Phosphate compounds in red cells of two dogfish sharks: Squalus acanthias and Mustelus canis

0300-9629 82’090135-06$03.00/0 0 1982 Pergamon Press Ltd

Camp. Biochrw. Phpsiol. Vol. 73A. No. I. pp. 135 to 140, 19X2 Printed in Great Britain

PHOSPHATE COMPOUNDS IN RED CELLS OF TWO DOGFISH SHARKS: SQUALUS ACANTHIAS AND MUSTELUS CANIS GRANT

Laboratory

for Comparative

R. BARTLETT

Biochemistry.

San Diego, CA 92109, U.S.A

Abstract 1. As part of a survey of the red cell hematology of birds, reptiles and fishes, two dogfish sharks, the spiny, Syutrl~~ ucumhius (suborder Syudoidri) and the smooth. M~r.srelus c’trnis (suborder Guleoidc4). have been selected from the elasmobranch fishes for a detailed study. 2. Investigations were carried out on embryos. new born. young and adults at the Marine Biological Laboratory, Woods Hole, Mass. 3. Studies on the changes of the red cell during early development of the two sharks, which have different modes of reproduction. the spiny ovoviviparous and the smooth viviparous, were facilitated by the ready availibility of embryos of convenient size for sampling of blood and testing of the red cells. 4. Phosphate components of the red cells were isolated by ion-exchange column chromatography and the turnover rates of the nucleotides measured by their incorporation of radioactivity after brief incubation with “P-labelled inorganic phosphate. 5. Of special interest to a comparison of the two dogfishes is the concentration of red cell guanosine triphosphate which is high in the smooth and low in the spiny.

INTRODUCTION

As part of a comparative study of metabolic properties of vertebrate red cells we have had the opportunity to carry out analyses of the phosphate components of red cells of a variety of fishes including several advanced teleosts, two lungfishes, an osteoglossid, the ratfish, some sharks and the cyclostomes, hagfish and lamprey (reported in a companion paper) (Bartlett, 1982; Bartlett, 1980). Among the sharks we have chosen two dogfish, the spiny dog (Syualus ucanthius) and the smooth dog (Mustrlus cunis) for an in depth study of their red cell hematology. There are several reasons for the choice of these two sharks and the Marine Biological Laboratories (MBL), Woods Hole. Massachusetts, for their study. The MBL is an ideal place for this research project; with a competent marine resources group for collection and holding of specimens; with lab space, supplies and equipment suitable for sophisticated physiological and biochemical experiments; and with an outstanding reference library. Laboratory space is usually available on short notice to qualified researchers outside the busy summer season. Both sharks are frequently abundant in the nearby sea during spring, summer and fall. Their size, ranging from about 1OOg at birth to 5 k for the adult, is convenient for experimental purposes acd samples of blood can be taken easily from the caudal vein with or without anesthesia and without harm to the fish. The two sharks, both of whom give birth to live young. are especially useful for studies of changes of the red cell during early development since samples of pure blood can be obtained throughout most of embryonic growth. Of special interest is the fact that Syualu.s acanthias is ovoviviparous and Mustelus canis, viviparous. Female fish caught in the vicinity of Woods Hole frequently contain embryos of different ages and often give birth in the holding tanks. An

important reason for looking at these two sharks in tandem is that though superficially similar in appearance and habits they have many striking differences, as would be expected from their distant relationship: Squulus is put into the Selachian suborder Squaloids while Mustelus is a member of the suborder Galeoids (Budker, 1971; Bigelow & Schroeder, 1948a; Bigelow & Schroeder, 1948b). The first part of the projected hematological study of the two dogfish sharks, the subject of this report, was to compare the pattern of phosphate compounds in red cells of the smooth and spiny dogfishes at different ages of development. Metabolic turnover rates of the phosphate compounds were measured by their incorporation of radioactivity from added 32P-labelled inorganic phosphate. Purine nucleotides of red cells were of special interest and were compared with their concentration in other tissues of the body.

