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Full text of "Studies on molluscan celomic fluid [microform] : effect of change in environment on the carbon dioxide content of the celomic fluid : Anaerobic respiration in Mya arenaria"

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CS ON MOLLUSC AN CELOMIC FLUID . 



F CHANGE IN ENVIUONMENT ON THE 
GWaBON DIOXIDE CONTENT OF THE 
CELOMIC FLUID 






ANAEROBIC RESPIRATION IN MYA ARENARIA 



BT 

J. B. COLLI P 



(Fhom thb Marine Biolooical Statio.v, Dui-AuruKt: Bay, Canada) 



. i 



RlFBINTHD FBOU 

THE JOURNAL OF BIOLOGICAL CHEMISTRY 
Vol. XLV, No. 1, December, 1920 



INprihtril fn.iii I in loi iiwi. m Hioi.oi.ical ('tit.MJ-i in, \..l MS . N.. 

IhllTlllKT, I<I20 



STUDIES ON MOLLUSCAN CELOMIC FLUID. 

EFFECT OF CHANGE IN ENVIRONMENT ON THE CARBON 
DIOXIDE CONTENT OF THE CELOMIC FLUID. 

ANAEROBIC RESPIRATION IN MYA ARENARIA. 

Hy .1. \i. COM.Il'. 

(From the Marine liiologicnl Stntinn. h(i„irhii,- li,,;,, Ciinivlit.) 

(Rccoivcd for public:iticm, Si-pUrriber 10. ISWI.) 

INTHODICTIOV. 

It was noted in a previous coimininicalidii fh tliat tlic con- 
font of conit)inc(l carlion dioxide of molhisean eclomic flui<l tends 
to ri.se when the animals are removed from llieir natiiralVin iron- 
ment whereas a fall was noticed in this factor in the case of fisii 
removed from their natural hal.itat. In order to determine what 
was the cause of this peculiar effect in the molhisean forms a 
series of experiments was undertaken, the results of wliicii are 
herein reported. 

KXl'KUIMK.VTAL. 

EjJcd of Exposure to Atmoxphiric Air on (lie Comhinnl Crhon 
Dioxide Contcitl of the C domic Fluid. 

The metliod of securinK samples of celomic fluid or "clam 
juice" from the various .specimens was the .same as that detailed 
previously (1). Specimens of seven species of pelecyixxl Mol- 
hisca were exposed to atmospheric air in a closed jtlass container 
for varying periods of time. One species of the Ani|.hineura 
and two si)ecies of the C.asfropoda were similarly studied. .Sev- 
eral non-mollu.scan forms were al.so exjiosed to ;itmospheric air 
under similar con<Iitions. These inchu'ed the calcareous shelled 
arthropod Bulnnu.s ufjuilh, the common hrachiopod Tinhrehlla 
Iranmraa, vaiious ("nistacea of the decapod typv, starfish, sea 
urchins, .and certain varieties of marine fish. The container 

23 



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Mollusejui CVlnniic F1ui<l 



used wiis of nood size and a full supi)ly nf oxyncii was assured. 
It vas k('i)t covorod to i)r('V»'iit Iciss of water li.v evaiioration. 
Filter paper nioistene<l in sea water was fre<iueiitly placed in the 
container with the specimens. Specimens which were exposed 
to air were kept in certain instances at a fairly constant tempera- 
ture l)y iiumersiiijj the coiitainer in sea water while in others 
they were kept in the laboratory and were thus subject to the 
temperature changes of the latter. Table I illustrates the efTects 
of ex)»osure to air for various j)eri(»<ls upon the combined carbon 
(lioxi<le content of the celomic fluid of the different species inves- 
tigated. Tile ra))id increase in the carbon dioxide content of the 
celomic fluid of the molluscan forms and the arthropol Bahniis 
(ifiuilla is very striking. That this increa.se is due to bicarbonate 
is evident .since the samples wen- e(|uilibrated with atmospheric 
air l)efore being submitted to analysis. The decapod cnistaoeans 
examined failed to show this reaction, while a very slight increase 
was in some instances manifeste<l in the echinoderms. As the 
latter reaction was not uniform it is of very doubtful significance. 
The sunival time for specimens exposed to atmospheric air 
varied greatly. It was early noted that .!///« nnuaria was pecu- 
liarly resistant to long exposure toatmosplu-ric air at the tempera- 
tures which prevailed in the surface wat(M- and the air at Depar- 
ture Bay during the summer months. It was for this reason 
used extt'nsively in lat<.>r investigation. It is regretted that no 
facilities were availal)lc which would enable one to keep si)ecimcns 
at a low as well as constant temperature. The results obtained 
will therefore have to be considered in the light of this condition. 
The greatest increase in the carbon dioxide content of the blood 
was in two sjiecimens of Mija airnaria which had l)een exposed 
f<ir !)() hours. The increa.se here was from ().5 volumes per cent 
in the controls kept in sea water to 105 volumes p«-r cent in the 
specimens cxpo.sed to atmospheric air a( the temperature of sur- 
face sea water. The container used was a (1 liter cylindrical 
glass mu.seum jar and it was opened daily both for the pm-pose 
of removing specimens for examination and to allow a change of 
air. The temperatme of the sea water in the vessel which was 
taken on the 4 consecutive days during which these specimens 
wen; exposed was li».S% I8.S", lit.:r, and 1S.«I"('. Slightlv over a 



