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PLEASE TYPE ABSTRACT DOUBLE SPACED BELOW
KINGSTON, ROGER S. (Hopkins Marine Station of Stanford University,
Pagific Grove, California). Anatomical and Oxygen Electrode Studies
of Respiratory Surfaces and Respiration in Acmaea (Mollusca: Gastropoda:
Prosobranchia). The Veliger
Colloidal carbon injection showed that blood flow is close to
the external surfaces in the ctenidium and the mantle facing the
mantle groove, and that blood flows through one ov the other of these
areas just before returing to the heart. A ciliary counter-current
was also found associated with each of these surfaces. Field
observations showed the mantle vessel network expands and ctenidium
withdraws when out of the water. The low intertidal species Acmaea
scutum,A. pelta, and A. limatula have larger ctenidia and smaller
mantle expansion capabilities than the high intertidal species
A. digitalis and A. scabra. Micro-oxygen electrode measurements
indicate that both the mantle and the ctenidium are respiratory
surfaces, and that the mantle is more effective in aerial
conditions and the ctenidium more effective in submerged conditions.
-Author.
-
C
ANATOMICAL AND OXYGEN ELECTRODE
UDIES OF RESPIRATORY SURFACES AND RESPIRATION IN
ACMAEA (MOLLUSCA,: GASTROPODA:PROSOBRANCHIA).
Roger S. Kingston*
Hopkins Marine Station of stanferd University
Pacific Grove, California
Roger s. Kingstor
The limpets of the genus Acmaea are found in abundance
in the rocky intertidal zone on the shores of the Monterey
sninsula, California. Individuals of the species Acmaea
scabra (Could, 1846) and A. digitalis Eschscholtz, 1833,
are found high in zones one and two described by Ricketts
and Calvin (1962). Other species common to this area, A.
elta Eschscholtz, 1833; A. limatula Carpenter,
1864; A. asmi
1833; are
Middendorff, 1849); and A. scutum Eschscholtz,
found under water much of the time lower down in zones three
and four.
These species differences in esposure suggested that
Amaea must be capable of differing derees of respiration
in air and water. The aim of this study was to determine
the site(s) of the specialised respiratory surfaces of
Acmaea, and to list their respiratory effectiveness when
posed to air and when submerged
ANATON
Initially the anatomy of the circulatory system wa
nvestigated. It was assumed that gas diffusion could
ceur across any body surface and that a specialized respir
atory area might be one where gas exchange was facilitated
blood flow close to the external body surface
ger S. Kingston
Individuals of the species Acmaea scutum, A. pelta
and A. digitalis from low, middle, and high intertidal zones
respectively, were taken as samples. The principal method
of study was injection of colored substances into the cireu
tory system.
Injections were carried out with twenty-six gauge
ypodermic needles or fine glass needles pulled from soft
glass tubing. A number of different injection fluids were
ried, including latex, vital stains dissolved in alcohol
or water, and commercial inks. Best results were obtained
with an aqueous colloidal suspension of carbon. The tissue.
remained relaxed during the carbon injections and the pa
ticles did not diffuse out of the vessels. Successful
njections were made on both live, fresh animals and on
animals relaxed in a solution of Mg Gl, isotonic with sea
water. Large areas of the circulatory system were colored
by injections into the heart or visceral cavity. For stud
of localized areas, injections were made into local vessels.
Blood flow direction was determined by observation of the
essels during injections of a dilute carbon suspension.
Gross blood flow, determined by the above methods,
shown in Figure 1. The results indicate two separate
ind distinct areas-the ctenidium and the mantle--where
there is a large amount of blood flow close to the animal.
external surface.
Roger S. Kingsto
The first of these areas, the ctenidium, is generall
considered the respiratory organ of Acmaea. Colloidal carbor
injections of this organ colored the major efferent and
afferent vessels and the capillaries of the right and lef
thirds of the etenidial filaments, but not the central por-
tions of the filaments. The filament vessels, however, wer-
stained in their totality with vital dyes.
These observa-
tions indicate that capillaries sufficiently fine to prevent
the passage of colloidal carbon connect the efferent an
afferent vessels.
The other area where a large amount of blood flow
ound close to the surface was the ventral side of the
mantle fold facing the mantle groove. Blood enters the
mantle fold from the visceral cavity through vessels whic
pass through the shell muscle fibers. In the area between
the base of the mantle groove and the circumpallial vessel
these vessels anastomose. The density of interconnecting
vessels in this network is extraordinary (see plate I, 1-3.)
Near the circumpallial vessel (Figure 2) the vessels of thi
network come together to form a highly branched pattern.