METHODS Smooth and spiny dogfish were caught in the sea within a few miles of Woods Hole by otter trawl, All fish were

kept in tanks of running, well-aerated sea water; adults at the end of the pier in front of the Lillie building and newborns and older. in their first year, in the Marine Resources building and in my lab. Blood was taken from newborn and older sharks by needle and syringe from the caudal vessel usually without anesthesia. Occasionally large sharks were lightly anesthetized by placing them for about five minutes in a separate tank of sea water containing 1OOmg per liter of tricaine. Immediately following sacrifice of a gravid female by a blow to the back of the head, the abdominal cavity was opened, the uterus removed and the embryos and surrounding tissue extruded. Samples of blood were obtained from embryos of the spiny dog by nicking a vessel on the large yolk sac and drawing blood into a ~1 pipet. The embryos of the smooth dog were attached by a long umbi35

GRANT

136

R. BARTLETT

lical cord to a small irregularly shaped yolk sac. The umbilical cord was cut next to the yolk sac and the embryo immediately placed on a platform with the cord running into a 1.5 ml heparinized conical polyethylene centrifuge tube. Blood dripped from the cord into the tube for several minutes with the help of the pumping action of the heart and in this way about 0.5 ml of blood could be collected from a 5 cm embryo. Hematocrit and microscopic analyses were carried out on most blood samples as were perchloric acid (PCA) extracts of red cells; in the case of the embryos from pooled blood. The PCA extracts were made as soon as possible after collection of blood to prevent any changes of the red cell phosphates. In our experience sharks have substantially more white cells in their blood than teleost fishes. It was not unusual for the volume of white cells (leukocytes and platelets) to be IO”, of the volume of red cells. as measured by the hematocrit. Therefore special care was taken to remove most of the white cells, which formed a layer on top of the red cells after centrifugation of the blood. The red cells were extracted with several volumes of 0.6 N PCA immediately after separation from the blood, the extract neutralized with potassium hydroxide and, after cooling. the precipitated potassium perchlorate removed. For microscopic observation whole blood was diluted wtth plasma (from the hematocrit separation when scarce) and examined at 40 x and 100 x plus oil. with and without staining with brilliant cresyl blue. A drop of a saturated solution of brilliant cresyl blue in ethyl alcohol was allowed to evaporate to dryness at the bottom of a 1.5 ml conical centrifuge tube. To this was added two drops of the diluted blood suspension, followed by mixing to put the dye into solution. After at least five minutes the stained cells were examined microscopically. The PCA extracts of red cells, I-log, from blood of newborn to adult sharks. were chromatographed on I x 25cm columns of Dowex I x 8 resin (200 to 400 mesh). Extracts of 500 mg or less of red cells from embryos were chromatographed on 0.5 x 15 cm columns of Dowex 1 x 8 resin (-400 mesh). This anion-exchange resin, purchased from BioRad and called by them AG l-X8 (minus 400 mesh). after treatment to remove large particles and fine flour, gave flow rates of at least 1 ml per min using a Sigmamotor peristaltic pump Model TM-20-2. These small columns were eluted with lOOmI of formate and then by various amounts of HCI depending on the requirements.

SPINY

UTP

GTP

collecting 2-3 ml fractions. Details of the methods used for chromatographic analysis of red cells are given elsewhere (Bartlett, 1968; Bartlett, 1978a). 32P-labelled sodium phosBuffered isotonic (human) phate was purchased from Mallincrodt. One tenth ml was added to each of two 20 ml portions of freshly collected blood from a spiny and a smooth dogfish and the mixture incubated with occasional shaking at room temperature (21 ). Half of the blood was removed at lOmitt, quickly cooled in ice, centrifuged, huffy coat discarded and packed red cells extracted with PCA. The other half of the blood was treated the same way after 30min of incubation. Eluate fractions from ion-exchange column chromatography were counted in a Packard Scintillation Spectrometer, Model 2002. To find out whether the ratio of GTP to ATP was unusually high in tissues of the smooth dog other than the red cell, 100 ml of blood was taken from the caudal vein of a large non-gravid female. She was then sacrificed and portions of intestine, skeletal muscle and liver removed and immediately frozen. The blood was used for isolation of white cells (a mixture of leukocytes and platelets) which were extracted by mixing with PCA in a glass tube. The organs were disintegrated in a small Waring blender while suspended in several volumes of PCA. The extracts were treated and chromatographed as described for red cells. RESULTS