♦*!*■: ',. 



J. B. CoUip 



29 



sixlccnfold im-rease in the coniluiied caibou dioxide in the blood 
was noticed in this instance. 

The incmiso in the caiLon dioxide in the small pelecypod 
Macoma secta from 11.2 to 48.() volumes per cent in 6 hours is 
noteworthy, as is also that observed in the gastropod Polynices 
lewisii following 80 hours exposun". The combined carbon 
dio.\ide rose in tliis latter instance from 12.5 volumes per cent 
in the control to 77.4 volumes per cent in (he ex|)o.sed si)ecimen. 
There were few forms wliidi would surA ive an eximsure to atmos- 
pheric air of more than 24 hours at the i)revailinK land and sur- 
face^ water temperatures. The amphineuran Vniptochiton, the 
cockle Vnnlium corbis. and the eaple barnacle BalnnuH aqnilla 
were \-ery .s>nsitiv(> (o exposure. 'J'he horse ••lam Schiznlhoerm 
niilMli, and the butler clam So.ridonius ijiqmdea withstood an 
exposure of 24 to 48 hours. The lit ( le neck clam Paphia sUnnincn 
and the edible form Mija tircnnrin were very resistant to exposure 
and in some instances sur\iv(Hl as long as T) days when placed 
in the air, the evaporation of water beinj; practically excliuled. 
The nudibranch Anisoihris was most .sensitive of all forms, dying 
shortly after being brought into the lal)oratory. The absence 
of a calcareous shell is probably a.ssociated with the lack of resis- 
tance to exposure to air in this f(.rm although the temperature 
factor must al.so be of great importjmce. 



Kfffd of Kxpimm' (o Mii,„xi>lin!c Mr on tin: Carhun Dioxide 
Ciiltncilij of (he Cdoniir Flin'il. 

Several samples of celomic tluid taken from specimens exjwsed 
to atmospheric air for varying periods weiv analyzed in the Van 
Slyke apparatus (2) when eciuiiibrated with atmos[)heric air and 
also when equilibrated with alveolar air of lli(< normal sui)ject 
after the manner described by Van Slyke and ('ullen (;{). Table 
II illustrates the results obtained in this .series of exp(>riinents. 
It will be noted that in every instance the carbon dioxide cai)acity 
of the sample was considerably in excess of the carbon dioxide 
content of the same when equilibrated with atmospheric air. 



30 



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31 



Effect of Exposure to Atmospheric Air on the Total Nitrogen Content 
of the Celomic Fluid. 

In order to determine if the increase in the carbon dioxide con- 
tent of molluscan celomie fluid brought about l)v exposure to 
atmospheric air was in any way due to an increase in its protein 
content the total nitrogen was determined in from 25 to 100 cc 
of composite samples of celomic fluid taken from several speci- 
mens. The estimation was made by the usual Kjeldahl method 
The results are expressed in Table III. As there was a slight 



TABLE III. 



No. 
uaed. 



1 
2 
3 

15 
8 

23 
8 
6 
6 

14 

11 
1 



Specimen. 



Total nitro- 

Ken prr 1(10 

cc. of fresh 

material. 



Schizothoerus nuttalli. 



Saxidomus gigantea 

Paphia staminea 

" u 

Cardium corbia 

Mya arenaria 

Polynices lewisii (fluid from foot). 



my. 

37 5 
34 

50.3 

70.0 

37,5 

33 6 



Total nitro- 
gen per 100 

cc. after 
exposure to 

atmos- 
pheric air, 



mg. 

34.4 

51.8 

61.6 

50.4 

40 7 
9.3 



Time in 
air. 



hrs. 




