Using ultrasound to understand vascular and mantle contributions to venous return in the cephalopod Sepia officinalis L.
1 Department of Biology, Dalhousie University, Halifax, NS,
Canada
2 Department of Radiology, Dalhousie University, Halifax, NS,
Canada
3 Department of Psychology, Dalhousie University, Halifax, NS,
Canada
4 Scripps Institution of Oceanography, La Jolla, CA 92093-0209,
USA
* Author for correspondence (e-mail: ajking{at}dal.ca)
Accepted 8 March 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: cardiovascular dynamics, venous return, ventilation, cephalopoda, mantle, circulation, systemic heart, branchial heart, vein, cuttlefish, Sepia officinalis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Our understanding of coleoid cardiovascular function is incomplete. One
outstanding issue is the relative importance of the mantle (the large muscular
body wall that forces water over the gills), the hearts, and the contractile
veins in driving venous return in intact coleoids. The mantle encloses the
large veins and the hearts (Tompsett,
1939), much as the mammalian thorax encloses the equivalent
organs. Mantle contractions influence intravenous pressures more noticeably
than contractions of other organs such as the renal appendages, and have been
credited with driving venous return within the mantle cavity
(Johansen and Martin, 1962
;
Bourne, 1987
). However, to move
blood between vascular areas within the mantle cavity, mantle contractions
must generate pressure differences between those vascular areas. The pressures
created by the mantle, although large at times, are probably applied equally
to all vascular areas within the mantle. Therefore they would not create the
pressure differences required to generate venous flow. Besides the intravenous
pulse caused by the mantle, a second, shorter, overlaid pulse also is usually
measured in the large veins. It is not usually considered to be propulsive,
and often is attributed to the contractions of organs bordering the veins such
as the gills or renal appendages (Johansen
and Martin, 1962
; Bourne,
1982
). Another possibility, however, is that the large veins
themselves might contract in vivo, creating this second pulse and
also contributing to venous return. Indeed, the large veins have been found to
contract in vitro (Williams,
1909
; Tompsett,
1939
; Schipp,
1987a
), in dissected octopods
(Smith and Boyle, 1983
), and
in anaesthetized octopods whose mantles were turned inside out
(Wells and Smith, 1987
). Early
studies on cephalopod cardiovascular systems concluded that the system was
driven solely by serial peristalsis between organs [Bert (1867), Fredericq
(1914) and Skramlik (1929), as cited in
Johansen and Martin, 1962
;
Wells and Smith, 1987
].
Understanding what generates the propulsive forces is important for our
understanding of cardiovascular function and energetics in cephalopods.
Despite our incomplete understanding of cardiovascular function in
coleoids, experiments tapered off in the early 1990s. This is probably partly
because coleoids are difficult to study using existing technology. Recent
experiments have usually measured intravascular pressure or blood flow by
implanting cannulae in the vasculature of unrestrained, unanaesthetized
octopods (e.g. Johansen and Martin,
1962; Wells, 1979
;
Wells and Wells, 1983
;
Wells et al., 1987
). Squid and
cuttlefish are less well studied. We know of only two studies investigating
pressures and flow through squid vessels (Bourne,
1982
,
1984
), and no studies
investigating pressure or flow in cuttlefish vessels. The three in
vivo studies on cuttlefish circulation measure only systemic heart
function (Mislin, 1966
;
Chichery and Chanelet, 1972a
;
Chichery, 1980
), possibly
because the large internal shells of cuttlefish and their other viscera impede
access to many of the veins (Chichery and
Chanelet, 1972b
).
Imaging technologies promise to improve our understanding of coleoid
cardiovascular dynamics. Ultrasound imaging was used by Tateno
(1993) to visualize octopus
mantles. We used ultrasound imaging to view the cardiovascular organs of
unanaesthetized, unrestrained cuttlefish (Sepia officinalis L.) in
real time. Ultrasound can be applied in any imaging plane and is non-invasive.
Consequently, we were able to view different combinations of organs
repeatedly, without harming the cuttlefish.
Our study sought to determine which organs contributed to venous return in resting cuttlefish, in order to clarify how cuttlefish circulatory power requirements are met. First, we established that we could reliably identify the large veins in cuttlefish using ultrasound imaging. Then, we determined whether the veins appeared to contract actively or to be compressed by other organs. We examined what role the veins might play in driving venous return. Finally, we evaluated the mantle's role in propelling the blood of resting coleoid cephalopods.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental set-up
We monitored physiological parameters using an ultrasound machine and a 5
MHz convex array ultrasound transducer (Ultramark 4 plus, Advanced Laboratory
Technologies, Bothell, Washington, USA). Ultrasound transducers emit high
frequency sound and interpret the reflected sound to create two-dimensional,
real-time images of the internal organs of animals. Our transducer created
images at approximately 17 frames s-1. We could resolve vessels
that were a few millimeters in diameter, which limited us to imaging the large
veins (the arteries were too small to see). The transducer was held under the
experimental tank and moved to capture the organs of interest. Ultrasound
images (sonograms) were recorded on Hi-8 movie tape.