Blood in these vessels goes to the edge of the mantle,
servicing the glands and pallial tentacles, returns to the
anterior afferent vessel, and thence to the heart. As in
the ctenidium, there were fine capillaries in the mantle
fold near the circumpallial vessel which could not be
Kooer S. Kingsteor
filled with suspended colloidal carbon, but which could be
solored by dissolved stains.
he above observations show that the ctenidium and
the mantle fold are the two areas through which blood passe
just before returning to the heart (Figure 1). Since, in
most organisms, blood is oxygenated at the respiratory sur.
these observa-
just prior to its return to the heart,
aces
fold and the
tenidium
ions sugoest that both the mantle
ratory surfaces.
The possible role of the mantle fold as a respirator,
surface suggested that the ciliary mantle currents, earli
described in Acmaea by Yonge (1962) and believed to be
cleansing currents, might also serve a respirato
role
To observe these currents, carmine particles suspended
sea water were placed in the mantle groove of over-turne
animals. A circular current in the mantle groove was found
moving in a plane perpendicular toithe side of the foot and
o the animal's substratum. This ciliary current moved
opposite to the direction of blood flow in the mantle and
was a counter-current of the type often associated with
respiratory surfaces (see Figure 3). A similar ciliar
urrent was found in the nuchal cavity moving opposite
This mantle
the direction of blood flow in the ctenidium.
found in Aemaea is similar to that found in
counter-current
the mantle groove of Lottia (Abbott, 1956)
Roger S. Kingston
Because of the large amount of blood flow close to
the surface, the type of veination, the position relative
to the heart in terms of blood flow, and the ciliary
sounter-currents, both the etenidium and the mantle are
here suggested as respiratory surfaces in Acmaea.
FTELD OBSERVATTONS
Acmaea scabra and A. digitalis from zones one and
two, A. pelta and A. limatula from zone three, and A. scutum
from zone four were observed in the field to record the
behavior of the mantle and ctenidium under dry and wet
conditions. The study was qualitative; observations were
made of animals on dry rocks, on splashed rocks, and in
quiet pools with the aid of a ten-power lens.
When an individual had been out of the water for e
ong enough period to have adjusted to the lack of water,
the following characteristics were usually evident. The
shell was pulled down onto the surface of the rock substr
(though not tightly clamped), the mantle fold was wet (as
found in Lottia: Abbott, 1956), and when the animal was
isturbed it clamped down tightly with some water often
exuding from the mantle groove. If the animal was taken
from the rock and turned over, the wet area of the mantle
fold between the foot and circumpallial vessel was notice-
ably swoilen, and the vessels appeared dilated and gorged
The ctenidium was found to be withdrawn in the
with blood.
246
Reger S. Kingston
nuchal cavity, and in A. scabra which had been dry for many
hours and whose nuchal cavity was no longer filled with
water, the ctenidium was hardly visible. The above char-
acteristics were observed in members of all species but
were particularly evident in A. scabra and A. digitalis.
When an Acmaea individual was under water, its shel
was elevated one to three millimeters off the substrate,
its mantle protruded somewhat beyond the edge of the shell
and a ciliary current in the nuchal cavity, demonstrated
with carmine particles in a quiet pool, flowed counter
tenidial blood flow. When an animal was overturned and
ompared with a member of the same species from a dry area,
the wet animal's mantle appeared flatter and the mantle
vessels seemed smaller in diameter.
in the laboratory where the ventral side of submerged
Acmaea could be observed through aquaria walls, the ctenidia
fA. scutum, A. pelta, and A. limatula were consistentl
found extended and lying partially in the right mantle
groove. In A. scabra and A. digitalis taken from high,
y areas and kept under water in aquaria for forty-eight
r more hours, the ctenidia were never seen to extend more
than one-half the distance from the back to the front of th
nuchal cavity. They are, therefore, apparently not suf-
iciently long to extend to the mantle groove, and are much
Koger S. Kingston
reduced in sise compared to the etenidia of A. pelta,
limatula, and A. scutum.
These field studies indicate that the mantle is the
tte of increased blood flow when the animal is out of the
water, whereas the ctenidium is usually the site of similar
increased flow when the animal is under water. Under waten
the elongated, filamentous ctenidium is a principal wetted
surface. Out of the water, however, the ctenidium contracts
and the surface of the mantle fold is kept wet at the expense
of water in the nuchal cavity, suggesting that the mantle
fold is the chief respiratory site in dry environments.
