on the buoyancy of some deep-sea sharks

On the Buoyancy of Some Deep-Sea SharksAuthor(s): E. D. S. Corner, E. J. Denton and G. R. ForsterSource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 171, No.1025 (Feb. 25, 1969), pp. 415-429Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/75799 .

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Proc. Roy. Soc. B. 171, 415-429 (1969)

Printed in Great Britain

On the buoyancy of some deep-sea sharks

BY E. D. S. CORNER, E. J. DENTON, F.R.S. AND G. R. FORSTER

The Plymouth Laboratory of the Marine Biological A8sociation of the United Kingdom

(Received 22 May 1968)

[Plates 17 and 18]

Fish of five species of deep-sea squaloids (Centrophorus squamosus, Centroscymnus coelolepis, Dalatias licha, Deania calcea and Etmopterus princeps) and one-deep sea holocephalan (Hydro- lagus afftinis) were all found to float when brought to the surface and placed in surface or laboratory sea water. However, by taking account of the effects of salinity, temperature and pressure differences between this seawater and that inwhich the animals lived, it is shown that all these animals must have been very close to neutral buoyancy at the bottom of the sea. Every one of these fish had an enormous oily liver and the lift which this gave almost exactly compensated for the weights in sea water of the rest of the animal. These livers contained large amounts of the hydrocarbon squalene which is not a convenient material to have as a metabolic reserve but which, with its low specific gravity (0 86), is particularly suited to give lift, being 80 % more effective per unit weight for this purpose than cod-liver oil. It is calculated that because of this unusual oil such fish not only obtain the lift needed for neutral buoyancy more economically in terms of the weight of oil required, but also in terms of the metabolic energy which has to be used to provide the oil-store responsible for buoyancy. It is argued that these fish must carefully regulate the oil content of their livers so as always to balance exactly the weight in sea water of their other tissues. The mechanism whereby they do this is not known.

INTRODUCTION

Neutral or near neutral buoyancy can obviously be of very great advantage to a mid-water animal and it is not surprising to find that many such animals, e.g. the cranchid squid and most gelatinous species, have specific gravities close to that of the sea water in which they live. Some of the most elegant buoyancy arrangements are found, however, in animals such as the cuttlefish and the conger eel, which are caught on or very close to the bottom of the sea. Although a fast-moving animal which is denser than sea water can easily obtain lift from its forward motion,* this would be much harder for an animal like the cuttlefish, which, although it sometimes buries itself, hunts its prey by swimming slowly and hovering over the bottom of the sea. Animals which do this must profit enormously by being able to swim and hover with a minimal disturbance of the surrounding water. In this paper we show that some deep-sea sharks of the family Squalidae which were caught on the bottom are as close to neutral buoyancy as any other group of animals.

* Many sharks generate a good deal of lift by using their pectoral fins as hydrofoils. For a giveni area of fin such a fish can, over a limited range of velocities, obtain constant lift by varying the angle of attack of its fins; increasing the angle to compensate for decreasing forward velocity. However, when this angle is increased beyond some critical value the lift falls instead of rising and the fish cannot avoid stalling if it goes too slowly. If a fish is to stay off the bottom when moving slowly it must either have a very large fin area, or a reduced weight in sea water or swim in a different way from the common sharks.

[ 415 ]



416 E. D. S. Corner, E. J. Denton and G. R. Forster

Many teleosts bring themselves close to neutral buoyancy by using gas-filled swimbladders; others gain a good deal of buoyancy from the fat which their tissues or sometimes swimbladders contain (Tressler & Lemon i95i; Marshall i960). Cartilaginous fishes do not have swimbladders but fat is often stored, particularly in the liver. Thus Hickling (1930) noted that the spur dog, Squalus acanthias L., and other sharks, must gain a good deal of lift from their livers, and Harrison Matthews & Parker (I950) that the basking shark, Cetorhinus maximus (Gunnerus), is only a little denser than sea water and obtains a great deal of buoyancy by the same means.

It has also been known for some time that the liver oils of certain sharks contain large amounts of the hydrocarbon squalene, but its role in these livers has not been defined. For example, Heller, Heller, Springer & Clark (I957) found that squalene accounted for 70% of the liver oil in Dalatias licha (Bonnaterre) and 90 % in Centrophorus nyato (Rafinesque), but could offer no explanation of its function. Here we examine quantitatively the buoyancy of some deep sea sharks and show they are all so close to neutral buoyancy, and their liver oils have such an unusual composition, that a principal function of the liver must be to give buoyancy and that of its oil content must be regulated to give neutral buoyancy. A brief account of this work has been given earlier (Corner, Denton & Forster, cited by Denton 1962).