32 


30 


28 


30 


28 
52 



mcrease m two species, Cardium corbis and Mya arenaria, prac- 
tically no change in Saxidomus gigantea, and a slight decrease in 
Schtzothoerus nuttalli and Paphia staminea, it is very unlikely 
that protein plays any appreciable part in the increase in the 
combmed carbon dioxide or alkali reserve of the celomie fluid of 
the mollusk which has been exposed to atmospheric air. 

Effect of Exposure to Atmospheric Air upon the Calcium and 
Magnesium Content of the Celomic Fluid. 

Calcium and in a few instances magnesium were determined 
in composite samples of celomic fluid taken from several speci- 
mens which had been exposed to atmospheric air for varying 



32 



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33 

periods of time. The method of McCn.ddon (4) was followed, 
the calcum bemg estimated l,y the titration of the oxalate with 
0.1 N potassiimi permunpanate. 25 cc. of celomic fluid were 

wnt/l '""^V"''r"r • . ^^'' ^'"' evaporated to dryness on the 
vu^ter bath fused dissolve. 1 hy tho aid of concentrated hvdro- 
chloric acid, and the calcium finally precipitated as the oxalate. 
The results are shown m Table IV. It will be noted that, whereas 

mollusk, the calcium increases to a great extent and also the 
increase m this latter constituent is more or less parallel with the 
increase ,n the combined carbon .lioxide. It is therefore evident 
that the great increase in the alkali reserve of the blood of mol- 
uscan forms when expose,! to atmospheric air is due toan increase 
m the concentration of bicarbonate which is balanced for the 
\fvlT-V?' '"" '""■''^'' '" ^^^ concentration of the calcium ions. 

oxiri/l^r/''rTi^"?"^ •'^' '"^- °f ^^'"""^ ^^alculated as 
oxide in the blood of Saxtdomus nuttalli and 107 mg. in ^chho- 
ihoerus nuttalli. He has also commented on the very high I 
cium content of moUuscan blood. I have failed to fin,l that the 
calcium content of the celomic fluid of fresh molluscan forms 
diflfei^ materially from that of sea water. It is onlv after expc^ 
sure to air that the calcium content becomes high. ^ 

^■^'It/f TT '« f '"^•^/''i-'-'^- ^'> ^Pon the Total Alkalinity 
and the Buffer T aluc or Reactivity of the Celomic Fluid. 

Lacking the means of determining the hydrogen ion concen- 
tration, a most important factor in these experiments a met .1 
|vas employed to determine approximately the total alkalinitv o" 
tne blood ami also its buffer value. The method adopted was 
^nuar to t^at previously described (1) based on the princ " 
made use of in the method of double titration for bica bona el 

P> n nould of course introduce an error but as has been shown 
t. .utem content of the celomic fluid ,loes not varv to any 
app.ociabIe extent an.l therefore approximately the same de'ree 
of error w-ould exist in all the titrations. The alkalinity was 
determine.! by noting the amount of 0.01 x alkali requ e. to 
produce a just noticeable pink tint when phenolphthalcin had 



THE JOrnN-AI, OF BIOLOUICAL CHEMi^ihY, V 



Ol, XLV, NO. 1 



34 



Molluscan Celoroic Fluid 



been added to the celoniic fluid. The reactivitj- of Moore and 
Wilson (7) or the buffer value of Sdrenson (8) was determined 
l)y titrating from the phonolphthaloin to the methyl orange point 
using w.i,. sulfuric acid. 0.2 x acid w.-is used in those instances 
where the reactivity was of large proportion. The results are 
shown in Table IV. It will be noted that the rate of increase in 
the reactivity of the celoniic fluid is in close agreement with the 
rate of increase of calcium and also of the comlnned carbon dioxide 
content of the same. 



Effect of Exposure to Air Followed by Submersion in Fresh Water. 

Table \ illustrates that, whereas exposure to air causes a rapid 
increase in the alkali reserve of the celoniic fluid, the subsequent 
immersion in fresh sea water causes a return to approximately 
the normal value for this factor. 

TABLE V. 



Specimen. 


COi content of 
lOOec.of fluid 
equilibrated 
with atmos- 
pheric air. 


Remarks. 


Mya arenaria. . 

U it 
« X 

ii t( 
<( « 


cc. 

8.2 
32.0 
23.3 
14 9 

9.2 


Fresh. 

72 hrs. in glass container in laboratory. 

Submerged 4 hrs. in fresh sea water. 

a ^Q i( (( a It it 
11 nj tt It tt it it 



Effect of Srtbmrrsion in Sea Water in a Sealed Container. 

