Comp. Biochem. Physiol., 1971, Vol. 40B, pp. 923 to 930. Pergamon Press. Printed in Great Britain

THE DIFFERENTIATION OF BLOOD PROTEINS DURING ONTOGENY IN SEPIA OFFICINALIS L.

W. DECLEIR," J. LEMAIRE and A. RICHARD

Institut de biologie maritime et rkgionale, Wimereux, France; and Instituut voor Omgevingswetenschappen, V.U.B., Brussels, Belgium

(Received 10 May 1971)

Abstract-1. Blood from embryos, young animals and adult Sepia oficinalis L. has been analysed by an ultramicroelectrophoretic technique.

2. During the differentiation of a very small embryo into a 2-month-old animal, we have found eleven different protein fractions.

3. All these proteins have been identified as hemocyanin by histochemical tests.

4. The possible origin of the embryonic, young and adult hemocyanins is discussed.

INTRODUCTION

UNTIL recently the site and mode of hemocyanin synthesis has remained a mystery. Only during the past few years have the first indications of the origin of this, the main respiratory pigment in molluscs and arthropods, been published.

Fahrenbach (1970) found in Limulus polyphemus a new type of leucocyte, rarely found in the circulatory bloodstream, but occurring in varying abundance in the sinusoidal spaces of the neural plexus of the compound eye. These leucocytes are probably formed in the hepatopancreas and are the site of biosynthesis of the hemocyanin. Hence these cells are called "cyanoblasts". Electron microscopic analysis has shown that the hemocyanin accumulates in the cytoplasm of these cells and aggregates into crystals, which are liberated into the hemolymph by rupture of the cyanoblast. The liberated crystals dissolve very slowly in the blood.

Schipp & Schafer (1969) found in Sepia oflcinalis and Loligo vulgaris ovoid endocrine cells situated among the muscle fibers in the wall of the branchial hearts. When examined with an electron microscope these cells were found to contain crystals with an inner pattern similar to that of the extracellular hemocyanin and it was suggested that the hemocyanin of cephalopods is synthesized in these cells.

In Crustacea, hernocyanin has a smaller molecular weight than in molluscs and Xiphosura. The intracellular hemocyanin is consequently more difficult to detect.

* Present address: Department of Physiological Chemistry, Casinoplein 21, B 9000, Gent, Belgium.

Decleir & Vercauteren (1967) found in the leucocytes of Cancer pagurus a pseudo- phenoloxydase activity comparable to that of hemocyanin and they concluded that this respiratory pigment might be synthesized in the leucocytes.

Without doubt many other hemopoietic sites in invertebrates will be found during the years ahead.

In a previous publication (Decleir & Richard, 1970) we have published electron micrographs of the blood in gills of the cuttlefioh S. oficinalis L. Thc adult animals showed linearly polymerized hemocyanin molecules with a pattern similar to the extracellular crystalline hemocyanin found by Barber & Graziadei (1965), Schipp & Schafer (1969) and also by Fahrenbach (1970). There is no such finding in young animals immediately after hatching. The site of hemocyanin formation is consequently felt to be different in young as opposed to adult S. oficinalis.

We have also shown that the electrophoretic pattern of hemocyanins from young and adult animals differs significantly (Decleir & Richard, 1969, 1970; Richard & Decleir, 1969). This is a well-known phenomenon in vertebrates where different hemoglobins are found to succeed each other during ontogeny. As far as we know this had not previously been described in invertebrates. [Parisi et al. (1964), with an immunological method, found hemocyanin to be present in em- bryos of S. oficinalis from stage 7 of Naef onwards (Naef, 1928). but they did not notice any difference between embryonic and adult hemocyanin.]

The present work is an electrophoretic study of the blood of embryos, young and adult specimens of S. oficinalis L. The purpose was to find out when hemo- cyanin is first synthesized in the embryo and how embryonic, young and adult hemocyanins succeed each other during ontogeny. Finally, the possible sites of biosynthesis of the different hemocyanins are discussed.

MATERIALS AND METHODS All the experiments were carried out with specimens of the cuttlefish Sepia oficinalis L.

Adult animals were caught in the coastal waters near the marine station of Wimereux. Eggs and young animals were kept in the laboratory in separate aquaria according to their age, as published earlier (Richard, 1968; Decleir & Richard, 1970).