MATERIAL

Most of the deep sea sharks used in the present experiment were caught by R.V. Sarsia by a long line in the Bay of Biscay (Forster I964). They were all of the family Squalidae. Those caught on a cruise in July 1960 were wrapped in several layers of Polythene sheet and stored in a cold room and studied later in the Plymouth Laboratory. Others, caught in May 1961 and May 1964 were weighed in sea water aboard ship before being stored in the same way in a cold room for later study ashore.

A few direct observations of the deep-living squaloids have been reported by Peres (I958) from the bathyscaphe F.R.N.S. III. The fish, a Centrophorus sp. and an Etmopterus sp. were observed swimming very close to the bottom. From experi- ence with deep line fishing Forster (I964) found that for successful catches the hooks should be touching the sea bed. It seems probable that those squaloids normally taken on the bottom in deep water do not range far up into mid-water levels, although Hickling (1928) reported one instance of a Centrophorus squamosus (Bonnaterre) taken by line 70 fm above the bottom in 460 fin, and one fish of this species was caught in mid-water, about 1000m off the bottom, from R. V. Sarscia in 1968.

METHODS

Weighings of whole animals under sea water

At sea, the sharks were placed in an improvised tank containing sea water continuously pumped from the surface of the sea. They were left for some time to reach the temperature of the surface sea water and they floated at the surface of the tank. To find the weight of such a shark in this sea water it was loaded with small



The buoyancy of some deep-sea sharks 417

pieces of lead until it just sank. These pieces of lead were then stored and later weighed in the shore laboratory. This is a very sensitive method of weighing; 1 or 2 g of lead will determine whether a fish weighing several kilograms sinks or floats and the movement of the ship does not significantly disturb the accuracy of the method.

The weighings in air were made using a calibrated spring balance at sea and checked on accurate balances in the Plymouth Laboratory. When animals were brought back to Plymouth they were taken from the cold room and allowed to warm to room temperature in their Polythene wrappings before being unwrapped and weighed in sea water of approximately the same temperature.

Weighings of parts of animals under sea water

The abdomens of these fish were opened in the Plymouth Laboratory and they were all seen to have enormous livers (figure 2, plate 17). One specimen of Centro- scymnus coelolepis (Bocage & Capello) was also carrying a number of very large eggs and these were very close indeed to neutral buoyancy. Every liver floated in sea water. To estimate the upthrust of a liver, pieces of lead were added until it just sank, and both liver and lead were then weighed under sea water on a sensitive torsion balance: in some cases the liver was placed in a Polythene bag for this purpose. From the weight of the lead in sea water and the weight of liver plus lead in sea water the upthrust of the liver could be calculated.

The animals without their livers were all appreciably denser than sea water. They were weighed under sea water with a calibrated spring balance.

Extraction and properties of liver oil

After the liver had been removed from the fish and weighed it was wrapped in a Polythene bag which was placed in an airtight jar and kept in a deep freeze until needed for analysis of the oil content. To illustrate the method used in extracting and analysing the fat the following detailed account is given for a specimen of Etmopterus princeps.

The liver was taken out of the Polythene bag, sliced and drained of oil. The residue was weighed, shredded and centrifuged at 6000 g for 5 min and the super- natant oil then added to the oil already drained from the liver. Droplets of oil which remained in the Polythene bag and jar were removed with chloroform which was evaporated and the residue added to the total yield of oil obtained by draining and centrifuging. The combined fractions were then weighed (169-6 g). The liver left after centrifuging was weighed, then admixed with sand and anhydrous CaSO4 and ground into a paste with a pestle and mortar. The paste was then placed inside a Soxhlet thimble and extracted with boiling light petroleum (b.p. 40 to 60 'C) until no more oil could be removed. The solvent was removed by distillation and the oil so obtained then weighed (111.9 g) and added to the combined samples extracted earlier. The total quantity of oil was found to account for 81 % of the liver by weight (figure 1). Itwas paleyellow in colour an.d transparent; and its specific gravity, as measured by the density-bottle method, was 0-88 at room temperature.

An accurately weighed sample of the oil (1.750 g) was saponified by refluxing




with 25 ml. of 0.5 N alcoholic KOH and the saponification number then deter- mined in the usual way (Nicholls I952). The values were extremely low (45-7 and 45 0 in duplicate determination), which implied that a considerable fraction of the oil was unsaponifiable -

..~~~~~~~~~~ 4.. ~ ~ ~ .. . . . . .

FIGUXRE 1. Centrifuged liver of deep-sea squaloid showing the very large fraction of oil which it contains.