Hematocrit determinations were made on blood of a large number of smooth and spiny dogfish of both sexes including embryos, neonates, fishes several months old in their first year and miscellaneous adults. The hematocrit values ranged between 20 and 30 with no correlation with species, age, sex or apparent state of health. Microscopic examination showed red cells of both smooth and spiny dogfishes to have a shape typical of the non-mammalian vertebrates, that is an oval plate with centrally located nucleus. The average dimensions of the red cells were 20 x 15 x 5 pm for length, width and thickness with at least 85% of the cells of uniform shape and staining characteristics. Five to loo/, of the cells had the staining properties of

SMOOTH

*PI

Fig. I. Phosphate compounds in red cells of adult smooth and spiny dogfish. Perchloric acid extracts of red cells were chromatographed on 1 x 25 cm columns of Dowex 1 x 8-formate (20&400 mesh) which were eluted with 1 1 of linear gradient O-5 M ammonium formate (4 parts formic acid to 1 part ammonium formate) followed by 1 I of linear gradient G-1 N HCI. The values for the phosphate compounds are given in Table I. Phosphorus. --ee; A at 260 nm. --o-e.

Phosphates Table

1. Phosphate

compounds

in shark

137

RBC’s

in red cells of smooth

and spiny dogfish*

AMP

ADP

ATP

GTP

Pi

XPl

XP2

Hct

g RBCt

Spiny dogfish Early embryo Adult Adult Adult Adult Adult

< 0.1
0.4 0.7 0.9 0.7 0.8 0.6

4.0 9.1 7.1 10.4 5.7 7.7

0.7 1.3 0.9 1.9 0.9 I.1

1.7 2.4 2.6 I.5 1.6 1.3

tr 0.7 1.3 0.4 I.0 I.1

0 0 0 0 0.5 0.4

28 22 24 26 25 24

0.4 2.1 1.4 I.9 I.7 4.8

Smooth dogfish Early embryo Newborn Adult Adult Adult Adult Adult


0.7 1.3 0.5 0.4 0.4 0.3 0.5

11.6 19.5 9.8 6.5 8.3 7.2 6.1

tr 6.1 14.3 9.6 13.6 12.0 13.3

2.1 1.9 0.9 0.7 0.7 1.4 1.2

0 0 0 0 0 0 0

0 0 0 0 0 0 0

20 23 27 22 25 20 28

0.8 0.8 2.0 2.x 1.9 1.6 8.1

* Values in micromoles of P per ml RBC i- Amount used for chromatography.