Several fresh specimens of Mya arenaria were immersed in a 
relatively small volume of sea water in a cylindrical glass con- 
tainer which was then tightly sealed. After varying periods of 
time the celomic flui<l of the specimens was examined. An 
analysis of the .«ea water vhich was used in the experiment was 
also made. The results are expre.>^.sed in Table VI. A few 
experunents were carried out in which lioiled out soa water was 
used in iilace of fresh sea water. In on' instance sea water, the 
buffer value of which had been greatly increased l)y the addition 
of 5 gm. of l)asie sodium i)hosphate per liter, was used. It will 



J. B. CoUip 35 

be noted that the J«>havior of Mya arenarm kept in a scaled con- 
taincr in either boiled or fresh sea water is ver^- similar to that 
ol.ser%ed when the specimens were kept in the air. The alkali 
resen-e and the calcium content of the celomic fluid mount 
steadily until the animal dies. The increase in the carbon diox- 
ide and calcium content of sea water is also considerable. The- 
values for these two factors in sea water are, however, lower in the 
case of the celomic fluid except after long submersion of the 
specimens in which instance there is a tendency for the concen- 
trations of these latter substances in the celomic fluid and sea 
water to equalize. The addition of basic sodium phosphate to 
the boiled sea water used in one experiment did not materially 
alter the results. A dense precipitate was formed in the sea 
water in this experiment which consisted for the most part of 
calcium phosphate. 

If one considers the increase in the combined carbon dioxide in 
the sea water used in an experiment one finds that there is very 
little difference in the rate of increase in this factor in specimens 
exposed to air and in specimens submerged in a relatively small 
yolmne of sea water. Thus in Experiment I. Table Vl an 
increase of 29.3 volumes per cent was noted in the combined 
carbon diox.de content of the celomic fluid, while an increase of 
fuJly 20 volumes per cent took place in the sea water. The 
total bulk of the eight specimens used in this experiment was 
550 cc. while the .volume of sea water used was 750 cc There 
was therefore an increase of 150 cc. of combined carbon dioxide 
due to the actiyity of the specimens over and above the increase 

d, *nt '°, «-''' ?"^' ^"^'^^ ^^^ ^^^"« «f *he eight specimens 
displaced 8o cc. of water. Not allowing for the water entrapped 
in the mantle cavities, there were 465 cc. of clam tissue present 
m this experunent so that the increase of 150 cc. of carbon dioxide 
found m the sea water would mean that 32 cc. of this carbon 
dioxide had resulted from the activity of each 100 cc. of clam 
tissue. This added to the carbon dioxide content of 100 cc of 
celomic fluid indicates that approximately 01.3 cc. of combined 
carbon dioxide resulted from the aclivitv for 40 hours of 100 cc 
of clam tissue, an amount which is in close agreement with the 
obsen;ed increase of the volume per cent of carbon dioxide in the 
celomic fluid of similar specimens exposed to atmospheric air in 
a closed vessel for a corresponding period of time (Table IV) 



36 



Molluscan Celomic Fluid 



I 



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J. B. CoUip 



37 





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MolliLscan Celomic Fluid 



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39 



KJTirl of Suhmimiun in l)ixtilli<f Wnlir. 

The rosiilfM of soruo cxprrinjcnts in which HjM'(irii«'ii,M oi \fya 
urcmrin woro hiiimierged in fresh (li.still«i water in a rUk* con- 
tainer and kept at the temperature «if surface sea water ap/war 
in Table VII. The conihined eMrLon dioxide and ealciiini f^n- 
tent of celoniie fluid rise after nnieh tlie xanie niaritier aH vas 
oh8er\-ed when specimens were placed in .-ither frexh or Unm\ 
sea water. Kxperiments I and II, Tal.lc VII, are of intercHt in 
that they show that a fall had taken place in the concentration 
of magnesium in the celoniie fluid of the specimens, while a riw 
occurred in the concentration of j-alcium. There i •.!.,» a niarkcH 
difTen-nce in the total alkidinity and in the read 
and the celoniie fluid. The chlorine content ol 
and of the water in which the specimens of .1/ 
suhmerKcd was determined in Kx|)erimeiits II 
VII. The manner in which the concentratioi, 
the celoniie fluid is kept :it a relatively liifth le\ 
eumstnnces ohtaining in iiose experiments is ion 
(9) has shown that the addtn-for muscle of tlK- 
nmria is jM-culiarly resistant to hypertonic ,^4»atir«,s ,rf ^,J,Iiw, 
chloride and to (knihie strength sea water, flie cnrrnti m^ 
sodiun» chloride -: Ini; to only ahout one-half that ,.f ti.^ ^„, 
rounding mediuia >!so found that the ..antic i:. it....^., 

imiK-rmeal.le to .sodiu,, .oride. The al.ilin ^^( M ,, ,.,,/« 
to withstand immersion iii water, the osinoii, „n-j^niv • Jieh 
is verj' low, is of interest in the lijjht of th. na' On ^ 