Generally blood samples were taken by puncturing the branchial hearts with a Pasteur pipette. The amount of blood obtained in this way varied between 0.1 and 1 p1 for embryos and between 1 and 3 p1 for young animals up to 14 days old.

In small embryos blood sampling was not always possible as explained. Therefore we proceeded as follows:

(1) Very small embryos with a mantle length of about 1 mm Blood could only be obtained from the large head sinus.

(2) Embryos of 2 mm mantle length Blood was obtained from the head sinus, the branchial hearts and the arterial heart. All

these samples showed an identical electrophoretic behavior.

(3) Embryos from 3 to 5 mm mantle length Blood was obtained from the head and the branchial hearts. It was not possible to

puncture the arterial heart because it is embedded in a thick yolk mass which occludes the pipette before it reaches the heart.

DIFFERENTIATION OF BLOOD PROTEINS IN SEPIA OFFICINALIS 925

(4) Embryos from 5 mm to hatching Blood could only be obtained from the branchial hearts. The head sinus was now too

small to be used as a source of blood.

(5) After hatching The yolk mass around the arterial heart diminishes quickly and puncturing the arterial

heart becomes possible again. Since we found no difference between the electrophoretic patterns of blood from either the branchial or the arterial heart, we preferred to take the samples from the branchial hearts because the punctures were easier and more quickly accomplished.

The electropherograms were obtained with the ultramicroelectrophoretic technique of Wieme (1959). The buffer used was a phosphate buffer, pH = 6.5 (p = 0.08), and the electrophoresis time was 60 min. Proteins were identified with amido black 10 B (Wieme, 1965), copper was determined with rubeanic acid (Decleir, 1961) and peroxidase activity was shown with o-dianisidine (Manwell & Baker, 1963).

RESULTS

The different protein patterns obtained at pH 6-5 with the blood from very small embryos to adult animals are summarized in Figs. 1 and 2. After hatching, distinction was made between animals fed on amphipods (which proved to be a very adequate food source) and starved animals (Fig. 2). The latter swam actively

FIG. 1. Electrophoretic patterns of the blood proteins from embryos and young S. oficinalis specimens at pH 6.5.

R e l a t i v e m o b i l i t y

( % )

120 - 100 -

8 0 -

60 -

40 -

20 -

0

L e n g t h 1

(mm) *

9 10 11 12 13 15 18 30

C

EMBRYO nrrcxlws YOUNG A N I M A L (FED)

,--... ! f l . 5.2

Fraction

number

'

88$&30 . ,..-.\ : . . m o m .--*

C-'; 8 8 a 0 :::;> ,:y< ,:.,. 0 0 <--; . _ .- ,..- .--.. ..-.: :.-----.? c--.-:; : : .*-.:

0 0 @:o @ 0 oio

QaQ

111 I1 I

1111

1111 lllC

IV

v

YI

YII

Vlll

during the first 3-5 days and afterwards they floated head downwards on the surface of the water, but stayed alive until a few days after all the yolk reserve was utilized (13-16 days).

The relative mobilities of the protein fractions are arbitrarily expressed as the percentage of the mobility of the adult hemocyanin fraction. The length of the animal is always expressed as the length of the mantle as explained in a previous publication (Decleir et al., 1970).

(1) Blood proteins in embryos As can be seen in Fig. 1 all the embryonic blood proteins have a very low

electrophoretic mobility increasing with the length (or the age) of the embryo. Embryos from 1 to 5 mm have one single protein fraction (VIII), which shows a positive peroxidase reaction and a very weak reaction with rubeanic acid. Prob- ably our rubeanic acid technique for the demonstration of copper was not sensitive enough for these small quantities of blood (Decleir, 1961; Decleir et al., 1970).

In 6-mm and larger embryos we found three new protein bands (VII, VI and V) with a positive reaction for copper and peroxidase. Some time before hatching another fraction with the same histochemical properties (IV), but with a higher electrophoretic mobility was found. This disappeared when the animals were about 14 days old.

FIG. 2. Electrophoretic patterns of the blood from S. oficinalis specimens during the first days after hatching at pH 6.5.

Fraction number

I

111 111

l l l d

1111 l l l C I Y

Y

Y I

Y l l

0 n

S U r V I ,-----.. y -.__..- I

--a- ..--... ,.---. _.--. ....-.-. ....-.: . ..-.. ?*

a

-----

I 8 12 14 18

4 C

YOUNG ANIMAL(STARVED)

Relative mobility

( % )

120 -

100 - t:::::>

80 -

a a ,----...

