100
400
600
800
1000
Time, second Fig. 5. Time courses for a change in oxygen saturation for early embryos (a) from Hb-poor Duphnia and that for adult animals 2 mm in body size (B). The embryos were initially exposed to anoxic water, then to air- saturated water (arrow a), and finally anoxic water (arrow b).
consumption by early embryos and the resultant utilization of oxygen which was bound to the Hb. This suggests that oxygen in early embryo is rapidly consumed by the Hb contained in the embryo. Changes in oxygen saturation animals and embryos
of Hb in adult
Hb in embryos also required a longer time for oxygenation and deoxygenation in both air-saturated water and anoxic water compared with adult animal Hb (Fig. 5A,B). The longer time required for deoxygenation of Hb in early embryos results from a higher concentration of Hb and the fact that oxygen is not circulated in early embryos due to the lack of a respiratory organ which exists in adult animals. Earlier studies comparing the rates of hatching between Hb-poor and Hb-rich animal eggs in low oxygen water have reported that eggs from Hb-rich animals could hatch in water of lower oxygen concentration (Kobayashi et al., 1987). However, no reference has been made to the reason why parthenogenetic eggs contain a higher concentration of Hb than their parents. When mature, Daphnia magna have respiratory
organs, such as gills, so that when exposed to oxygen depletion in water they are capable of responding to their own demand for oxygen by floating up to the water surface which abounds in oxygen. Embryos develop in a brood pouch and are thus sub-jetted environmentally to the same oxygen conditions as surround their parents. A great number of parthenogenetic eggs are laid in a parental brood pouch. The presence of as many as 108 eggs in a single brood pouch has been reported (Green, 1955). It is presumed that the oxygen environment in such a brood pouch is poorer compared to that which surrounds their parents. For embryos that are forced to act passively in a brood pouch it seems possible to endure oxygen deficiency and continue development, if they can maintain respiration by utilizing oxygen which is bound for a longer time to their own Hb than oxygen bound to parental Hb.
References Calvalho G. R. (1984) Hemoglobin synthesis in Daphnia maze Straus (Crustacea: Cladocera): ecolo&al difkentiaion betwe& neighbouring po&ation< Fkeshwarer Biol. 14, 501-506.
In vivo oxygenation of Daphnia Hb
Dresel E. I. B. (1948). Passage of haemoglobin from blood into eggs of Daphnia. Nature 162, 736737. Fox H. M. (1948) The hemoglobin of Daphnia. Proc. R. Sot. Land. (Biol.) 135, 195-211. Fox H. M. and Phear E. A. (1953) Factors influencing hemoglobin synthesis by Daphnia magna. Proc. R. Sot. Land. (Eiol.), 145,. 179-189. Fox H. M., Hardcastle S. M. and Dresel E. I. B. (1949) Fluctuations in the hemoglobin content of Daphnia. Proc. R. Sot. Land. (Biol.), 136, 388-399. Green J. (1955) Studies on a population of Daphnia magna. J. Anim. Ecol. 24, 84-97.
Hoshi T. and Kobayashi K. (1972) Studies on physiology and ecology of plankton. XXV. Iron-content and millimolar extinctioncoefficient of the Daphnia-haemoglobin. Sci. Rev. Niiaata Univ. Ser. D. (Biol.) 8, 65-68.
Hoshi T. and Yahagi N. (1975) Studies on-physiology and ecology of plankton. XXIX. In vivo equilibrium of oxygen with blood haemoglobin of Daphnia magna. Sci. Rep. Niigara Univ. Ser. D. Biol. 12, l-7.
131
Kobayashi M. and Gonoi H. (1985) Horizontal movement of pale and red Daphnia magna in low oxygen concentration. Physiol. Zool. 58, 19&196. Kobayashi M. and Hoshi T. (1984) Analysis of respiratory role of haemoglobin in Daphnia magna. Zool. Sci. 1, 523-532. Kobayashi M. and Tanaka Y. (1991) Oxygen-transporting function of hemoglobin in Daphnia magna. Can. J. Physiol. 69, 2968-2972.
Kobayashi M., Hayakawa H. and Ninomiya M. (1987) Hatchability and hemoglobin of Daphnia magna embryo. Physiol. Zool. 60, 507-512.
Kobayashi M. and Nezu T. (1985) Variation of hemoglobin content in Daphnia magna. Physiol. Zool. 69, 35542. Kobayashi M., Kato S., Ninomiya M., Fujiki M., Igarashi Y., and Kaiita A. (1990) Hemoglobin in the narthenogenetic eggs of D>phr& mug&. J. exp. 2001. 254, 18-24. Teissier G. (1932) Les pigments des ouefs de Daphnia pulex. C.R. Rot. Biol. Paris, 109, 813-816.