A known weight of the oil (21 9 g) was first saponified by refiuxing with 250 ml. Of 0*5 N KOR, and the ulnsapomifiable material then extracted from the mixtureF with di-ethyl ether, using the procedure described by Nicholls (1952). The quantity of oil so obtained (17.2 g) was such that 78*5 %/ of the starting material was unsaponifiable, and knowing this, the saponification number of the saponifiable fractions could now be calculated as 209 to 212, a value slightly higher than that reported for fish oils (cod-liver oil, 180-190; dogfish-liver oil, 170 (Tressler &r Lemon ' 95'I) .

Isolcation of sqv,alene fromn the ubnsaponiftable fraction

The pale yellow starting material was absorbed over a column of alumina which was then eluted with benzene. The eluate, when freed from solvent, yielded a colourless oil. A known quantity of this oil (4 34 g) was dissolved in acetone (20 ml.) and treated with dried HOl gas at 0 00 until a copious crystalline mass had formed. This was removed by ifiltration and the ifiltrate again treated with HOl gas until further quantities of crystals were obtained. This process was repeated until no more crystalline material was formed. The combined yield of crystals was washed with acetone and dried to constant weight and weighed. The total yield was 2s785 g of crystalline material. This product was recrystallized twice from acetone, finally being obtained as colourless needles which were dried thoroughiy in a desiccator at room temperature. Melting point 126 to 127 00 (not depressed by admixture with the derivative prepared from a purified sample of squalene (specific



Corner, Denton & Forster Proc. Roy. Soc. B, volume 171, plate 17

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The buoyancy of some deep -sea sharks 419

gravity 0.86) supplied by L. Light and Co. Ltd., m.p. 128 to 132 'C.) Found: C, 57-6; H, 941; Cl, 33-5 %. Squalene hexahydrochloride (C30H56C16) requires 0, 57-2; H, 8-9; Cl, 33-7 %. (Microanalyses were made by Drs Weiler and Strauss, Oxford.)

From the amount of squalene hexahydrochloride obtained it was calculated that squalene accounted for 42 <)/O of the oil. This, however, is a minimum value because of losses encountered during the organic preparation.

In addition to squalene, the liver oil contained a high proportion of material which formed pale yellow crystals at room temperature. At first it was thought that cholesterol might be present, and some evidence of this was obtained with the Liebermann-Burchard test. However, because of the presence of squalene, the colour changes characteristic of cholesterol were difficult to discern with any confidence; moreover, attempts to extract cholesterol as the acetate derivative from Etmopterus liver oil (the only material which gave any sign of cholesterol with the Liebermann-Burchard test) were unsuccessful. It seems unlikely, therefore, that cholesterol was present to any considerable extent in the various liver oils examined, and the yellow crystalline material observed in the oil from Etmopterus remains unidentified.

MeIasurements of the specific gravities of the oil extracted from livers

Specific gravities were measured both by density bottle and by the method of Dulong and Petit. In the latter method the oil is placed in one arm of a U tube and balanced against water in the other. The heights of the columns of oil and water above the junction between oil and water were measured with a cathetometer which was very kindly given to the authors by the late Dr T. H. Oliver. The ratio of these heights gave the specific gravity of the oil. Very broad tubes (about 1 cm in diameter) were used so that surface tension effects could be disregarded. The method of Dulong and Petit is very convenient for measurements of the specific gravity of oil over a range of temperature because the columns in the U tube can be measured in warm and cold rooms without handling the oil.

Corrections for differences in temperature, salinity and pressure between the sea water in which the animals lived and the sea water in which weighings were made

Although the intact sharks were found to be fairly close in density to the sea water in which they were mneasured, the weights of these animals in the environment from which they were taken can only be predicted after correcting the measured weights for differences in salinity, temperature and pressure.

Let Sa, Ta, Pa be salinity, temperature and pressure respectively of the sea water in which weighings were made and Sb, Tb, Pb be the corresponding quantities for the sea water from which the animals were taken. Approximate values of these quantities for the measurernents in 1960 were

Sa 327 %On Sb = 35-1 %O;

Ta 18-10C, Tb =50C; Pa, = 1 atm, Pb = 155 atm.

27 Vol i7i. B.




The corrections, which were made independently of each other, are listed below. (1) Correction of the whole animal for salinity difference. If V be the volume of

the animal in ml. at 17-5 0C (a standard temperature used in tables) and pressure Pa; da be the density of sea water Sa at 17-5 0C and 1 atm pressure; and db be the density of sea water Sb at 17-5 ?C and 1 atm pressure, then the approximate correction in grammes to be applied to the upthrust of the animal because of salinity difference will be V(da - db). (The densities can be found from the salinities using Knudsen's Hydrographical tables ( 9gO I)).