40 -

20 -

0

Age (days)

-__ _.- ::::>

a a

----.---

1 8 12 14 1.8

L

YOUNG ANIMAL (FED)

DIFFERENTIATION OF BLOOD PROTEINS IN SEPIA OFFICINAL.IS 927

(2) Blood proteins in young animals Young animals showed a quickly changing electrophoretic protein pattern

from hatching to the age of 2 months. The slowest protein bands disappeared whilst new bands with higher electrophoretic mobility appeared. All the protein bands obtained during this period showed a positive reaction for copper, peroxidase and phenoloxidase and may be considered to be hemocyanin (Decleir & Richard, 1970). I t was only after this period that a non-hemocyanin fraction appeared in the blood. This fraction showed a negative reaction for copper, peroxidase and phenoloxidase but a positive reaction for glycoprotein. All animals older than 2 months had only one hemocyanin fraction (fraction I) and one non-hemocyanin fraction (fraction 0).

When no food was given to the animals after hatching, the blood protein patterns showed striking differences. Figure 2 compares the blood proteins between fed and starved animals of the same age. The mantle length is no longer a good criterion since starved animals grow only 1 or 2 mm in 2 weeks. As shown in the Figure the blood proteins I11 A, I11 B and I11 C also appeared in starved animals, but their appearance was much retarded as compared to normally fed individuals. The most characteristic feature of starved animals, however, was the persistence of the protein fractions IV and VI, which disappeared soon after hatching in fed animals.

DISCUSSION

During the ontogeny of S. oficinalis L., we have found eleven different protein fractions in the blood. At pH 6.5 the slowest migrating protein fraction appears first and then new fractions arise with higher and higher electrophoretic mobility whilst the slowest fractions disappear. All these fractions are numbered from I to VIII (see Figs. 1 and 2) and they all have the same histochemical properties, which resemble the histochemical properties of adult hemocyanin on electro- pherograms. Therefore the blood proteins found during ontogeny in S. oflcinalis are all copper proteins with "hemocyanin-like" properties. I t is well known that when hemocyanin is present in the blood, this protein always forms the bulk of the protein ranging from 80 to 100 per cent of the total blood proteins (Goodwin, 1960; Ghiretti, 1966). The first protein fraction with no hemocyanin-like proper- ties is found in animals older than 2 months.

All these results point to the probable existence in the blood of S. oficinalis L. of embryonic, young and adult hemocyanins. Additional proof for this would be the determination by an ultra-micro-method of the oxygen dissociation curves of the different hemocyanins. Very probably the different respiratory pigments are adapted to the different oxygen needs of the animal and to different outside oxygen pressures during ontogeny.

The fact that we have found eleven different protein fractions does not mean that there must be eleven different hemocyanins replacing each other during ontogeny. Several protein fractions may be subunits belonging to the same complex protein molecule. Hemocyanins are copper proteins with a very high

molecular weight (several million in molluscs) and they easily split into their com- ponents at pH values remote from the isoelectric point. Where and how the different hemocyanins are formed cannot as yet be said with certainty. However, when we compare the differentiation of the blood proteins with the differentiation of other systems and organs we are able to build up a very plausible explanation for the origin of the first embryonic hemocyanins.

During more than half the embryonic life (embryos from 1 to 5 mm) we can find only one protein fraction, which must be the first hemocyanin. In a previous publication (Decleir et al., 1970) we have shown that the total amount of copper in the initial yolk sac is the same as in the newly hatched individual and that the copper must be transferred from the yolk sac to the embryo. The work of Portman & Bidder on Loligo (Portman & Bidder, 1928) and our work on Sepia (Lemaire & Richard, unpublished results) have thrown some light on the way in which the yolk is absorbed by the embryo proper. In the early embryo there exists only an outer yolk sac, which is entirely surrounded by a blood space, which lies between the perivitelline membrane and the extra-embryonic ectoderm (Fig. 3a). More- over, there exists a complete embryonic blood circulation between the embryo and the outer yolk sac. Later, part of the outer yolk sac enters the embryo and becomes the inner yolk sac. This inner yolk sac is still entirely surrounded by a blood sinus, which will later become a part of the circulatory system. Meanwhile the blood circulation of the outer yolk sac has lost its function. It has been interrupted by the growth of the arm musculature of the embryo. The yolk itself is transformed by the perivitelline membrane and is then transferred to the blood. The protein fraction which is formed by the transformation of yolk by the outer and inner vitelline membrane must be our electrophoretic fraction VIII.

