CIHM Microfiche Series ({Monographs) ICIMH Collection de microfiches (monographies) Canadian Institute for Historical Microraproductions / Institut Canadian da microraproductions historiquas Technical and Bibliographic Notes / Notes techniques et bibliographiques The Institute has attempted to obtain the best original copy available for filming. Features of this copy which may be bibliograpliically unique, which may alter any of the images in the reproduction, or which may significantly change the usual method of filming are checked below. D D n n D Coloured covers / Couverture de couleur Covers damaged / Couverture endommag^e Covers restored and/or laminated / Couverture restaur^e et/ou peiiicul^e ^ Cover title missing / Le titre de couverture manque Coloured maps / Cartes g^ographiques en couleur Coloured ink (i.e. other than blue or black) / Encre de couleur (i.e. autre que bieue ou noire) Coloured plates and/or illustrations / Planches et/ou illustrations en couleur Bound with other material / Relid avec d'autres documents Only edition available / Seule Edition disponible Tight binding may cause shadows or distortion along interior margin / La reliure serr^e peut causer de I'ombre ou de la distorsion le long de la marge int^rieure. Blank leaves added during restorations may appear within the text. Whenever possible, these have been omitted from filming / II se peut que certaines pages blanches ajout^es lors d'une restauration apparaissent dans le texte, mais, lorsque cela 4tait possible, ces pages n'ont pas ixi fiimees. L'Institut a microfilm^ le meilleur exemplaire qu'il lul a 6\6 possible de se procurer. Les details de cet exem- plaire qui son! peut-£tre uniques du point de vue bibli- ographique, qui peuvent modifier une image reproduite, ou qui peuvent exiger une modifk:ation dans la m^tho- de normale de filmage sont indiqu^s cl-dessous. I I Coloured pages / Pages de couleur I I Pages damaged/ Pages endommag6es □ Pages restored and/or laminated / Pages restaur^es et/ou pellicul^es 0 Pages discoloured, stained or foxed / Pages ddcolordes, tachet^es ou piqu^es I I Pages detached / Pages d6tach6es p^l Showthrough / Transparence □ Quality of print varies / Quality in^gale de I'impression Includes supplementary material / Comprend du materiel suppl^mentaire Pages wholly or partially obscured by errata slips, tissues, etc., have been refilmed to ensure the best possible image / Les pages totalement ou partiellement obscurcies par un feuillet d'errata, une pelure, etc., ont 6t6 film6es k nouveau de fa^on k obtenir la meilleure image possible. Opposing pages with varying colouration or discolourations are filmed twice to ensure the best possible image / Les pages s'opposant ayant des colorations variables ou des decolorations sont film6es deux fois afin d'obtenir la meilleure image possible. D D D Additional comments / Commentaires suppl^mentaires: Pagination is as folloMs: p. 23-%9. i : Si This item Is filmed at the reduction ratio checked below/ Ce document est filme au taux de reduction indiqui ci-destous. lOx 14x 18x 22x 26x 30x 7 1 1 12x 16x 20x 24x 28x 32x Th« copy filmed h«r« has b««n raproduead thanks to tha ganarosity of: Blacker-Wood Library of Biology HcClli University, Montreal Tha imagas appaaring hara ara tha baat quality possibia eonsidaring tha condition and lagibility of tha original