In vivo oxygenation of Daphnia magna Michiyori Kobayashi Department
0
of hemoglobin
0300-9629/94 $6.00 + 0.00 1993 Pergamon Press Ltd
in early embryos
and Yukio Takahashi
of Biology, Faculty of Science, Niigata University, Niigata 950-21, Japan
The concentration of hemoglobin (Hb) in early embryos of Daphnia magna is higher than that in hemolymph of adult Daphnh magna.The oxygen tension in ambient water at which Hb in early embryos was 50% oxygenated was 30 torr in eggs of &poor animals and 17 torr in those of Hb-rich animals. These agreed with the data of in uiuo oxygenation of the Hb in the hemolymph of their respective parents. Hb in early embryos of I-&rich animals required a longer time for deoxygenation than that in early embryos of Hb-poor animals. A longer time was required for early embryos Hb than for adult animals Hb to be deoxygenated in an anoxic environment. Key words: Hemoglobin;
Daphnia magna.
Comp. Biochem. Physiol. 107A, 127-131,
1994.
Introduction The
concentration
of hemoglobin (Hb) in influenced by the concentration of oxygen dissolved in water and undergoes marked variations with the oxygen concentration in a living environment (Fox and Phear, 1953; Hoshi and Kobayashi, 1972; Calvalho, 1984). Hb-poor animals living in water of high oxygen concentration are colourless and Hb-rich animals living in low oxygen concentration are bright red. Daphnia magna usually reproduces by parthenogenesis. It is known that Hb also exists in parthenogenetic eggs and that its concentration varies depending on the concentration of Hb in the parental hemolymph on the concentration of Hb in the parental hemolymph (Teissier, 1932; Fox et al., 1949; Kobayashi and Nezu, 1985). No difference is found between the molecular weight of parthenogenetic egg Hb and that of parental Hb. Purified Hb from parthenogenetic eggs has a similar oxygen affinity to its respective parental Hb (Kobayashi Daphnia magna is acutely
Correspondence to: M. Kobayashi, Department of Biology,
Faculty of Science, Niigata University, Niigata 950-21, Japan. Received 18 February 1993; accepted 26 March 1993.
et al., 1990). Parental Hb is considered to have transferred into the parthenogenetic eggs (Dresel, 1948). The physiological functions of Hb in adult Daphnia magna have been investigated in relation to the amount of oxygen consumption (Kobayashi and Ho&i, 1984) swimming activity in a low oxygen environment (Kobayashi and Gonoi, 1985), oxygen saturation in uivo (Hoshi and Yahagi, 1975; Kobayashi and Tanaka, 1991), etc. for both Hb-poor and Hb-rich animals. Little attention has been directed to the significance of egg Hb of Hb-poor animals. For Hb to demonstrate its function in viva, Hb should not stay saturated with oxygen but should release the oxygen. Absorption spectral changes are induced by the release of oxygen from Hb. Measurement of the absorption spectrum of Hb in living early embryos is possible by means of microspectrophotometry. In this paper, the relationship between the oxygen saturation of Hb in early embryos and the oxygen concentration in environmental water was investigated and compared with the corresponding data for parental Hb in order to characterize the physiological functions of early embryos Hb. 127
128
Michiyori Kobayashi and Yukio Takahashi
Materials and Methods We used female Daphnia magna and their early embryos. Rice bran and Chlorella were provided as food. Hb-rich animals were reared in a low oxygen environment and Hb-poor animals in an air-saturated environment. Of the eight stages of embryonic development of Daphnia magna according to Fox (1948), stages 1 and 2 were used as early embryos. Adults measuring 2.5 mm in body length were used. In determining the Hb content, crushed early embryos or hemolymph was collected on a microslide (0.05 mm in light path) to read the absorbance of light. In vivo oxygenation of Hb To examine the absorption spectrum of Hb in a living body, an adult animal was immobilized by fixing its carapace to the tip of a tungsten wire with instant-drying reagent (Aronalpha, Konishi Co., Osaka), and the animal inserted into the absorption cell. Both sides of the cell were then sealed with glass cover slips by means of Araldite adhesive (Konishi Co.). Embryos were removed from the maternal brood pouch and placed in a 0.3 mm-square mesh of a net in an absorption cell. Water of a known oxygen concentration was allowed to flow into the cell by means of a peristaltic pump at a flow rate of about 2 ml/min. The absorption spectrum was read using a microspectrophotometer (Zeiss UMSP 80). The absorption spectra were measured at the dorsal region of the adult animal and at the center of the early embryo. A spot of monochromatic light of diameter 0.08 mm was illuminated. The hemolymph of a single animal was placed on a microslide (0.05 mm light path), and its absorption spectrum was measured at 400-450 nm with a microspectrophotometer. The Hb concentration was calculated using an oxyHb extinction coefficient of 126.6 mM-’ cm-’ at 414 nm (Hoshi and Kobayashi 1971). The Hb concentration in hemolymph from Hb-rich and Hb-poor animals were 0.92 and 0.25 gHb/lOOmm hemolymph, respectively. The oxygen partial pressure in the ambient water was monitored with an oxygen electrode (Yellow Springs Instrument Co., Ohio). Experiments were carried out at 25°C.