Effect of Kj-posiiir to (I llii<lro(i< Alitios/,i„ 

Several specimens of .1///,/ anmnin were kept in a MM:.^ ue 

of .sea water for 2 hour- in order that the ow^en .-..nt.ni ..jr 

tissues should he redm, 1 to a low level. ( h.e .specim. i . ifien 
hied as a control, tlu -thers were placed in a hvdron. atmo.s- 
sphere over alkaline pyiofrallic acid, tw.. separate containers 
hemg u.sed; One uroup of three was exposed for 2(> lioiir^- the 
other for 48 hours. The results of the analy.s(.s of the .vl'omic 
(huds of these specimens are shown in Tal.le VIII. ' Tl.. maxi- 
mum and minimum temjieratures for the 2 days durinfi which 



the wat*^ 
•iniic fluid 
**iri(i were 
'fl, 'I iihie 
hlori(i in 
the ir- 



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40 



Molluscan CVlomic Fluid 











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J. P Collip 



41 



thi.H experiment wjim jwrfoniKHl were on the lut day 24.4» and 
lO.ft'C, on the 2n.| duy 2a.9' und 11.4"f'. It will l.e noticed 
that the incnmse ohwem-d in the concentration of l)icttrl)onate in 
the cclomie fluid is very nuirketl. It iM not, however, so Rreat 
as that which is found when tlie siH'ciniens areexpose«l toatn»o«- 
piieric air. The increase in tlie calciuni and magnesium is com- 
parable to that ol)8er\-cd for bicarbonate. 

Effect of Exposure to a Nitrogen Atmosphere. 

Three Bpecimena of Mya arenaria were placed in a small 
desiccator "ontaining a concentrated solution of pyrogallic acid 
in 40 i)er cent sotlium hydroxide. A glass tute was so attached 







TABLE IX. 


No. 
iMed. 


Sp«cimrn. 


COiper 

lOOcc.of 
tiuid <><iuili- 
brstnl with 

■tmiM- 
pheric air. 


Remark •. 


3 
3 

3 


Mya arenaria 
•< II 

II II 


ee. 

SO 
34 8 
17.8 


Fresh control. 

25 hrs. in glass container in laboratory 
25 " " desiccator over alkaline pyro 
gallol. 



to the exhaust cock that as oxygen was ab.sorl)ed the air which 
enten'd first bubbled through the alkaline pyrogallol in the bottom 
of the desiccator. Three other s|)eciinens of like size were placed 
at the same time in a gla-ss container which was kept at the same 
temiierature as the desiccator for the duration of the experiment. 
After 25 hours the specimens were bled and an analysis was 
made of the composite .samples of celoniic fluid. The results 
are expre.s.sed in Table IX. It will be observe<l that the com- 
bined carbon dioxide of the celomic fluid did not increase to 
the same extent in the sjiecimens wi.icli wore kept in the desic- 
cator over alkaline pyrogallol as it did in the controls which were 
kept in the air. 



42 



MoUuscan Celomic Fluid 



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J. B. Collip 



43 



Comparison of the Carbon Dioxide Content of Cclomic Fluid and 
Other Fliiidfi Obtained from Mollusca. 

A comparison was made between the carbon dioxide content 
of the celoinic fluid and other fluids of ilifforcut iloUusca. The 
rcsuhs of this study are shown in Tabic; X. It was found that 
the carbon dioxide content .f the fluid which exuded from the 
exhalant siphon of fresh Mi/a arenaria was just sUghtly higher 
tlian that of sea water. >\lien specimens of this species were 
exposed to atmospheric air for some time the l)icarl)onate content 
in the fluid of the mantle cavity closely approximated that in the 
•■elomic fluid. Somewhat similar observations were made on 
the auiphincuran form Crijptochiton. The fluid in the foot of 
the large gastropod Pohjnicen leirisii l)ears somewhat different 
relation to the blood and celomic fluid of this form as far as the 
bicarbonate content is concerned from that which holds in the 
fluid between the mantle cavity and the blood and celomic fluid 
of a pelecyi)od such as Mija arenaria. The combined carbon 
dioxide content of the fluid of the foot of Poli/nices leirixii approx- 
imates the value obs(>r\-ed for tlu; celomic fluid. The drawing 
in, therefore, of the foot in this form causes a considerable decrease 
in the total alkali reserve of the animal. It is of interest in this 
connection to note that this animal does not withdraw its foot 
imless subjected to rather violent irritation. 