(2) Correction for change in temperature on the upthrust of the liver oil in sea water. Let: W be the weight of the oil in grammes; dl, and d2 be the densities of the oil at Ta and Tb respectively; and dA and dB be the densities of sea water Sb at Ta and Tb respectively; then, with pressure held at 1 atm, the effect of temperature change alone on the upthrust of the oil in sea water will be

w (dB dA) - CW g

For the actual sea waters given above SA = 351 and for an actual oil, going from 18*1 to 5 ?C, a was found to be 0-0091. The oil had a greater temperature coefficient of expansion than sea water.

(3) Correction for the change in weight of the oil in sea water caused bv the pressure change PA to PB. From the International critical tables (I 927) we find that the compressibilities of natural oils are not usually very different from that of sea water and, in general, differ little amongst themselves. Thus the fractional con- traction (C) [unit volume]-' [atmospheric change in pressure (at 14 5 'C)]-l is 60 x 10-6 for sperm oil, 50 x 10-6 for castor oil and 55 x 10-6 for rape oil; from Ekman (i9io) we find that C for sea water of salinity 3541 is 45 x 10-6. Assuming that the C for the liver oil is 55 x 10-6 then, on going from PA- to PB, the sea water will be compressed less than the oil and the upthrust in grammes which the oil gives will be diminished approximately by the volume of the oil multiplied by 105 (PB -PA) (density of sea water). This will give only a small correction in the present experiment.

(4) Corrections for the effect of temperature in the weight of the shark minus the liver. A 'steak' of dogfish (Scyliorhinus canicula (L.)) weighing 123 g was weighed in sea water, using a sensitive torsion balance, while the temperature of the sea water was changed several times between about 20 and 8 'C. These measurements gave an average change in weight of 0- 13 g, with an increase in weight in sea water as the temperature fell. We have assumed that the tissues of a shark, apart from its liver oil, would on the average have given similar weight changes. We have, for example, taken the approximate effect of a temperature change from 18 to 5 'C to be an increase in weight in sea water of 0.00117 g/g of tissue. This correction is small; so therefore are the errors involved in the approximation.

(5) We have found no relevant information which would allow us to estimate the effect of pressure on the weights of animals minus their liver oils in sea water. Their most important component, which might have a compressibility different from that of sea water, will be the protein. The volume of protein for the whole fish



The buoyancy of some deep-sea shark7s 421

would, in these animals, have been less than the volume of oil in the liver so that, unless the compressibility of protein is very different from that of sea water, this cannot be an important correction.

It will be noticed that the method of cumulative corrections described above assumes that the effects of temperature and pressure are independent of one another. In the estimation of small corrections, such as those found here, the assumption will not lead to any significant errors.

RESULTS

In table 1 we summarize some results for two specimens of Centroscy1nnus coelolepis caught in July 1960 and in table 2 we give calculated corrections to these animals' weights in sea wa,ter and estimate approximately the weight which they must have had in their original environment at the bottom of the sea. These corrections are small and the net correction for temperature, salinity and pressure differences together is only about + 0 15 % of the weights of the animals in air.

TABLE 1. EXPERIMENTS ON FISH CAPTURED IN JULY 1960

Centroscymnus coelolepis fish 1 d fish 2 d

wt. of fish in air (g) 5260 3195 wt. of liver in air (g) 1550 790 vol. of liver (ml.) 1718 870 density of liver 0 904 0.908 vol. of fish (ml.) 5149 3117 wt. liver (% total wt.) 29-5 24-7 vol. liver (% total vol.) 33.4 28 0 wt. animal in sea water (Plymouth) (g) - 18 -3 wt. (animal-liver) in sea water (g) 190.5 98

wt. (animal - liver) in sea water 100 xK 5*15 4*06 wt. (animal-liver) in air

wt. of liver analysed as % of total liver wt. 94 94 wt. of oil in liver as % of wt. of liver 82.2 803 vol. of oil in liver as % of vol. of liver 84.9 83.2

* Note. Negative weights mean that the fish floated in sea water.

In table 3 we give the results of weighing on specimens of four other sip ecies o Squalidae obtained on a cruise in May 1961. We have not calculated corrections for the individual animals in this group but the net corrections would, for all these animals, have given an increase in their weights in sea water by about 0.1 00 of their weights in air. For the animals caught in 1961 the salinity, temperature and pressure differences to be allowed for were all smaller than the corresponding differences involved in the weighings of 1960. We may conclude from the results of tables 2 and 3 that the Squalidae of all five species studied must have been very close indeed (generally within 04 I %) to neutral buoyancy.

In table 4 we summarize some results on liver oil and its relation to the whole animal.