The laboratory and field observations show that ctenidi
longation and mantle swelling may be controlled by the
water or air environment, and also suggest an evolutionar
reduction in the ctenidium and a corresponding increase in
mantle capacity from low to high intertidal species
Acmaea
IT. POLARCRAPICSTUD
Polargraphic methods were used to substantiate the
spiratory functions of the ctenidium and mantle indicated
nthe previous studies. Electrodes of the recessed type
irst deseribed by Brink and Davies (1942) were used to
measure the difference in the oxygen tensions in different
parts of the body of Acmaea.
doger S. Kingston
sperimental apparatus consisted of several platinum
cathodes, one silver -- silver chloride anode, a 0.8 volt
.c. power source, a Keithley ammeter of 10-9 ampere sen-
sitivity, and a chart recorder. Cathodes were made by
sealing 26 gauge (Bäs) platinum wire in hand-pulled soft
glass capillaries. The wire was first heated to white
incandesence in a flame to drive off the surface adhering
gases, and the capillary then fused around it. Next the
capillary was cut to extend one millimeter beyond the
polished end of the platinum wire. The recess thus forme
as filled with distilled water and covered with a col-
odion membrane to prevent the entry of proteinaceous
material into the recess.
After construction each cathode was calibrated.
First the interval required for equilibration of oxygen
within and without the electrode's recess was determined
by comparing successive readings from a constant environment.
Next the optimum time duration for voltage application was
sought, this being the shortest interval vielding a linear
amps. vs. /02/ relationship. By comparing the electrode
output at different time intervals in sea water samples of
lifferent oxygen concentrations, the reading at eighteen
seconds was found to be the shortest time yielding such a
linear relationship (rigures 5 and o). In the final
Roger S. Kingst
calibration step, an amps. vs. /02/ curve (Figure 6, line
was determined for each cathode, using three to five sea
water samples of known O, content (by Winkler method).
These cathodes were very stable, with little or no changes
evident in the calibration curve from day to day.
Al experiments and calibrations were performed at
To compensate for minor temperature fluctuations
during an experiment (a one degree change resulted in as
much as a ten per cent change in current flow), the 0
ension at two-three different sites on the animal were
imultaneously measured with two-three different cathodes.
neach experiment the animal, previously kept in
he desired environment for a minimum of four hours, was
placed upside-down and the cathodes inserted into the
esired tissue or circulatory vessels, through holes pre
iously made with a dissecting needle. The cathodes wer-
left in place and supported in small ring stands, whereas
he anode was placed on the tissue only at the time
measurement.
In the first experiments, comparisons were made o
lood oxygen in the visceral cavity, the anterior afferen
vessel, and the pericardial sinus. The results (Table 1
show that the O2 tension is higher in the pericardial sinus
in both aerobic and aquatic conditions. Comparing the
visceral cavity with the anterior afferent vessel showed
Roger S. Kingston
that the O, tension was higher in the latter area. Because
lood flows from the visceral cavity to the anterior af-
ferent vessel through the vessels of the mantle fold,
gaseous exchange must occur at the mantle-fold surface.
In the next group of experiments the ctenidial
respiration was eliminated, and the respiratory effici
of the mantle fold determined. Small lead clamps, cut
from thin sheets of lead and bent in "V" shapes, were
inched around the ctenidial afferent and efferent
vesse
fanimals relaxed in Nocl,. The treated animals, kept
vernicht in either wet or dry environments, were tested
during the following days and then sacrificed and examine
ascertain whether the clamps were still in place and
funtioning.
twenty animals kept under water, nineteen were
till alive one day after the clamping operation. Testing
f ten of these animals showed (Table 1) the Op tension of
the pericardial sinus to be somewhat higher than that of
the visceral cavity, although the differences were le
than in normal, unclamped animals.
The second day after the operation the remaining
nine limpets kept under water were dead. This experiment
was performed twice, and each time all the animals were
dead by the second day.
Roger S. Kingston
All twenty animals with clamped ctenidia kept under
dry conditions were still alive one day after the clamping
operation. Testing of ten of these animals showed the
tension of the pericardial sinus to be higher than that of
the visceral cavity, this difference being greater than in
submerged animals with clamped ctenidia, and about the same
as in normal animals under dry conditions.
The second day after the clamping operation two of
the ten dry animals had died. Testing of the remaining
sight showed the Og tension of the pericardial sinus
till be greater than that in the visceral cavity, although
the absolute tension in both were slightly lower than the
previous day.
tour lines of evidence indicate that the mantle fold
serves a respiratory role in the limpet Acmaea. These are
1) the presence of a capillary system close to the surface
fthe mantle fold, (2) a counter-current ciliary system
assing over the mantle fold, (3) the dilation of the
mantle fold with blood, and the concomitant decreased size
of the ctenidia, when the animal is dry, and (4) the polaro-
graphic evidence that the O, tension of the blood is higher
er S. Kingstor
after passage through the mantle fold, and before pa
through the ctenidium.