Effect of Submersion of Dead Specimenx of Mya arenaria in Sra 

Water. 

The results of two experiments are shown in Table XI. It is 
evident that the bicarbonate content of sea water in which ilead 
clams are immersed rises quite rapidly once decomposition has 
set in. It will be noted, however, that there is little change 
during the first 21 hours submersion. The behavior of pelecypod 
mollusks exposed to air or submerged in a relatively small vol- 
mne of water is therefore quite distinct from that of dead clams 
which are undergoing decomposition. 



44 



AloUuscan Celomic F. lid 



TABLE XI. 



Specimen. 


Total COi 
per 100 re. 


Remarks. 


Experiment I. 
4 Mya arenaria (175 cc.) 

Sea water (boiled) 


cc. 

3 3 
4.7 

42.0 

55.0 
07.0 
72.0 

2.8 

3 3 

11 7 

35.0 


Placed in 275 cc. of boiling sea 
water. 


«< i( 11 


After 24 hrs. No decomposi- 
tion of clam tissue. 

After 48 hrs. Decomposition 
clearly manifested. 

After 72 hrs 


i( u u 


« tl it 


It tt tt 


" 96 " 


tt tt tt 


" 144 " 


Experiment II. 
10 Mya arer" ia (500 cc.) 

Sea water (boiled) 


Placed in 700 cc. of boiled sea 
water containing 9.5 per cent 
alcohol. 


U It tt 


After 24 hrs 


l< 11 It 


" 48 " .Milky, decompo- 
sition evident. 
After 96 hrs 


« (1 tt 







DISCUSSION. 

As has already l)cen indicated the marked increase in the 
l)icarl)onate content of the celomic fluid, and therefore in all 
probal)ility of the hlood and tissues of the calcareous shelled 
pelecypod moUusks and the arthropod Balnnm aquilla on expos- 
ure to air is quite opposite to the effect ohserv'ed in fishes when 
the}' are removed from their natural habitat. This phenomenon 
is undoubtedly associated with the presence of a calcareous shell 
the calcium carbonate of which furnishes an alkali reserve which 
is added to that of the body fluids and tissues, and which it 
appears can be readily utilized. 

As specimens of Myn arrnnrin appear to remain practically 
normal even after long exposure to atmospheric air there is no 
reason to supjwse that any material change has been effected in 
their metabolic proce.s-ses as a result of the change in environment. 
If one assumes, therefore, that combustion still i)roceeds in the 



^■^ft:.. 



J. B. CoUip 



45 



exposed specimens, then the increase in the bicarbonate content 
of the body fluids can be explained according to the equation 

CO2 + H2O + CaCO, t=; Ca(HC05): 

The carbon dioxide resulting from the respiratory process 
would, l)y shjihtly increasing the hydrogen ion concentration, 
dissolve calcium carbonate from the shell and the concentration 
of the calcium ions and of i)icarbonato ions would therefore 
steadily rise as combustion in the tissues proceeded. The amount 
of carbon dioxide actually excreted from the specimens in the 
gaseous form was not determined. If this factor were known 
one could calculate the intensity of metaljolism in these forms 
by considering the amount of carbon dioxide excreted in addition 
to the amount retained as l)icarbonate. It would appear that 
50 per cent of the increase observed in the carbon dioxide content 
of the celomic fluid is due to carbon dioxide formed by combus- 
tion in the tissues, while the remaining 50 per cent results from 
the solution of calcium carlionate of the shell. 

The degree of alkalinity of the celomic fluid determined by 
titration is l)y no means an indication of the hydrogen ion con- 
centration, but the ratios observed between the alkalinity figures 
and the reactivity values suggest that no marked increase in the 
hydrogen ion concentration takes place during the early part of 
the exposure at least. The reactivity or buffer value is, in nearly 
every instance, in close agreement with the calcium content and 
the carbon dioxide concentration of the celomic fluid. 