Red cells of the youngest embryos were more spheroidal and had different staining particles in the cytoplasm. Red cells of the embryos in the latter half of gestation and new borns had assumed the oval disc shape but 2(t40”/<, of them were reticulocytes. The reticulocytes had almost disappeared in the several month old fishes. Typical ion-exchange chromatographic analyses of phosphates in red cells of adult smooth and spiny dogfish are given in Fig. 1. Red cells of both fish contained a relatively large pool of ATP. However the smooth dog had in addition a high concentration of GTP, about twice as much as the ATP. Another difference was the appearance in the spiny dog of small amounts of two phosphates called XPI and XP2, which were eluted by the HCI gradient in about the same positions expected for inositol penta- and hexaphosphates. Because of the small quantities available we are not positive of their identification. However a color characteristic of inositol. but not as much as expected, was obtained by a relatively unspecific calorimetric method (Lornitzo. 1968; Bartlett. 1978b). Despite their low concentration these substances are of special interest since we have not seen them in chromatographs of red cells of other fishes including the smooth dogfish. Red cells of the spiny dog contained a small amount of GTP, about 150, of the ATP. ADP and Pi were also prominent in the chromatographs of both sharks along with small amounts of several other phosphates. The concentrations of important phosphates are given in Table 1. The metabolic turnover of ATP in human red cells has been measured by the rate of incorporation of radioactivity from added 3”P-labelled inorganic phosphate (Bartlett, 1968). The same technique was used in this study to compare sharks with each other and with man, and also to find out if the large pool of GTP in the smooth dog was significantly different than the ATP. Blood from each shark was incubated for 10 and 30 min after addition of a trace of 32P-labelled inorganic phosphate. At the end of the incubations the red cells were separated, extracted with PCA and the P compounds, isolated by ionexchange column chromatography, were assayed for reticulocytes.

radioactivity. Figure 2 shows the result of the chromatography of extracts of cells from smooth and spiny dogfish which had been incubated for 10min. With both sharks there was a large drop in specific activity of inorganic phosphate between plasma and red cells showing a strongly restricted movement of this compound across the membrane. The necessity for rapid extraction of red cells following the incubation of the blood, and therefore our choice not to wash the red cells. undoubtedly led to an error in the specific activity of the intracellular inorganic phosphate on the high side due to contamination with extracellular phosphate. The specific activity (counts per min per nmole of P) for ATP and GTP was 6 and 5 after 10min of incubation and 12 and 10 at 30min for the smooth dogfish, and 6 and 4 at 10min and 14 and 10 at 30 min for the spiny dogfish. The SA of the intracellular inorganic phosphate (Pi) was variable with a minimum value of 110 which undoubtedly still had substantial contamination by the high SA (1200) Pi in the plasma. Because of the uncertainty of the value for intracellular Pi the actual fraction of this Pi incorporated into the nucleotides cannot be determined. The results show however that the metabolic turnover of ATP was essentially the same in the two sharks and that the GTP in each was almost the same as the ATP. Chromatographic analyses of red cell phosphates were carried out on two sets of embryos from each shark. The embryos from the smooth dogfish were 5 and 8 cm long and therefore less than half way through gestation (Ranzi, 1932; Ranzi, 1934). Embryos of the spiny dogfish were 6 and 15 cm, early in gestation and about half way through (Scammon, 1911). Chromatograms of the youngest embryos of each fish are shown in Fig. 3 and a summary of the amounts of phosphates found given in Table 1. The predominant phosphate constituent of red cells of both fish was ATP. Of special interest was the lack of GTP in embryos of the smooth dogfish and of the HCI-eluting phosphates in the spiny. Analyses for phosphates were carried out on PCA extracts of the following tissues of an adult smooth

GRANT

138

R. BARTLETT

Fig. 2. Phosphate compounds and radioactivity in red cells of adult smooth and spiny dog, from blood phosphate. Perchloric acid extracts of red cells incubated for 10min at 21 with added “P-inorganic were chromatographed on 1 x 25cm columns of Dowex 1 x g-formate (20@400 mesh) which were eluted with I I of linear gradient S-5 M ammonium formate (4 parts formic acid to I part ammonium formate) followed by I I of linear gradient t&l N HCI. The values for the phosphate compounds are given in Table 1. Phosphorus, --t&; A at 260 nm. -O-O-; radioactivity, -A-A--m.

dogfish: white cells, intestine, liver and skeletal muscle. Although the tissues were removed and frozen immediately following sacrifice of the fish still some loss of ATP would be expected. Nevertheless the results show that these tissues have the normal low ratio of GTP to ATP (0.1-0.2) in marked contrast to that found in the red ceil. DISCUSSION