0.3
0.2 !I
% 0.0
0.6
0.4
400
450
550
500
wavelength,
500
nm
Fig. 1. Absorption spectra of Hb in early embryo (a) and parental hemolymph (b) of Hb-poor and those (c, d) for Hb-rich Daphnia magna.
lymph and early embryos. The absorbance should directly reflect the Hb concentration, since the absorption spectrum was read in a microslide of light path 0.05 mm. The figure shows that early embryo from both Hb-poor and Hb-rich animals contain several times as much concentration of Hb as their parental hemolymph. Absorption spectrum of Hb in a living body
Figure 2 shows the absorption spectra of early embryo from Hb-rich animal which was subjected to water of different oxygen concen-
Results and Discussion 510
Concentrations of Hb in early embryos and in parental hemolymph
Figure 1 compares the Hb concentrations from the absorption spectra of parental hemo-
530
170
550
Wavelength,
590
mn
Fig. 2. In uiuo recorded absorption spectra in early embryo removed from Hb-rich Daphnia mama. (a) deoxygenated Hb, (b-e) partially deoxygenated Hb, (f) oxygenated Hb.
In oivo oxygenation of Daphnia Hb
129
Oxygen saturation of Hb in vivo
dp d E
o 100
s
50
0 0
30
oxygen
60
90
pressure,
120
150
torr
Fig. 3. Effect of the ambient oxygen pressure on in uiuo oxygen saturation of Hb in Daphnia magna. Open circles:adult Hb-rich animals, closed circles: adult Hbpoor animals, open squares:early embryos from Hb-rich animals, closed squares:early embryos from Hb-poor animals.
trations. The absorbance was measured at the alpha and beta bands. The oxygen saturation of Hb in early embryo from Hb-rich animals was obtained from the change in absorbance at 562 nm where the absorbance varied greatly (Fig. 2), and that in embryo from Hb-poor animal from the change in absorbance at 420 nm.
L 0
The relationship between the oxygen tension and oxygen saturation of Hb in early embryos is shown in Fig. 3. Deoxygenation of Hb in early embryo from Hb-poor animal (open squares) started at an oxygen tension below 120 torr, and the oxygen tension at which Hb was 50% deoxygenated being approximately 38 torr. Deoxygenation of Hb in early embryo from Hb-rich animals (closed squares) started at a lower oxygen tension (approximately 70 torr). The oxygen tension at which Hb was 50% deoxygenated in early embryo was also lower (approximately 17 torr). These data were similar to those obtained from adult Hbpoor animals (closed circles) and Hb-rich adult animals (open circles) that were collected from the same populations. This agreed with the finding that purified Hb from early embryos and parental Hb has the same affinity for oxygen. Deoxygenation of Hb in early embryos
The time required for early embryo Hb to be deoxygenated by consumption of oxygen by the early embryo itself was measured in a small air-tight vessel filled with anoxic water (Fig. 4A,B). Deoxygenation of Hb was completed in a relatively short time, but Hb in embryo from Hb-rich animals required a little more time for deoxygenation. However, completion of deoxygenation in relatively short times, seems to be attributable to the diminution of dissolved oxygen due to
I
200
400
600
Time,
600
1000
1200
second
Fig. 4. Time courses for a change in oxygen saturation of Hb for Hb-poor embryos (A) and that for Hb-rich embryos (B) of Daphnia magna in anoxic water.
