J. mar. biol. Ass. U.K. (1964) 44, 2°3-2°7 2°3 Printed in Great Britain THE COMPOSITION OF GAS IN THE CHAMBERS OF THE CUTTLEBONE OF SEPIA OPPlelNALIS By E. J. DENTON AND D. W. TAYLOR The Plymouth Laboratory and the Department of Physiology, University of Otago Medical School, Dunedin, New Zealand (Text-figs. I and 2) Denton & Gilpin-Brown (1961 a-c) and Denton, Gilpin-Brown & Howarth (r96r) have argued that the liquid which fills a newly formed chamber of the cuttlebone is pumped out by some active 'osmotic' process. A space is left behind which contains gas under very low pressure and into this space gases from the tissue slowly diffuse until the gases in the space and tissue come to equilibrium. This communication gives an account of analyses of gas from various chambers in the cuttlebone for 02' CO2 and N2 and shows that these gas compositions are consistent with this hypothesis. MATERIALS AND METHODS The cuttlefish, Sepia officinalis (L.), was freshly killed and the cuttlebone carefully dissected out taking care not to scratch its surface. The gases were extracted using the apparatus shown diagrammatically in Fig. 1. The cuttle- bone was punctured by the hypodermic needle (h) through a rubber patch stuck over its soft ventral surface. This hypodermic needle was connected through a two-way tap (t) to a long thin capillary tube leading to a manometer (m). The tip of the needle was slightly bent inwards to avoid its becoming blocked by the material of the cuttlebone. The needle, the tap and part of the capillary were initially filled with mercury. The cuttlebone was driven on to the hypo- dermic needle slowly and once the tip of the needle was sealed into the rubber patch the tap was turned so as to connect the needle to the capillary tube. The time when the needle penetrated the first chamber containing a gas space was known because such a chamber contained gas at a pressure appreciably less than atmospheric and so the mercury began to be pulled into the cuttlebone. The arm (a) of the mercury manometer was then lowered until the pressure in the manometer became lower than that in the chamber and the mercury was pulled back into the capillary. The penetration of successive chambers could be noted by successive displacements of the mercury in the capillary tube. When the required number of chambers had been punctured more gas was drawn into the capillary tube and then pushed, under mercury, through the 204 E. J. DENTON AND D. W. TAYLOR cup (C) of the tap into a gas pipette. The gas was transferred to a Scholander gas analysis apparatus and the fractions of CO2 and 02 found. The residual gas was assumed to be N2• The hypodermic needle was finally withdrawn and the cuttlebone cut into two and shaved down until the path of the needle could be seen. The number of punctured chambers was then counted. m Fig. 1. Diagram of apparatus used to extract gases from the cuttlebone. RESULTS One unexpected and interesting result came out of these experiments. The experiments were first tried on mature Sepia in April and only gas from the older chambers could be analysed for it was found impossible to extract sufficient gas for analysis from the most newly formed chambers. This was because their walls were very close together and therefore the chambers were of small volume. A diagram of such a cuttlebone is shown in Fig. 2A. The experiments on the newly made chambers were made on younger animals in December; a diagram of a cuttlebone at this time of year is shown in Fig. 2 B. It can be seen that in April there are two sets of closely spaced chambers (a) and (b). The older set (a) is that described by Adam (1940, p. 89). It was suspected that the two sets of closely spaced chambers in April represented periods of slow growth in two successive years and that the chambers of the cuttlebone could be used to give a very accurate index of the growth of the cuttlefish. This has been used by one of us (E.J.D.) to study the life history of the cuttlefish in the Channel. It is hoped to present these results together with earlier ones obtained by Dr D. P. Wilson in a later paper. The results of the gas analyses are given in Table I. COMPOSITION OF GAS IN CUTTLEBONE 205 a B A Fig, 2, Diagram of cross-section of typical cuttlebones, (A) caught in April, and (B) younger animals caught in December, a and b mark two sets of closely spaced chambers. TABLE 1 No. of gas- filled chambers punctured %02 %C02 %N2* 27 1'9 