The increase in the alkali reserve as indicated by an increase 
in bicarbonate concentration in specimens exposetl to atmospheric 
air is due for the most i)art to increase in the calcium content. 
Magnesium, which in the normal animal in its natural habitat 
exceeds calcium in the degree of its concentration, and therefore 
balances a greater proportion of bicarbonate ions than does 
calcium, increases only slightly as comparei! with calcium when a 
specimen is exposed to air. It is prol)al)le that the relative 
increase in calcium and magnesium concentrations under these 
circumstances is somewhat similar to the relative amounts of 
these substances in the shell from which solution of bicarbonate 
is taking place. It is of interest to note here that no increase 



46 



Molluscan Celomic Fluid 



was ohsorvcd in the concentration of n.aRnosiuin in the cockle 
(tardiiini corhis) on exposure to air. 

As specimens suhnierRed in boiled sea water and kept in a 
sealed container cntinne to develop an increased carbon dioxide 
content, calcunn concer.t ration, and buffer value, after much 
the same manner as specimens exposed to atmospheric air, and 
since a similar effect is manifeste.1 by specimens kept in an atmos- 
phere of hydrosen or nitrogen, one is led to ask the question "Can 
anaeroluc respiration be manifested l>v these forms?" 

If one considers the results of an experiment recorded in Table 
Mil, one fimls that after 48 hours in a hydrogen atmosphere 
the combined cari)on dioxide ro.sc from 8.2 to 45.5 volumes per 
cent, or an observed increase of 37.3 volumes per cent. If one 
a.ssumes that aerobic respiration was taking place and that car- 
bohyilrates were I,eing burned, then a volume of oxvgen equiva- 
lent to the volume of carbon dioxide produced would l,e required. 
It ;)0 per cent of the obser^-ed increa.se in the combined concen- 
tration of carbon ilioxide is indicative of the amount of this sub- 
stance prmluced due to combustion then an amount of oxygen 
equivalent to .",0 ,,of cent of 37.3 volumes per cent, or 18 05 
volmnes per cent, would be required. As the specimens used 
in this e.xijerunent were kept in a small volume of sea water for 
2 hours before they were transferred to a hydrogen atmosphere, 
one fails to see how any appreciable amount of oxvgen covld be 
contamed m the tissues of the specimens. As there is no apparent 
source lor 18.05 volumes per cent of oxygen in these clams it is 
therefore evident that thej- must be respiring anaerobically or 
ese the increase in the carbon dioxide, calcium, and buffer value 
of the celomic fluid is due to some other cause than that suggested 
ear her in the paper. Decomposition of the clam tissue can be 
exclu, ed smee the specimens were very active, resijonding to 
s inmlation like normal animals, after they were removed from 
the hydrogen atmos[)here. 

There is the possibility of (he solution of the calcium carbonate 
ot the shell due simply to the .solvent action of the tissue fluids 
containing free carbon dioxide. This would result in the forma- 
tion of calcumi carbonate the solubility „f which is considerablv 
increased by an excess of carbon dio.xide in the water (10) In 
dealing with a closed system, however, s.ich as the individual 



• '--.m 



J. B. Collip 



47 



clam in an atmospliprc of hydroKon, tlio solution of calcium car- 
bonate due to the solvent action of the free carbon flioxidc would 
require a constant supply of the latter if the process is to con- 
tinue; otherwise etiuilibrum would be established between the 
dissolved bicarbonate, the calcium carbonate of the shell, and 
the free carl .on dio>dde, and no further increase would be mani- 
fested unless this balance were disturbed. It will be noted that 
the rate of increase in the carbon dioxide content mid the calcium 
concentration of the blood is for a considerable period practically 
constant. If respiratory activity account.s for the increase in 
bicarbonate concentration of the tissue fluids, then this uniform- 
ity in the rate obseri-ed would fit in well with the fairly constant 
rate of metabolism which might be expected under such circum- 
stances, anaerobic respiration being possible. 

The fact that the rate of increase in the bicarlfonate concen- 
tration, the calcium content, and the buffer value is greater in 
air than it is in either hydrogen or nitrogen would indicate that 
absence of oxygen does exert an influence on the intensity of the 
metai)olic processes but by no means causes a complete cessation 
in the respiratory function. 

The extreme sensitivity of the cockle and the horse clam to 
expo.sure to air at the prevailing summer temperatures made 
experiments with these forms of the same type as were conducted 
with Myn arcnaria temporarily impossible. Neither of these 
forms is normally submitted to the same degree of low oxygen 
tension as is Mija airnaria. It is hoped that experimental work 
of a similar nature to that carried out with Mya aremria may be 
done on other forms at a more favorable time of the year. 