Studies on the phosphate metabolites of vertebrate red cells can be useful for two reasons: (1) to provide information about the nature of the energy metabolism which helps to keep the cell intact and functional in the circulation and (2) to reveal metabolites which may participate in the regulation of the oxygen affinity of hemoglobin. The oxygen affinity of red cells of adult vertebrates is frequently found to be significantly lower than that of the corresponding purified hemoglobin, and this has been shown in a number of cases to be due to the interaction of some small molecule constituent of the red cell, usually an organic polyphosphate, with hemoglobin to modify its binding to oxygen (Bartlett, 1980). The oxygen affinity of the mammalian red cell, with the exception of cats and bovinoids, is controlled to a large degree by 2,3_diphosphoglycerate and that of birds by inositol pentaphosphate. There are interesting differences among the reptiles. Red cells of the crocodilians have a low concentration of nucleotides and other phosphates but it has been reported recently that their hemoglobin is unusually sensitive to carbonate which may act as a specific affinity regulator in this group of animals (Perutz et al., 1981). All of the snakes and lizards so far examined have had unusually high red cell concentrations of ATP and this pool may be divided in function between energy exchange and regulation of oxygen affinity. Turtle red cells have an intermediate concentration of ATP and a low concentration of IP5, too small to have a significant effect on oxygen affinity.

The finding of a relatively high concentration of ATP in red cells of several fishes led to suggestions that it might act as a regulator of oxygen affinity (Gillen & Riggs, 1971, 1972, 1973a, b; Wood & Johansen, 1972; Okazaki ct (II., 1974; Weber & de Wilde, 1975; Weber et u/., 1976b; Powers, 1980). It was then discovered that red cells of the Anguilla eel had very high concentrations of GTP and evidence was obtained to suggest that GTP rather than ATP was an important control of oxygen affinity in this fish (Weber et (II., 1975. 1976a: Kaloustian & Poluhowich. 1976; Peterson & Poluhowich, 1976). There followed reports of analyses of nucleotides of red cells from a variety of fishes which showed that some had a high concentration of GTP, higher than the ATP, some were intermediate and some had a concentration of GTP which was only a small fraction of that of ATP (Bartlett, 1980). There was no apparent relationship of level of red cell GTP to taxonomy or habitat of the fish. One of the fishes found to have a high concentration of GTP was the smooth dogfish. Must&s canis (Borgese et al.. 1978) and one with a low concentration the spiny dogfish, Squalus ucunthius (Borgese & Nagel. 1978). In view of the ready availability of these two kinds of dogfish at different stages of development it was decided to carry out a more detailed study of their phosphate metabolites and to examine other aspects of their red cell hematology. Mustelus cunis, the smooth dogfish, is a member of the Triakidae, one of several families of the large suborder, Galeoidei, which contains many of the best known sharks. Squulus acunthius, the spiny dogfish, belongs to the family, Squalidae, of the much smaller suborder, Squaloidei. Both fishes are similar in appearance and size with two prominent dorsal fins, Squalus with a spine in front of each and without an anal fin. The two sharks are greyish in color above and greyish white to creamy below with Syuulus having an irregular row of whitish spots along the side, especially in younger fish. Lengths and weights of both species range from about one foot and 100 g at

Phosphates

SMOOTH

DOGFISH

in shark

I ~_

1 ATP:,

EMBRYO

Fig. 3. Phosphate compounds in red cells of early embryos of smooth and spiny dogfish and of a newborn smooth dogfish. Perchloric acid extracts of red cells of the smooth dogfish were chromatographed on I x 25 cm columns of Dowex 1 x 8-formate (20@400 mesh) which were eluted with 11 of linear gradient &5 M ammonium formate (4 parts formic acid to I part ammonium formate) followed by I I of linear gradient O-1 N HCI. The red cell extract of the spiny embryo was chromatographed on a 0.5 x 15 cm column of Dowex I x 8-formate (-400 mesh) which was eluted with 100 ml of linear gradient 0 to 6 M ammonium formate (4 parts formic acid to 1 part ammonium formate) followed by 30ml of 1 N HCI. The values for the phosphate compounds are given in Table I. Phosphorus. -o--t: A at 260 nm. -O--O-.