27-2




Some of the other deep-sea sharks caught on these cruises were used for experiments on their visual pigments (Denton & Shaw I963). One of these turned out to be interesting in connexion with the present experiments because it contained a number of large eggs (see figure 2, plate 17). Although its head was chopped off at sea and its eyes dissected out for another experiment, the results obtained on its buoyancy would not thereby be seriously disturbed. It was a large specimen of

TABLE 2

Centroscymnus coelolepis fish 1 fish 2

weight of fish in Plymouth sea water (g) -18 -3

corrections to be added

For salinity difference for whole fish (g) -9.3 - 5.6

for temperature difference on liver oil (g) + 11.5 + 5.9

for temperature difference on animal + 41 + 2 7

minus liver oil (g)

for pressure difference on liver oil (g) + 2.6 + 1.3

net correction for salinity, temperature +9 + 4.3 and pressure (g)

net correction as a percentage of wt. of + 0.17 + 0.14 fish in air

calculated wt. of fish in its natural -9 + 1.3 environment (g)

100 X wt. of fish in natural environment M - 0.17 + 0.09 wt. of fish in air

TABLE 3

wt. of

wt. of animal animal in sea uncorrected wt. of in surface sea water in wt. in sea water

animal in air water at sea Plymouth wt. in air species, number and sex (g) (g) (g) (%)

Deania calcea (Howe)

3 V 2686 -2-7 +3 5 -0 1 4 2663 -I +1 -0-04 5 9 2565 - 2-8 - -01

Centrophorus squamosus 6 S 7530 -10.5 0 -0-13 7 3 8650 - 27-3 - 0-32

Etmopterus princeps Collet

8 S 1215 - 0 3 - 0 03

Centroscymnus coelolepis (fish 9). It weighed (with head but without eyes) 10670 g in air. When freshly caught it floated in sea water and it was found later ashore to have a weight of -37 g in sea water, i.e. relative to its size it was about as close to neutral buoyancy as was fish 1 of table 1. Its liver weighed 2270 g in air, and its

eggs 2191 g in air. The eggs were very close indeed to neutral buoyancy. The ratio of weight of liver to the weight of the whole animal minus its eggs was 26 7 % i.e. intermediate between the corresponding value for the two male fishes 1 and 2. The ratio of the weight of the animal minus liver and eggs in sea water to its




weight minus liver and eggs in air was 4-2 % which is again a value intermediate between the corresponding values for fishes 1 and 2 (table 1).

To summarize, this aninmal had laid down over 20 % of its weight as eggs: these eggs were neutrally buoyant: the animal without its liver was about 4 % denser than sea water but its weight in sea water was almost exactly balanced by the lift given by oil in its liver.

TABLE 4

Etmo- Centroscymnus Centrophorus pteru-s

species coelolepis Deania calcea squamosus princeps __ _ __ _ _ __ _ _ ___ `

r

number and sex 1 C 2& 3 ? 4& 5 ? 6& 7& 8& wt. of liver as % of wt. of 29.4 247 20.5 20.9 20-2 21.4 21.4 19.5 animal (in air)

wt. (animal-liver) in sea water (%) 5.1 4*06 3.8 3*84 3*9 3.8 4*05 3.53 wt. (animal - liver) in air

specific gravity of liver 0 904 0 908 0 89 0 895 0 886 0 896 0 884 0.891

liver oil as % of wt. (irn air) of 82.3 80.3 86 - - - 81 liver

wt. of liver oil as % of weight of 24-2 19.6 17.6 - - 15.8 animal (in air)

specific gravity of liver oil (room 0.877 0.882 0.876 - - 0866 - 0*875 temperature)

% of liver oil which is 68 67 - - - 79 unsaponifiable

specific gravity of unsaponifiable - 0*861 - 0*868 oil

In December 1963 a specimen of the shark, Dalatia8 licha, of the family Squalidae, was caught off the Cornish coast near Polperro by a local trawler. This fish was interesting in that it was, unlike those of tables 1 to 3, caught in winter and in shallow water. It weighed 15 9 kg in air but only 13 g in sea water; its liver accounted for 24-3 % of its weight in air and it contained 950 g of eggs which were all very close to neutral buoyancy.

Again, in April 1968, another specimen of D. licha was caught close to Plymouth by trawling in about 35 fm. It died after living 2 days in the Laboratory's aquarium, a time sufficiently long for its tissues to come to the temperature of the aquarium sea water (12 ?C instead of 8 0C in the sea) and for its body fluids to come into equilibrium with this sea water (salinity 34 %O instead of 35 %O) The change in the weight of the animal in sea water caused by the change in the sea water would only be such as to increase its weight in sea water by a few grammes. Shortly after its death the fish, which weighed 18 015 g in air, weighed only -9 g in the aquarium sea water, i.e. - 005 % of its weight in air. Its liver accounted for 20 % and its egg-filled oviducts for 23 0 of its weight in air. The animal without liver and oviducts weighed 10 190 g in air and 485 g in sea water, a ratio typical of a muscular animal without any special buoyancy arrangement.