These results demonstrate that better gascous exchang
ccurs at the mantle surface in air than in water, and that
Acmaea has physiological and behavioral adaptations which
allow it to better expose the mantle in air and the ctenidium
in water.
That Acmaea uses both etenidium and mantle fold a.
tespiratory organs is evolutionarily interesting, pointin
similarities with the related limpets Lottia
gigante:
Sowerby, 1843, which respires with ctenidium and pallial
gills (Abbott, 1956), and Patella,
which has no etenidium
and respires solely with ciliated flaps fringing the margin
of the circumpallial vessel
(Yonge, 1962
oger S. Kingston
UDMARI
The circulatory systen of Aemaea was injected with
olloidal carbon. Two areas - the ctenidium and the mantle
were found where a large amount of blood flous close
the animals' external surfaces. Blood flows through one o
the other of these surfaces immediately before it returns
o the heart. A ciliary counter-current was found associ
with each of these surfaces.
When observed in the field, the mantle fold was
un to expand and the ctenidium to contract when the
animal was out of water. Conversely the ctenidium elongates
and the mantle fold flattens under water. Low intertidal
species of Acmaea have larger ctenidia and smaller mantle
respiratory capacities than higher intertidal species.
Oxygen polargraphy was used to measure the oxygen
tensions of the blood in different parts of Acmaea. These
measurements indicate that both the mantle and the ctenidium
are respiratory surfaces, and that the mantle is more ef-
fective in aerial conditions and the ctenidium more effec
tive in submerged conditions.
ACKNOWLEDGEMENTS
This work was made possible by Grant GY 806 from the
Undergraduate Research Participation Program of the National
Science Foundation.
The author also wishes to thank Drs. David Epel and
Donald Abbott for their encouragement and help during the
study and Dr. Lawrence Blinks for his suggestions and mate-
rials used in the polargraphic study.
Roger S. Kingston
LITERATURE CITED
Abbott, Donald P.
1956. Water circulation in the mantle cavity
of the owl limpet Lottia gigantea Grey.
Nautilus 69(3): 79-85.
Davies, Philip W. and Frank Brink, Jr.
1942. Microelectrodes for measuring local oxygen
tension in animal tissues. Review Scientific
Instruments 13(12):524-533.
Ricketts, Edward F. and Jack Calvin
1962. Between Pacific Tides. 3rd. ed. Stanford
University Press, xi + 518pp. Stanford,
California.
Yonge, C.M.
1962, Ciliary currents in the mantle of
species of Acmaea. The Veliger 4(3):
119-123.
26
Footnote on
Page 1.

*Permanent address:
Footnotes
PHOTO PAGE TITLES
Photo 1: Colloidal carbon injection of mantle (beginning
injection).
1.
glass needle
2. mantle supply vessel
3.
side of foot
4. bottom of foot
5. edge of mantle
Photo 2:
Colloidal carbon injection of mantle (finished
injection). Respiratory vessel network is filled.
Note the "standard" veination in the foot (1).
Photo 3:
Roof of nuchal cavity partially injected.
Injec¬
tion was made in mantle groove at left.
Note
network pattern of vessels (1), anterior afferent
vessel (2), and head pinned back against foot (3).
Photo 4: Functional end of the oxygen cathode.
recess filled with distilled water
2.
glass cover
3. platinum wire
288
FIGURE LEGENDS
Figure 1: Circulatory system of Acmaea. Solid lines
represent know relationships; broken lines
represent supposed relationships.
Figure 2:
Blood flow and respiratory surfaces in Acmaea.
(ventral view)
= anterior afferent vessel
aav
cay =
ctenidial afferent vessel
cey
ctenidial efferent vessel
ctenidial gills
cpy
- circumpallial vessel
foot removed
mantle edge
msy -
mantle supply
vessel
myn -
mantle vessel network
nuchal cavity
ne
pev
posterior efferent vessel
PV
pallial vessels
visceral cavity
ver
visceral cavity removed
Respiratory ciliary current in the mantle groove.
Figure 3:
Solid lines represent blood flow direction;
broken lines represent ciliary current direction.
cpy - circumpallial vessel
foot
mantle
ve - visceral cavity
Figure 4: Circuit for oxygen electrode.
257
Figure Legends, page 2
Figure 5: Recorder tracings at three different oxygen tensions.
A z 2 cc 02/1; B z 1 cc 02/1; C s 0.5 cc 021. The
dotted line at 18 seconds indicates the amperage valve
used for construction of the 0, calibration curve.
Figure 6: Output of the oxygen electrode at different O2 tensions
and different times after closing of circuit. A - 3
seconds; B - 9 seconds; C « 18 seconds.
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