birth to four feet and five kilos for large adults. Adults of around 100 cm and 3 to 4 kg are commonly caught by the MBL collectors. Both sharks are abundant in waters off New England arriving in spring and leaving in late fall, Mustelus staying in large numbers around Cape Cod and Squ&s moving further north to return again in November on their way south (Bigelow & Schroeder, 1948a; Bigelow & Schroeder, 1948b). Squalus acmthias is ovoviviparous. Two to three embryos usually develop in each uterus from very large yolky eggs. The uterus provides little if any nutrient to the embryo but probably is important in the exchange of respiratory gas. Gestation is about 20 months. Late embryos delivered in the lab, still carrying a large yolk sac, survived in an aquarium of running sea water with gradual resorption of the yolk. Mustehs canis is considered to be truly viviparous. Five or more embryos develop in each uterus. There

RBC’s

139

is a small yolk sac but most nutrient for the embryo comes from the blood of the mother through a spongy highly vascular placental-like tissue under the uterine case and surrounding the developing embryos. Gestation is about 10 months and the new born carry no yolk sac (Hoar, 1969). Gravid females of both species containing embryos at different ages of development are commonly caught in the sea near Woods Hole by MBL. Phosphate analyses were carried out on extracts of red cells of embryo, neonate, young and adult specimens of Mustelus cunis and Squu1u.s ucmthius. The results on adult fish confirm those reported previously (Borgese & Nagel. 1978; Borgese ~‘t (I/., 1978). There was a high concentration of GTP, about 1.5-2 times that of the ATP, in red cells of adult smooth dog and a very low concentration of GTP, about 0.1~0.2 of the ATP, in red cells of the adult spiny dog. The ratio of GTP to ATP was found to change markedly during early development of Mustelus, being very low in the embryo, a little higher in the new born and approaching the high level of the adult in fish estimated to be in the last half of the first year of life. The ratio of GTP to ATP was low at all stages of development of squu111s. The ratio of GTP to ATP is normally maintained at a low level in living tissue (Henderson & Paterson, 1973; Mandel, 1964). red cells of some fishes being the only known exception. The greatly increased ratio of GTP to ATP in the fish red cell implies alterations in the usual enzymes of purine metabolism probably in those responsible for the conversion of inosinic acid to guanylic acid. If the sole function of the large pool of GTP is to control oxygen affinity of hemoglobin, then one might anticipate that the turnover rate of this nucleotide would be different, probably less, than that of ATP. As one approach to a measure of this turnover. blood of an adult smooth dog was incubated briefly with a tracer dose of 32P-labelled inorganic phosphate followed by analysis of radioactivity in ATP and GTP. isolated from the red cells by ion-exchange chromatography. A substantial amount of radioactivity was incorporated into both nucleotides and each was found to have the same specific radioactivity, in other words the same rate of turnover of their phosphate groups. We have carried out a similar study with the human red cell and found the turnover of the labile phosphates of ATP and GTP to be identical with the phosphorus attached to the ribose not labelled (Bartlett, 1968). It is possible that there are different metabolic turnover rates for the adenylate and guanylate portions of the shark nucleotides while the labile phosphates are equilibrated with each other through the action of purine nucleosidediphosphate kinase. When it was discovered that the concentration of GTP was very high in red cells of the American eel, Anguilla rmtratu, the question was raised as to whether the ratio of GTP to ATP might be elevated in other tissues. We compared the phosphate metabolites of red cells, skeletal muscle and liver, confirming high GTP for the red cells and finding the usual low ratio of GTP to ATP for muscle and liver (Bartlett, 1979). It was thought that it would be of interest to do the same experiment on Mustelus canis and so phosphate metabolites were isolated by ion-exchange