0'28 97'82 23 2'1 0'36 97'54 16 4'4 0'27 95'33 12 10'3 0·22 89'48 6 17'7 0'58 81'72 5 9'7 0'65 89'65 '----.r-----' 4 7'5 * By difference. DISCUSSION In the sea and in the tissues of animals the partial pressure of nitrogen is always about 0,8 atm. no matter what the depth. We should expect, therefore, on the hypothesis advanced by Denton & Gilpin-Brown, that the equilibrium pressure of nitrogen in the chambers of the cuttlebone would be 0,8 atm. It can be seen from Table I that in the older chambers the gas is largely nitrogen with a small percentage of oxygen, and so we should expect the total partial pressure of gases in these chambers of the cuttlebone to be a little above 0,8 atm., and this is in good agreement with the value of 0·83 atm. given for cuttlebone from cuttlefish of density 0·62 (the average value for freshly caught Sepia, Denton & Gilpin-Brown, 1961 a). These results obtained on the older chambers confirm those found by Paul Bert (1867). He ground cuttlebones under water, collected the gas released and analysed it. He found that this gas contained traces of CO2 and 2-3 % 02' 206 E. J. DENTON AND D. W. TAYLOR the rest being azote. He hazarded the guess that this composition would vary with circumstances in a way analogous to that found by Armand Moreau in the swim-bladder. This is certainly not true. The proportion of oxygen is higher in the newest chambers than in the older ones. This was to be expected because the diffusion constant of oxygen in water is about twice that of nitrogen and so oxygen will go towards equili- brium at twice the rate of nitrogen. Since the total pressure of gas in the newest chambers is appreciably lower than atmospheric the highest value of 17'7 % oxygen will represent a partial pressure of oxygen of only approximately 0'12 atm. and will not be higher than the value possible for the tissues about Sepia. In the sea the partial pressure of oxygen will be 0'2 atm. The partial pressure of oxygen in the newer chambers is, however, higher than that in the older ones and this probably arises either because the newer chambers will be close to more active metabolic tissues which must be well supplied with blood and oxygen, or because the older chambers contain a little living tissue which holds the residual oxygen at a low level. The partial pressure of carbon dioxide is everywhere low. It will attain diffusion equili- brium much more quickly than either oxygen or nitrogen for it is so much more soluble in water than nitrogen that, for a given partial pressure difference, it will diffuse about 40 times faster. In the older chambers the partial pressure of CO2 is only about 3 mmHg, a very low value compared with the value of approximately 45 mmHg found in human venous blood. It probably represents, however, a low partial pressure of CO2 in the blood of Sepia and emphasizes that the regulation of the respiration of Sepia must take place in a very different way from that of the mammal. SUMMARY Gases from the chambers of the cuttlebone have been analysed. Their partial pressures were never higher than those expected in the tissues of the animals. In the older chambers the gas was about 97 % N2• The pressure of the gas in these chambers is always close to 0,8 atm. These results are in accord with the theory that the gases play an unimpor- tant role in the mechanism of the cuttlebone and merely diffuse into spaces created by forces other than gas pressure. Observations have been made which can be used in studies of the life history and the rates of growth of cuttlefish in the Channel. COMPOSITION OF GAS IN CUTTLEBONE 2°7 REFERENCES ADAM, W., 1940. Resultats scientifiques des Croisieres du Navire Ecole BeIge 'Mercator'. IV. Cephalopoda. Mem. Mus. Rist. nat. Belg., Ser. 2, Fasc. 21, pp.82-171. BERT,P., 1867. Memoire sur la physiologie de la Seiche. Mem. Soc. Sci. phys. nat. Bordeaux, T. 5, pp. 114-38. DENTON, E. J. & GILPIN-BROWN,J. B., 1961a. The buoyancy of the cuttlefish, Sepia officinalis (L.). J. mar. biol. Ass. U.K., Vol. 41, pp. 319-42. -- 1961b. The effect of light on the buoyancy of the cuttlefish. J. mar. biol. Ass. U.K., Vol. 41, pp. 343-50. -- 1961c. The distribution of gas and liquid within the cuttlebone. J. mar. biol. Ass. U.K., Vol. 41, pp. 365-81. DENTON,E. J., GILPIN-BROWN, B. & HOWARTH, V., 1961. The osmotic mechanism J. J. of the cuttlebone. J. mar. biol. Ass. U.K., Vol. 41, pp. 351-64.
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