Michiyori Kobayashi and Yukio Takahashi
130
100
400
600
800
1000
Time, second Fig. 5. Time courses for a change in oxygen saturation for early embryos (a) from Hb-poor Duphnia and that for adult animals 2 mm in body size (B). The embryos were initially exposed to anoxic water, then to air- saturated water (arrow a), and finally anoxic water (arrow b).
consumption by early embryos and the resultant utilization of oxygen which was bound to the Hb. This suggests that oxygen in early embryo is rapidly consumed by the Hb contained in the embryo. Changes in oxygen saturation animals and embryos
of Hb in adult
Hb in embryos also required a longer time for oxygenation and deoxygenation in both air-saturated water and anoxic water compared with adult animal Hb (Fig. 5A,B). The longer time required for deoxygenation of Hb in early embryos results from a higher concentration of Hb and the fact that oxygen is not circulated in early embryos due to the lack of a respiratory organ which exists in adult animals. Earlier studies comparing the rates of hatching between Hb-poor and Hb-rich animal eggs in low oxygen water have reported that eggs from Hb-rich animals could hatch in water of lower oxygen concentration (Kobayashi et al., 1987). However, no reference has been made to the reason why parthenogenetic eggs contain a higher concentration of Hb than their parents. When mature, Daphnia magna have respiratory
organs, such as gills, so that when exposed to oxygen depletion in water they are capable of responding to their own demand for oxygen by floating up to the water surface which abounds in oxygen. Embryos develop in a brood pouch and are thus sub-jetted environmentally to the same oxygen conditions as surround their parents. A great number of parthenogenetic eggs are laid in a parental brood pouch. The presence of as many as 108 eggs in a single brood pouch has been reported (Green, 1955). It is presumed that the oxygen environment in such a brood pouch is poorer compared to that which surrounds their parents. For embryos that are forced to act passively in a brood pouch it seems possible to endure oxygen deficiency and continue development, if they can maintain respiration by utilizing oxygen which is bound for a longer time to their own Hb than oxygen bound to parental Hb.
References Calvalho G. R. (1984) Hemoglobin synthesis in Daphnia maze Straus (Crustacea: Cladocera): ecolo&al difkentiaion betwe& neighbouring po&ation< Fkeshwarer Biol. 14, 501-506.
In vivo oxygenation of Daphnia Hb
Dresel E. I. B. (1948). Passage of haemoglobin from blood into eggs of Daphnia. Nature 162, 736737. Fox H. M. (1948) The hemoglobin of Daphnia. Proc. R. Sot. Land. (Biol.) 135, 195-211. Fox H. M. and Phear E. A. (1953) Factors influencing hemoglobin synthesis by Daphnia magna. Proc. R. Sot. Land. (Eiol.), 145,. 179-189. Fox H. M., Hardcastle S. M. and Dresel E. I. B. (1949) Fluctuations in the hemoglobin content of Daphnia. Proc. R. Sot. Land. (Biol.), 136, 388-399. Green J. (1955) Studies on a population of Daphnia magna. J. Anim. Ecol. 24, 84-97.
Hoshi T. and Kobayashi K. (1972) Studies on physiology and ecology of plankton. XXV. Iron-content and millimolar extinctioncoefficient of the Daphnia-haemoglobin. Sci. Rev. Niiaata Univ. Ser. D. (Biol.) 8, 65-68.
Hoshi T. and Yahagi N. (1975) Studies on-physiology and ecology of plankton. XXIX. In vivo equilibrium of oxygen with blood haemoglobin of Daphnia magna. Sci. Rep. Niigara Univ. Ser. D. Biol. 12, l-7.
131
Kobayashi M. and Gonoi H. (1985) Horizontal movement of pale and red Daphnia magna in low oxygen concentration. Physiol. Zool. 58, 19&196. Kobayashi M. and Hoshi T. (1984) Analysis of respiratory role of haemoglobin in Daphnia magna. Zool. Sci. 1, 523-532. Kobayashi M. and Tanaka Y. (1991) Oxygen-transporting function of hemoglobin in Daphnia magna. Can. J. Physiol. 69, 2968-2972.
Kobayashi M., Hayakawa H. and Ninomiya M. (1987) Hatchability and hemoglobin of Daphnia magna embryo. Physiol. Zool. 60, 507-512.
Kobayashi M. and Nezu T. (1985) Variation of hemoglobin content in Daphnia magna. Physiol. Zool. 69, 35542. Kobayashi M., Kato S., Ninomiya M., Fujiki M., Igarashi Y., and Kaiita A. (1990) Hemoglobin in the narthenogenetic eggs of D>phr& mug&. J. exp. 2001. 254, 18-24. Teissier G. (1932) Les pigments des ouefs de Daphnia pulex. C.R. Rot. Biol. Paris, 109, 813-816.