GRANT R. BARTLETT

140

chromatography from PCA extracts of skeletal muscle, liver, intestines and white cells. All tissues had a low ratio of GTP to ATP (O.l-0.2), more evidence that high GTP is unique to the red cell. IP5 has been reported to be present in red cells of the spiny dogfish (Borgese & Nagel. 1978). During our chromatographic analyses of extracts of red cells of young and adult spiny dogfish, elution with HCI revealed two small phosphorus peaks in approximately the expected positions for inositol penta- and hexaphosphates. Although these two phosphates gave a positive reaction in a chemical test for inositol, the method is insensitive and unspecific, and the amounts of compounds available so small, that we are not yet certain about their identification. Inositol polyphosphates have not been found in red cells of other fishes examined with the remarkable exception of the osteoglossid, Arupmimu giyus, an air-breathing fish in the Amazon, which was found to have a high concentration of inositol pentaphosphate (Isaacks or ml.. 1977; Bartlett, 1978b). A~I,nor~ledgenlrnt~~~Supported by Grant HLB-6950 from the National Heart, Lung and Blood Institute.

REFERENCES

BARTLETT G. R. (1968) Phosphorus compounds in the human erythrocyte. Biochim. hiophys. Actu 156, 221-230. BARTLETT G. R. (1978a) Phosphate compounds in reptilian and avian red blood cells; developmental changes. C’omp. Biochem. Ph!siol. 61 A, 19 l-202. BARTLETT G. R. (1978b) Phosphates in red cells of two South American osteoglossids: Arupuimu gigus and Osteoglossum bicirrhosum. Can. J. Zoo/. 56, 878-88 1, BARTLETT G. R. (1979) Phosphate compounds in red cells. liver and skeletal muscle of the American eel. Anguillo roAtrutu. Cop& 2, 36&363. BARTLETT G. R. (1980) Phosphate compounds in vertebrate red blood cells. Am. Zoo/. 20, 103-l 14. BARTLETT G. R. (1982) Phosphates in red cells of a hagfish and a lamprey. Camp, Biochem. Physiol. 73A. 141-145. BICELOW H. B. & SCHROEDEKW. C. (1948a) Mustelus canis. In Fishrs of t/w Wesrrrn North Atluntic. No. 1. Part I. pp. 244255. Sears Foundation for Marine Research, New Haven. BIGELOW H. B. & SCHKOEDEKW. C. (1948b) Syuulu.s ucunthius. In Fishes of the Westrrn North Atlantic. No. 1. Part I, pp. 455-473. Sears Foundation for Marine Research. New Haven. BORGESE T. A. & NAGEL R. L. (1978) Inositol pentaphosphate in fish red blood cells. J. rxp. Zool. 205, 133-140. BORCESE T. A.. NAGEL R. L., ROTH E.. MURPHY D. & HARRINGT~N J. (1978) Guanosine triphosphate (GTP): the major organic phosphate in the erythrocytes of the elasmobranch Mu.sre/us canis (smooth dogfish). ~o,np. Biochem. Physiol. 60B. 3 17-32 I, BUDKER P. (197 1) Classification. In Tlrr L@ o!f Shurks. pp. 7-21. Columbia University Press, New York. GILLEN R. G. & RIGGS A. (1971) The hemoglobins of a freshwater teleost. Cichlusomu c~unogutfurum (Baird & Girard). I. The effects of phosphorylated organic compounds upon the oxygen equilibria. Camp. Biochem. Physiol. 38B, 585 595.

GILLEN R. G. & R~cc;s A. (1972) Structure and function of the hemoglobins of the carp. Cyprirnrs wrpio. J. hid. Chrm. 247, 6039-6046.