In May 1964 a specimen of the Chimaeroid, Hydrolagus affinis Capello, was caught on the bottom at a depth of about 2400 m. Like other animals it was stored




in a cold room and was studied later in Plymouth. Its weight in air was 13 3 kg; its corrected weight in sea water was only 003 %0 of its weight in air; its liver, which floated in sea water, accounted for 17 3 %0 of the animal's weight in air but the animal without its liver had a weight in sea water of only 1 6 %0 of the weight in air; this is a low value compared with that of the other animals studied here.

Histological observations Figure 3, plate 18, shows a section through the liver of one of these deep-sea

Squalidae (B) and it is compared with a corresponding section of the very fatty liver of the common dogfish, Scyliorhinus canicula (A). The very large fat spaces, and the very large proportions of the livers which are fat, can be clearly seen.

DISCJUSSION

We have seen that all eleven specimens studied, including animals from five species of sharks of the family Squalidae and one deep-sea holocephalan, were all a little less dense than the sea waters in which they were weighed. When corrections were made for the buoyancy changes which must have been caused by the differences in temperature, pressure and salinity between these sea waters and those of their natural environments we find that in life the specimens must all have been very close to neutral buoyancy; closer indeed than most animals which possess acknowledged arrangements for regulating their buoyancy.

Without their very large livers the sharks would have had weights in sea water varying between 3 and 5 % of their weights in air, values characteristic of fairly muscular animals with no special provision for buoyancy control. The upthrusts in sea water of the livers of these sharks were given by the large amounts of oil which they contained. Whilst there was a good deal of variation between the densities of these animals when their livers were removed, the whole animals-including their livers-were all so close to neutral buoyancy that the conclusion seems inescapable that these animals regulated the amount of oil in their livers in order to balance exactly the excess weight in sea water of the rest of their tissues. This implies a control of fat reserves altogether different from that found in most other animals. Normally fat is used as a metabolic reserve, being laid down when food is plentiful; sometimes in large amounts before a long migration, e.g. in the freshwater eel, Anguilla anguilla (L.). In the sharks of the present study, fat metabolism must be exactly regulated in relation to the metabolism of other components (protein, carbohydrate and minerals) so that the animal remains always neutrally buoyant, in particular the ratio of fat to protein in the animals must remain approximately constant. We can illustrate the kind of control of fat metabolism needed by giving a numerical example. A typical shark of this kind would have about 250 g of oil in its liver for every 1000 g of body weight and would be within 0-1 % of neutral buoyancy; it would differ by more than this 0 1 % if it laid down only about 5 g too much or 5 g too little oil.

The composition of the liver oil in these sharks is very interesting. Our experiments conifirm earlier work (table 5) which showed that these livers contain a great




deal of the hydrocarbon squalene. Squalene has a specific gravity of about 086, a particularly low value for a fish-liver oil; cod-liver oil has a specific gravity of about 0-926 (values of 0 910 to 0-935 are giveni in the Handbook of Chemistry and Physies (1954)). The difference hetween 0-86 and 0-926 may not seem very great, but it makes a big difference to the lift which these oils give in sea water. Thus 1 ml.

TABLE 5. LiVER OIL ANALYSES OF VARIOUS DEEP-SEA SHARKS

unsap. characteristics of liver oil matter

liver hydro- wt. oil content content hydrocarbon

as % of liver of unsap. carbon content species body wt. (%) sp.gr. matter content (%) reference

Centrosoymnus owstoni 22-8 80-6 0-8829 61-1 48.5 79*9 Higashi, Kaneko & (Garman), 32 Y + 42 6 Sugii I953a

Detnia (spp.), 18-8 80-5 0-8846 54-5 40 8 65-9 Higashi et al. 1953b 25?+ 126

Centrophorus (spp.), 24-5 81-4 0-8668 8545 77-3 90-8 Higashi et al. I953c 2?+206

C. scalpratus McCulloch - 86-9-90-7 - 77-2-82-6 - -

Hydrolacgus ogilbyi _ 900 _ 34-2 - Cowper & Downie (Waite) I 957

Scymodon plunketi - 93-4 - 28-8-294 -

(Waite) Dalattias licha 20'8 76-8 0-8787 67-5 50'8 73-1

(Bonnaterre), 3 l Somniosus pacificus 17-4 56-7 08987 29-8 7-2 24-4 (Bigelow & Schroeder), Higashi et al. I954

2?+IS Heteroscymnus longus 14-9 72-6 08915 43*4 18-6 41-7

(Yanaka), 2 ? + 2 X

of squalene will give a lift of 0-166 g in sea water (specific gravity 1-026), whereas 1 ml. of cod-liver oil will give a lift of about 0 1 g. The difference is even greater in terms of equal weights of oil, for I g of squalene will give a lift of 0-194 g, whereas I g of cod-liver oil gives a lift of only 0 108 g. We see, therefore, that although the livers of these sharks are enormous they would have to be very much larger if they achieved neutral buoyancy by storing an oil like cod-liver oil.