Later on, yolk absorption occurs in a more complicated way in which the liver and perhaps also the pancreas are involved. Both organs arise as "hepatopancreas rudiments", a pair of diverticula, given off from the junction of the fore- and mid- gut. This hepatopancreas grows forward between the yolk sac and the body wall. The anterior part which lies against the yolk sac will become the liver; the posterior part will become the pancreas. When the liver first develops the blood space between the yolk sac and liver disappears and later the mesoderm cells which separate the liver and inner yolk sac also disappear. The perivitelline membrane and the liver cells come into close contact with each other and from now on the liver takes an active part in the yolk absorption. The yolk sac becomes smaller and smaller while the liver develops (Fig. 3b). "The yolk sac which began its existence almost entirely surrounded by blood ends it almost entirely surrounded by liver. The function performed at first by the blood is later fulfilled by the liver" (Portman & Bidder, 1928).

The change from yolk absorption by blood to yolk absorption by the liver very probably corresponds with the disappearance of the blood protein fraction VIII and the appearance in the blood of the protein fractions VII, VI, V and IV. After hatching the liver continues its development (Fig. 3c), which allowed us to recog- nize twelve different stages as published earlier (Richard & Decleir, 1969). These

FIG. 3. a. Meridional section through the outer yolk sac of an embryo (mantle length = 0.5 mm). b. Frontal section through an embryo (mantle length = 6.0 mm). c. Transversal section through a young animal 5 days old. a.l., is the anterior lobe of the internal yolk sac; ex. e., the extra-embryonic ectoderm; 1, the liver; p.s., the perivitelline sinus; y, the yolk; y.s., the yolk syncitium.

DIFFERENTIATION OF BLOOD PROTEINS IN SEPIA OFFICINALIS 929

stages correspond with different blood protein patterns (appearance in the blood of protein fractions I11 C, I11 B, I11 A, I1 By I1 A and I).

Starved animals which depend entirely on their yolk reserve do not lose the protein fractions VI and IV. Therefore the uptake of food must play an important part in the differentiation of blood protein fractions in young animals.

We have tried other electrophoretic techniques, but only agar gel electrophoresis proved to be an ideal supporting medium for our experiments. Electropherograms obtained with starch or cellogel showed the same evolution of protein bands with increasing electrophoretic mobility, but in these gels the difference between the lowest and the highest mobilities was much smaller than in agar gel. In these conditions it is often very difficult to decide if the protein bands obtained are composed of one or several protein fractions.

CONCLUSION We have found in S. oficinalis L. different blood proteins which replace each

other during ontogeny in such a way that the slowest migrating electrophoretic fractions appear first and are gradually replaced by faster protein fractions. These proteins are probably all hemocyanins. Consequently we find embryonic, young and adult hemocyanins just like these embryonic, foetal and adult hemoglobins found in vertebrates. The first embryonic hemocyanin can be found in the smallest embryos. I t is then the only protein fraction in the blood and is produced by the perivitelline membrane which transforms the yolk into blood protein. Later on, this role of the vitelline membrane is taken over by the liver. This corresponds with the appearance of other hemocyanins in the blood.

In young animals still other hemocyanins appear in the blood. The uptake of food after hatching enhances the formation of these young hemocyanins. In starved animals some embryonic fractions remain in the blood. In 2-month-old animals and older ones we only found the adult hemocyanin and a glycoprotein, which does not contain copper.

Without doubt hemopoiesis in invertebrates, just like in vertebrates, is a very complex physiological system, wherein many different organs may be involved. Therefore the hemopoietic function of the perivitelline membrane and the liver in the embryo may be taken over by different organs in the adult. This is a subject for further research.

Acknowledgements-We wish to thank Professor Defretin (University of Lille) and Professor Vercauteren (University of Ghent) for their continued help and interest in this work and for offering ideal working conditions. We also thank Miss Coulombez for valuable technical assistance. Finally, we are very indebted to the Nationaal Fonds voor Weten- schappelijk Onderzoek for a grant to one of us (W. D.).

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Key Word Index--Hemocyanin; blood proteins; embryonic hemocyanin; Sepia oficinalis; differentiation o f blood proteins; physiology o f cephalopods.