copy and in kaaping with tha filming contract spacificationa. Original copias in printad papar eovara ara fllmad baginning with tha front covar and anding on tha last paga with a printad or illustratad impraa- sion, or tha back covar whan appropriata. All othar original copiaa ara filmad baginning on tha first paga with a printad or illustratad impraa- sion, and anding on tha laat paga with a printad or illustratad impraaaion. Tha last racordad frama on aach microflcha shall contain tha symbol ^-^ (maaning "CON- TINUED"), or tha symbol V (maaning "END"), whiehavar appiias. Maps, platas. charts, ate. may ba filmad at diffarant raduction ratios. Thosa too larga to ba antiraly includad in ona axposura an filmad baginning in tha uppar laft hand cornar. laft to right and top to bonom, as many framas as raquirad. Tha following diagrams illustrata tha mathod: L'axamplaira filmi fut raproduit grica k la gintrosit* da: Blacker-Wood Library of Biology McCin University, Montreal Las imagas suivsntas ont M raproduitas avac la plus grand soin. compta tanu da la condition at da la nattat* da I'axampiaira film*, at an conformity avac las conditions du contrat da filmaga. Las axamplairas originaux dont la couvartura an papiar aat ImprimAa sont film«s an commandant par la pramiar plat at an tarminant soit par la darniira paga qui comporta una amprainta d'imprassion ou d'illustration. soit par la sacond plat, salon la cas. Tous las autras axamplairas originaux sont filmis an commandant par la pramiAra paga qui comporta una amprainta d'impraasion ou d'illustration at an tarminant par la darniira paga qui comporta una talia amprainta. Un das symbolas suivants spparaitra sur la darnlAra imaga da chaqua microfiche, salon la cas: la symbols — ^ signifia "A SUIVRE". la symbols ▼ signifia "FIN". Las cartas, planchas. tableaux, etc.. peuvent itre film«s A des taux da reduction diff«rents. Lorsque la document est trop grand pour itra reproduit en un seul clich*. il est film« A partir da I'angia sup«rieur gauche, de gauche A droite, et de haut an bas. en prenant la nombra d'imagas nAcessaira. Las diagrammes suivants illustrent la mAthoda. 1 2 3 1 2 3 4 5 6 MICIOCOPV lESOlUTION TiST CHART (ANSI and ISO TEST CHART No. 2) 1.0 2.5 "^1- I ■ 5 0 ""^^^ IIHII 1^ Im 112.2 12.0 1.8 ^ /APPLIED IIVMGE Inc ^^ 1653 East Main Street S^S Rochester. New Tork U609 USA I^S (ne) *82 - 0300 - Phone ^aS (716) 28S - 59S9 ~ Fa» •.I I 2^'MLt I UNIV2S5??y ' ^ mju. 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, . e c a S^C -( i, a ~ gi, s 5? « CI a i •y » k % ■a bi; M e s M •4 e <« B •- ■^ ■■" •c ■M (- S" 5 o- c3 ST a ■Ji ;^ ;^ t i^ !* e SI oi S» 0 50 CI-->-fC-f-l'S — ««•«»< J. H. Collip 2A 1= e •fl • " " - (^ k> V 4< «« «« «rf :: s * * 1 § i t .s a .5 ,s 1 ^ J 1 «i fi 2. n 03 £. s. s. c h In U V o u e a a e a a n « «rf s 8 e a 8 s 8 — « il 1 1 I^ -t a ■3 ^t. ^i Z * bc a a .£ c = c s a s *« ■s £ ^ B. 01 3 t^ ?• s o a S-^ :i \^ '^ •> ■«» 'us o i •X 1- o" z o c •-■; c o .f; i.' ^1 ss ;5 g .« ^ « ri t>. "- M c c c o '■'■?; s § 5 s? o ^ $ ri s o o a « « 31 jp ®t'?i ® 2J « ?; s s 'jr. Molliisnin Crlomir Flni«l 9 c s e s 3 s •5'^ St S - ' i«= 5 r — CM C S r, r; I- c: X '" o ">■ ri c •■5 ?i a; «i t. s. 1 g* "" — ?i ri S cc f5 c -r c ?; 9 ."^ Tj ® 2 s; o « as .1. n. Coiiip 27 i B 41 s I s (9 ■5 e b 'S ■3 .5 4 ■J. £ « »* fi x ?i « '■^ L-: c c c s c s - ^ "* — S 2 £ ® « --r i«. t- c -c t il i£ i £• S ^ - I- ci g c -r e ae c ->•- r. c y s -,. ^ ... s z c r — «. — = 3 •!