It has long been known that animals show a very unequal 
resistance to lack of air. liunge (11) in his work upon respira- 
tion of intestinal parasites and nmd-dwelling organisms showed 
that parasites in the intestine of warm i)looded animals must 
live practically in the absence of o.xygen, while wonns living in 
the nmd were also subject to similar conditions, decomiwsition 
processes, with the formation of reducing substances, keeping 
the oxygen absent. Packard (12) found that worms and mud- 
dwelling Crustacea are resistant to the lack of o.xygen for some 
time. 



48 



MoUuscan Celomic Fluid 



The ability of an animal to resist a lack of oxygen may or 
may not he connected with an anaerobic respirator^' mechanism. 
If one finds complete evidence of metabolism in an animal exposed 
to anaerobic conditions, then anaerobic resjuration woidd be 
indicated. Such seems to be the condition in the ca.se of Mya 
arffiario. Crustacean types which were exposeil to the air or 
submerged in boiled sea water died within a few hours. The 
carbon dioxide content of the blood was, however, practically 
unaltered by such procedures. These forms do not use the 
calcium carbonate of their carapace as a protective measure when 
removed from their normal habitat. 

In the light of the results of the experiments which have so 
far been conducted upon Myp armaria the writer has tentatively 
to conclude that indiviihials of this species behave as facultative 
anaerobic organisms. It is realized, however, that in these pre- 
liminary experiments absolutely anaerobic conditions were not 
secured. It is the intention to continue this work at another 
time when it is hoped to <letcrmine the hydrogen ion concentra- 
tion of the celomic fluid and the rate of oxygen consumption 
under various conditions. 



SUMMARY. 

1. Calcareous shelled pelecypod Mollusca and the arthropod 
Balanus aquilla have in the calcium carbonate of their shells a 
potentially great alkali reserve. 

2. I'Aposure of these forms to atmospheric air causes a marked 
increase in the combined carbon dioxide of the celomic fluid. 

3. There is imder these circumstances a parallel increase in the 
calcium concentration and the buffer value of the celomic fluid. 

4. Various other marine forms studied did not so react. 

"). There is no increase in the total nitrogen of the celomic fluid 
of the pelecyiiod Mollusca exposed to atmospheric air. 

G. Mya arenaria is particularly resistant to long exposure to 
atmospheric air. 

7. When specimens of Mya arenaria are placed in a relatively 
small volume of fresh sea water, boiled sea water, distilled water, 
or in a hydrogen or a nitrogen atmospliere much the same reac- 
tion is observed as when specimens are exjiosed to atmospheric 
air. 



J. B. CoUip 



49 



8. The rate of increase in the content of carbon dioxide, the 
calcium concentration, and the l)uflfer value of the celomic fluid 
under all the above conditions is, during the first period of sev- 
eral hours, constant. 

9. The rate of increase in the rate of combined carbon dioxide, 
the concentration of calcium, and the buffer value is not so great 
in a hydrogen or nitrogen atmosphere as it is in air. 

10. It is suggested that Myn armaria is a Tacultative anaerobic 
organism which continues to produce carbon dioxide under 
anaerobic conditions. 

In conclusion I wish to exiiross my thanks to Dr. C. McLean 
Fraser, the Curator of the Biological Station, Departure Bay, 
British Columbia, for his cooperation during the carrying out of 
the experimental work reportc' in this communication. 

My thanks are also due to the Biological Board of Canada, 
by whom the exjicnses in connection with this investigation 1 e 
been dcf raved. 



BIBLIOGRAPHY. 

1. Collip, J. B.. J. Biol. Chem., 1920, xliv, 329. 

2. Van Slyke, D. D., J. Biol. Chem., 1917. xxx, 347. 

3. Van Slyke, D. D., and CuUen, G E., J. Biol. Chem., 1917, xxx, 289. 

4. McCrudden, F. H., J. Biol. Chem., 1911-12. x, 187. 

5. Myers, R. G., J. Biol. Chem., 1920, xli, 119. 

6. Brown, H. T., and I']8combe, F., Phil. Trans. Roii. Soc, 1900, cxciii, 

289. 

7. Moore, B., and Wilson, F. P., Biochem. J., 1906, i, 207. 

8. Sorensen, S. P. L., in Moore, B., Prideaux, E., and Herdman. G., 

Trans. Biol. Soc. Liverpool, 1915, xxix, 174. 

9. Meigs, E. B., Am. J. Physiol., 1914, xxxiii. p. xxii. 
10. Reuter, M., Z. anorg. Chem., 1898, xvii, 170. 

11; Bunge,G.,Z./)A.vsiV»/.C/icm.,lS83-t^4.viii,48:188S.xii,565;1890,xiv, 318. 
12. Packard, \V. H., .4m. J. Physiol., 1905-06, xv, 30. 



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