To reduce disruption to the cuttlefish during experiments, the experimental tank was divided into an inner and an outer compartment (Fig. 2). During experiments, we placed a single cuttlefish in the cylindrical (27 cm diameter, 14.5 cm high) inner compartment, where water depth was approximately 8 cm. This inner compartment had rigid plastic walls and a flexible, thin, white, plastic membrane across the bottom, upon which the cuttlefish could settle. Water entered the inner compartment through perforated airline tubing that ran around its bottom edge. Water then flowed through approximately 120 small holes (6 mm in diameter) in the walls of the inner compartment to the outer compartment, from which water was drained by a siphon. Water remained well aerated in the inner tank when a cuttlefish was present (>90% O2 saturation).
|
The outer compartment was made of flexible opaque plastic that hung from a rigid wooden frame, 29 cmx30 cm. To exclude sound-reflecting air from between the convex surface of the transducer and the surface of the outer compartment, consumer grade hand cream was applied to the transducer, which was then pushed into the soft plastic of the outer compartment. The water-filled space between the outer and inner compartments allowed the transducer to be operated without disturbing the cuttlefish.
The cuttlebone (the calcified skeletal structure found inside the dorsal body wall of the cuttlefish) is opaque to ultrasound. To avoid it, we insonated cuttlefish from below, through the acoustically transparent plastic bottoms of both the inner and outer compartments.
To visually isolate the cuttlefish from the rest of the room an opaque plastic curtain surrounded the experimental tank. A camcorder (CCD-TR910 NTSC, Sony, Tokyo, Japan) above the tank and connected to a remote monitor (Trinitron, Sony), enabled us to monitor the cuttlefish and to record its behaviour on Hi-8 movie tape during experiments. The camcorder movie and sonograms were synchronized using audio cues recorded on both tapes.
Experimental protocol
Each trial comprised seven 10 min physiological readings. The first reading
started 30 min after the cuttlefish had been transferred into the experimental
tank; before 30 min, cuttlefish moved too much to obtain the required
sonograms. Subsequent readings started every 15 min, the last reading starting
2 h after the transfer. During each reading, we attempted to image one of the
five organ groups described below for at least 20 s. Several trials were
performed on the same cuttlefish, each separated by at least 2 days.
Circulatory organs are labeled in Fig.
1. Where possible, our nomenclature follows common usage
(Williams, 1909;
Tompsett, 1939
;
Hill and Welsh, 1966
). We have
added a region that we call the `branch point' (BP). Although this point is
probably not physiologically distinct from the lateral venae cavae, it
clarifies subsequent discussion to note its location between the anterior vena
cava and the arms of the lateral venae cavae. To measure contractions of the
anterior vena cava, we identified two points along its length: Point A, near
the opening of the mantle, and Point B, adjacent to the opening of the
anus.
During each trial, we attempted to record images that contained the following organs simultaneously for at least 20 s (planes along which these sonograms were made are shown in Fig. 1): (1) A midsagittal section of the anterior vena cava and the mantle; (2) A midsagittal section through the branch point, the ventricle and the mantle; (3) A transverse section through the branch point, the efferent branchial vessels, the gills and the mantle; (4) A transverse section through the lateral venae cavae, the ventricle and the mantle; (5) A roughly transverse section through the ventricle, a branchial heart and the mantle.
In some trials, not all organ groups were imaged. Organ groups were not always imaged in the same order during a trial. Examples of sonograms can be viewed online as part of this article (see Movies 1 and 2 in supplementary material).
Data analysis
Movie segments were eligible for analysis only if a stable image of one of
the organ sets listed above was visible for at least 20 s. From these, we
excluded movie segments in which cuttlefish were moving or showing non-resting
body patterns (Hanlon and Messenger,
1996). For each day and each cuttlefish, we performed the analysis
below on the single longest remaining movie segment for a given organ set. The
entirety of this movie segment is referred to as an observation in the
results.
We recorded the times t (±1/15 s) of maximal contraction
and maximal expansion for each organ of interest in the selected movie
segment. Maximal contraction and expansion were determined by visually
assessing the diameter of the vessel, heart or mantle. We calculated the phase
shift between contractions of organs that were visible simultaneously and
contracted at the same rate (differed by less than 4%) as follows:
![]() | (1) |
where tn is time of contraction of organ n and pn is period of organ n.