GILLEN R. G. & RIGGS A. (1973a) The hemoglobins of a freshwater teleost. Cichlusomu cyrnoguttutum (Baird & Girard). II. Subunit structure and oxygen equilibria of the isolated components. Arch. Biochem. Biophys. 154, 34&359. GILLEN R. C. & R~c;c;s A. (1973b) Structure and function of the isolated hemoglobins of the American eel. Anguillu ros!ruttr. J. bid. Chem. 248, 1961-1969. HENIXRSON J. F. & PATERSON A. R. P. (1973) Nudrotide Mettrholism. Academic Press, Inc.. New York. HOAK W. S. (1969) Reproduction; vivaparity and gestation. In Fish Ph!~.siolog~. Vol. 3 (Edited by HOAR W. S. & RANDALL D. J.), pp. 20-30. Academic Press, New York. KALOUSTIA~~;K. V. & POLUHOWICF~J. J. (1976) The role of organic phosphates in modulating the oxygenation behavior of eel hemoglobin. Camp. Biochem. Physiol. 53A, 245-248. L~RNITZO F. A. (1968) A method for calorimetric assay of inosltol and some of its phosphate derivatives. Anul. Bio~hW1. 25, 396 405. MANDEL P. (1964) Free nucleotides in animal tissues. Prog. Nucleic Arid Rrs. 3, 299.-334. OKAZAKI T., MISAWA J. & SHLJKC’YAR. (1974) The effects of organic phosphates on the oxygen equilibria of two distinct hemoglobins of the eel, Anguillu juponicu. Biothem. biopk~.s. Rrs. Commun. 56, 1031-1037. PERLTZ M. F., BACER C., GROS G.. LECLERCQ F.. VANDECASSERI~C., SCHNEK A. G., BRAUNITZER G., FRIDAY A. E. & JOYSFY K. A. (1981) Allosteric regulation of crocodilian haemoglobin. Nuture (London) 291, 682-684. PETERSON A. J. & POLUHOWITH J. J. (1976) The effects of organic phosphates on the oxygenation behavior of eel multiple hemoglobins. Camp. Biochem. Phj,.siol. 55A, 351-354. Powt~s D. (1980) The molecular ecology of teleost fish hemoglobins: strategies for adapting to changing environments. Am. Zoo/. 20, 139-162. RANZI S. (1932) Le basi fisio-morfologiche dello sviluppo embrionale dei Selaci. Parte I. Pubbl. Stun. Zoo/. Nupoli 12, 209~ 290.

RANZI S. (1934) Le basi fisio-morfologiche dello sviluppo embrionale dei Selaci. Parti II e 111. Pubhl. Stu:. ZOO/. Nupoli

13, 331-437.

SCAMMoN R. E. (1911) Normal plates of the development of Squulus ucunthius. Normentufeln zur Entwicklunqs upschickte der Wirbeltiere 12, 14 p.. Jena Verlag. ” ”

WEBER R. E. & DI: WlLDt, J. A. M. 11975) Oxvgenation properties of haemoglobins from ;he flat-f% plaice (Plruronrcte.s plutes.su) and tlounder (Plurichrhys ,flr,~s). J. Camp. Physiol. IOlB, 99 I IO. WEBER R. E., LYKKEaoE G. & JOHANSEN K. (1975) Biochemical aspects of the adaptation of hemoglobinoxygen affinity of eels to hypoxia. Life Sci. 17, 13451349. WEBER R. E.. LYKKLB~E G. & JOHANSEN K. (1976a) Physiological properties of eel haemoglobin: hypoxic acclimation. phosphate effects and multiplicity. J. e.up. Biol. 64, 75-88. WEBER R. E., WOOD S. C. & LOMHOLT J. P. (1976b) Temperature acclimation and oxygen-binding properties of blood and multiple haemoglobins of rainbow trout. J. exp. Bid. 65, 333-345. WED S. C. & JOHANSEN K. (1972) Adaptation to hypoxia by increased HbO, affinity and decreased red cell ATP concentration. Nuturr New Bid. 237, 278--279.