It is not known whether these sharks are able to synthesize squalene either from acetate or any other precursor, and the possibility cannot be excluded that the source of the hydrocarbon iis dietary, although Blumer (T967) has observed that squalene, which accounts for 11P8 to 20-7 % of the liver oil in the basking shark, Cetorhinus maximus is present in only trace amounts (0- 14 % of the total lipids) in the zooplankton on which this animal feeds. Assuming that an animal could synthesize either squalene or cod-liver oil from a central pool of acetate we calculate that the synthesis of 1 g of squalene will require 2-6 g of acetic acid, whereas to obtain the same lift the fish would have to use 3'5 g of acetic acid in making cod- liver oil (according to data for the composition of cod-liver oil provided by Guha, Hilditch & Lovern 1930). Thus if the function of the oil is to give buoyancy, it is




much more economical-in terms of acetate-to make squalene in place of the more common fish oils. No previous work appears to have been done on the synthesis of squalene in sharks. Bloch (I965) mentions an attempt to study the problem using [14C]-labelled acetate, but concludes that 'experiments with the intact sharks or with the lipid-rich shark liver posed considerable technical difficulties'. However, a great deal of work has been done to elucidate the synthesis of squalene in mammals, and a good summary of the data is given by Wright (i 96I). Briefly, squalene is biosynthesized by (1) condensing three molecules of acetyl-CoA to form ,G- hydroxy-fl-methylglutaryl-CoA; (2) three molecules of ATP are then used in a stepwise conversion of this compound into the isoprene unit dimethylallyl pyrophosphate; (3) three such isoprene compounds then combine to form a C15 product, farnesyl pyrophosphate; (4) two of these C15 compounds condense to form squalene. Thus, the biosynthesis of squalene involves 18 molecules of acetyl-CoA and 18 molecules of ATP. By contrast, the biosynthesis of the C18 oleic acid requires 9 molecules of acetyl-CoA and 8 of ATP (assuming that one molecule of acetyl-CoA combines with 8 of malonyl-CoA, each formed from one molecule of acetyl-CoA, C02 and ATP). Acetyl-CoA and ATP are compounds of the sort described as 'energy-rich' and the AG values for their hydrolysis are roughly the same (- 8 2 kcal for acetyl-CoA and - 77 keal for ATP -> ADP; Segel I 967) . It follows, then, that the synthesis of 1 g of squalene requires about 0-7 kcal of energy, whereas the synthesis of 1 g of oleic acid requires 05 kcal of energy. These energies are small relative to the calorific values of the acetic acid used as starting material :* moreover, squalene gives so much more lift per gramme than oleic acid that even in terms of energy of synthesis it is still more economical to make squalene for buoyancy.

It seems unlikely, from work with mammals, that squalene, once formed, can be converted back into 2-carbon units. Thus, Langdon & Bloch (1953a) showed that when [14C]-labelled squalene was administered to rats, no labelled carbon could be detected in the fatty acid fraction: had the hydrocarbon been broken down into 2-carbon units these should subsequently have been incorporated into fatty acids. If the sharks resemble mammals in not being able to convert squalene into 2-carbon units but only into sterols (Channon l926; Langdon & Bloch I953 b) the argument that the hydrocarbon is laid down by deep-sea sharks as a means of obtaining buoyancy is greatly strengthened but the fine regulation of buoyancy would then be more likely to depend on the other lipids than squalene.

It is known that elasmobranchs which are denser than sea water support their weights in sea water by dynamic lift given by their tails and pectoral fins (Grove & Newell I936; Harris I936; Alexander I965). We might, therefore, expect neutrally buoyant elasmobranchs to have tails and pectoral fins which differ from others either in form or in motion. We have not made any observations on the motion of these fishes but we have studied their form. Since these fish, in common with sharks denser than sea water, possess heterocercal tails, it is evident that this form

* The heat of combustion of acetic acid, calculated from values for individual bond energies cited by Roberts & Caserio (I965), is roughly 3-7 kcal/g. Some 10 kcal of energy as acetate will therefore be used for each gram of squalene synthesized.




of tail need not generate lift. However, in figure 4 we show that the pectoral fins of the neutrally buoyant sharks are relatively very much smaller than those of denser sharks of roughly the same shape. This, together with the fact that the sharks studied were caught at various times of year, i.e. in April, May, July and December, supports the idea that these animals are normally close to neutral buoyancy and not neutrally buoyant only in some particular season.

