J. mar.   biol. Ass. U.K. (1964) 44, 2°3-2°7                                      2°3
          Printed in Great Britain

                          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
   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.


        Fig. 1. Diagram of apparatus used to extract gases from the cuttlebone.

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




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-
                      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
                           4                   7'5
                                      *   By difference.

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.


Gases from the chambers of the cuttlebone have been analysed. Their
partial pressures were never higher than those expected in the tissues of the
   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

ADAM, W., 1940. Resultats scientifiques des Croisieres du Navire Ecole BeIge
   'Mercator'. IV. Cephalopoda. Mem. Mus. Rist. nat. Belg., Ser. 2, Fasc. 21,
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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