• o -3 ■"^''-'-"— — — — — _ 28 Mollusejui CVlnniic F1uir 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 fnsitiv(> (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 Molluscan Celoniic Fluid a o £ u ^ ■2 o. t ^ a j^ u ^^ <= rr <- : : .= Suitoi;^ ?. ?;t:i?5 ''i-'gg?! S cc c< ri r r -r /: a -r r r: c c; o -r o o ci x u3 c X 55 -- rj ~ •'^ 2; S ^ S = v^ c M M ic i(j ■» M ci —' — -I- C -r '^ C3 t^ -1 X = T- I, I, <- •-■ <^^~:x 21 — c ri o so *C O «D ^ *C OOpOOO 0»C^05t>.«D K = s. "; v> ^ S g oc ~ i^ CI CC ■5~ ^ £> 5 O C O ."3 «5 S = ■3 « a b "■• i S3 iJ " 73 J= *" i 3'^, » z^ C CO o c c I is OS o 5- 3r fi ^ — rt ri 8) : c - t 3 el E ■3C ri a§ o s o q = 000 o c c o — ?i ?i O O :^ ^1 o ?J c I- s I- !iC — ->! t >> >> ^ 5 es gs k 0 0 . ,a -; -^ j: * •f «» u g.S 2.S (53 ^ rs * •3 « 3 .5 X . X S t £ £ c — 0 — "i^?: -?. ■s - 0 a- k V C v u. ;*; fe Ji: I I O C = O >•? S.; c5 Ji c o I I X CI sg oc' a: E.2 K • M — J. B. Collip 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^.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 - 2 id S8 C9 • •<= 5 * e 8 -•TOO . C O 9 li^.s a, ^r ^ •■■ ■S * ^ -^ 5 £ <5 «M 2 a as o "5 — • j= s -6 '^ ^ X o s o 5 U5 O O M O s o 1 2730 i o c 5 i o o 1-4 o ^1 o ■«r sis o *-< CM o c c* J. B. CoUip 37 H . B S s & |5! 5 ?s 5 * .a « « 85 S . a u 4) ... Is. 11=1 i>« i-s-^S ii Pii fa^ 1^ fes ^ o o o wo o 8§ o 28 2S M O o o U3 S8 1 1 1 ^ a» us 1 1 o o 3 s o " o s s o o o o SSJ s s ^^ «Q CO ;o t>> US to 1< *8 §5 eo (b t>; oi i ^^ s 8 O r^ ^-^ N— ' h h ki^ O V s •*J ■W ^ C3 >■ i" el a «3 O e3 1 '3 s 1 o^ 38 MolliLscan Celomic Fluid ii % a " •S ^ "^" ?= ^ = i! . = . — " « 'r — .2 _ " e § •- .i ? = 1 - o j; 5=' -? r,~ - " - C 4^ e ^ 24. c _ •a .£ - — .o y a ^ « s - « a ^ a o c c 8 5 I I o in c 8 "i - * « |0 .BJf M 2 5.2 » « C ^ ~ 2 -c .^ ^' 8~ fi 3 C i s 1- ?5 i u I ^^ C o o 9 Is C L* o c o ^s £ s X5 o Rs :2 - c O o O c 5§ li J? 3 S S l' lO 1-H S.i-5 L 7 = *8'f i-t o c 1(5 !•; o i."5 t o rl ^3 t>- o 'J" ?, a £ 5 S.^- o a S E o o * i^l CI © ■^S¥r:, J. B. follip 39 KJTirl of Suhmimiun in l)ixtilli!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 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- i 40 Molluscan CVlomic Fluid 1 i £ c >» >1 a e. is V e o el *« ^ , ^ fc b c i- ^ S ■s ^ - ^ s * t £ £ si Z n. S a 1 S B 1 •s s =: c i 2 i g a -p ji -9 -3 8 2 i .£ -S ■^ a T s ♦- *- «d 4^ a. a. a a ei a. C J« M a M a ;< -x ■X X s 1 r o o a _ < s 1 i E e 7 s o c ?i 1 o 1" . ^ i 1=1 «■= u in '■5 •r o X ri't .o c "C w E 1. I : 1 X 5i , ; : ^ M n f z 1 ^K; 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 <> a i n t> a > > > o o o JS ^ Ji 4 93 se s S u u O u > o > > o o c ^ K Jl X si U a 03 m E C3 in as eS cj e tn a C o u o B ,; a C « ^ in ^ 5 o D. g. 00 m « <» ^ u & S "^ ■* E s « ■s d a y, ^ * •Jl Oi J2 O o E V E 93 s 01 CO X 2 3 o 03 e S E a: a c a S 03 £■ u V Hi s = - b C4 CI bi •I" Tf ,^ ?* If -. CI CI Q C CJ M ?l M p^ O i.~ ?5 « irf o C«3 tC O O » f 05 !0 o o 1^ — C'.'^CSL'tSO— "•-•b-O^tMX . 5 . >, M i; X ^ c .£■ c •* S c S ^ i "S i 5 "5 "x -S S ■s a* ■:: = S = £ . c = w £* I^ Im = i s E ' -o ^ .-2 ^ E E "w * "^ f2 ^3 ^ *G *"*— -IS *— -Z *-■•=*** 'S "^ *3 **-• *3 •-"*= **^ li l^lllll||:2|:ii:=i- OUitOP^ wUt ^U^w c 3 o 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