S.c.

M.a. S.c. &M.a.

4 -Sc..O S.s. 0' S.S. ]?g *S.a.

~0-2 -

C.c. C.c. *C.S. C D.L.

Cc. C.m.

OL - -- I b 2 4 6 log1oW

FIGuRE 4. This shows that sharks close to neutral buoyancy (group B) have pectoral fins relatively smaller than those of similarly shaped fish that are appreciably denser than sea water (group A). S, area of one pectoral fin (cm2); W, weight of animal (g); Wi is roughly proportional to (linear dimension of fish)2. Group A: S.c., Scyliorhinus canicula; S.s., Scyliorhinus stellaris (L.); Mf.a., Mustelus asterias Cloquet; S.a., Squalus acanthias; P.g., Prionace glauca (L.). Group B: E.p., Etmopterus princeps; C.c., Centroscymnus coelolepis; C.s., Centrophorus squamosus; C.m., Cetorhinus maximus; D.l., Dalattias licha. We are indebted to Dr Q. Bone for the finding that the weight of Cetorhinus maximus in sea water is < 05 % of its weight in air; and to Dr B. L. Roberts for measurements on two specimens of Squalus acanthias, and on the very small dogfish, Scyliorhinus canicula.

The use of low density lipids by some other animals Basking sharks

It has been claimed (Lovern I962: citing the work of Pliva & Sorenson 1950) that pristane always occurs in relatively small amounts in the livers of certain elasmobranchs that contain squalene. Thus, the liver-oil of the basking shark is reported to contain 7 to 45 % squalene and 2 to 7 0 pristane (Schmidt-Nielson & Toft-Erickson 1945: cited by Lovern i962). A later study by Blumer (i967)




confirms the presence of squalene in the liver-oil of the basking shark, Cetorhinus maximus, the values in this case being 11b8 to 20-7 % (squalene) and 1-11 to 1-31 00

(pristane). Blumer finds that these hydrocarbons also occur together in zooplank-

ton, but the actual concentrations are much lower and it is pristane that is present in larger amounts in these animals (pristane, 1 0 00; squalene, 0'14 00 of the total

lipids).

Copepods

Blumer, Mullin & Thomas (I963) have made the interesting observation that

pristane, a hydrocarbon resembling squalene in being derived from isoprene units

(in this case via phytol), is present in certain zooplanktonic animals, particularly the copepods Calanus hyperboreus and C. finmarchicus, and they argue that it aids

regulation of buoyancy. The specific gravity of pristane (0.78) is lower than that of

the other lipids in these animals (0-88); and Blumer et al. claim that if the non-

metabolizable hydrocarbon pristane is used for buoyancy purposes, a relatively larger quantity of lipids of a metabolizable kind will be made available as an energy

source. It should be pointed out, however, that the highest concentration of

pristane found was in C. hyperboreus where it accounted for only about 3 0 of the

other lipids (and less than 1 % of the dry body weight of the animal). This means

that even with its low specific gravity pristane only provides about 5 % of the lift

given by these other lipids: an insignificant contribution to buoyancy compared with that of the squalene in sharks.

Whales

There are great differences in the properties of different whale oils. Thus the

specific gravity of the blubber oil from the whale, Balaena mysticetus has a specific

gravity of 0-917 to 0-924 (at 15 00) whilst the specific gravity of sperm oil is 0-878

to 0.884* (Handbook of Chemistry and Physics 1954). We have, therefore, exactly the same kind of difference between these blubber oils as between the liver oils of

the cod and the deep-sea Squalidae. The whale oils of low specific gravity do not

contain squalene but semi-solid waxes of low specific gravity. Tressler & Lemon

(I95 i) have noted that these waxes come principally from the sperm, bottle nose

and beaked whales and that they are also found in other toothed cetaceans especi-

ally those which dive deeply for squid. We can well understand why there should be differences between the two groups

of whales. The sperm and similar whales dive so deeply that the gas in their lungs must be compressed to a volume which would make it unimportant in giving lift:

the only effective buoyant component is, therefore, the oil; and it is clearly advantageous to achieve the necessary lift in the least volume. Blubber oils must

also give the baleen whales appreciable lift but, since they feed relatively close to

the surface of the sea, the gas space in their lungs must always be an important variable determining their specific gravities.

* The spermaceti, from the extraordinary head of the sperm whale, has a specific gravity of 0905 to 0O910. The function of the spermaceti is not known.




REFERENCES

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on the buoyancy of some deep-sea sharks

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