This calculation was performed for each contraction for a given organ pair
of a given cuttlefish on a given day (i.e. for that observation). Using
standard methods for circular statistics
(Zar, 1999), we then
calculated one average phase shift for each organ pair for that day and that
cuttlefish (i.e. for that observation). The average phase shift measures the
relative timing of the two organs' contractions; an average phase shift of
approximately 0° or 360° indicates that the two organs tended to
contract simultaneously, whereas an average phase shift of approximately
180° indicates a half-beat delay between the two contractions. To measure
the consistency of average phase shifts, we used the circular statistic
r (Zar, 1999
).
Let 1 be the phase of contractions of organ 1, i.e.
![]() | (2) |
and similarly for 2. Now r2 is the
squared correlation for a linear regression (forced through zero) between the
complex variables
z1=ei
1/180 and
z2=ei
2/180 [where
i=(-1)1/2], and resembles the coherence of conventional
spectral analysis (Priestley,
1981
). We calculated the statistical significance of average phase
shifts from r2 and the sample size using equation (1.3) of
Greenwood and Durand (1955
).
Significance indicated that the phase shifts were more similar than we would
expect by chance.
The Friedman test for trends (Zar,
1999) was used to test significance when order of contractions,
rather than similarity of phase shift, was of interest.
If we could not see the contractions of organs that have been reported in the literature to contract, we measured the cross-sectional area of the organ using the public domain NIH Image program (version 1.62, developed at the US National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/) to ensure we were not overlooking contractions that were not large enough to be visible. For each cuttlefish, we made 21 measurements of the organ both when it was most likely to be contracted, and when it was most likely to be expanded. This was repeated three times for the same 21 contraction cycles. The mean difference between contracted and expanded organs was taken for each cycle. The standard deviation (S.D.) between the three replicates was taken for each cycle. Then we took the mean of the difference and of the S.D. over the 21 contraction cycles. The mean S.D. was taken as the measurement error. If the mean difference between expanded and contracted measurements was not greater than the measurement error, then we assumed there was no difference, i.e. that the organ did not contract.
Histology of vascular valves
To find valves in the anterior and lateral venae cavae, we injected blue
tracing medium into the vasculature of an anaesthetized cuttlefish
via the peribuccal sinus. Tracing medium (following
Tompsett, 1939) was prepared
by dissolving 60 g of melted gelatin and 6 g of potassium iodide in 60 ml of
glycerol and 240 ml of 0.2% Alcian Blue (dissolved in 30% glacial acetic acid
and 70% ethanol). Functionally, we identified a valve as a place where the
tracing medium could be manually advanced along the vessel, but not pushed
backward. We then dissected a 1 cm section of vasculature on either side of
the putative valve and fixed it in neutral buffered formalin.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Organs were identified by their anatomical placement and connections. In the anterior part of the mantle cavity, the anterior vena cava was obvious in transverse views (Fig. 3A). In midsagittal views, it could be viewed in its entirety from the anterior end of the mantle to the branch point at the posterior end of the digestive gland (Fig. 3B; see Movie 1 in supplementary material). Anterior to the branch point, the anterior vena cava became small and indistinct in transverse views. The branch point was conspicuous in transverse view and its contractions were obvious (Fig. 3C and Movie 2 in supplementary material). It was harder to find in longitudinal section (Fig. 3B; see Movie 1 in supplementary material). The lateral venae cavae were distinguished by their thick walls (Fig. 3D). Their apparent wall thickness was likely due to their renal appendages. The ventricle appeared oblong and bent in transverse view (Fig. 3D). The shape and placement of the branchial hearts differed among cuttlefish. The efferent branchial vessels appeared as thin-walled structures on the distal edge of the gill (Fig. 3C).
Variability of mantle and ventricle contraction rates
Two hours after moving a cuttlefish to the experimental tank, we estimated
the ventilation rate (mantle contractions) and heart rate (ventricular
contractions) by counting the number of complete contractions in 10 s. At
21°C, the mean ventilation rate was 49.5±10.4 breaths
min-1, and the mean heart rate was 39.0±4.8 beats
min-1 (N=4 cuttlefish). The same four cuttlefish kept at
15°C had means of 33.0±6.6 breaths min-1 and
22.5±1.9 beats min-1, respectively, giving a Q10
of 1.97 for ventilation rate and 2.50 for heart rate. However, this
Q10 must be interpreted with caution, because the cuttlefish were
older and larger when kept at the lower temperature.
When the mantle and ventricle were observed simultaneously, their
contraction rates were not well correlated at a given temperature (linear
regression: 21°C: r2=0.112; 15°C:
r2=0.005). At 15°C, the mantle usually contracted
faster than the ventricle (30/37 observations, five cuttlefish). In 5/37
observations on five cuttlefish, the mantle and the ventricle shared the same
contraction rate. Even when they had the same rate (5 observations, two
cuttlefish), however, the phase shift between mantle and ventricle was not
consistent within (4 observations, one cuttlefish,
r2=0.00015, NS) or between animals (two cuttlefish,
r2=0.1557, NS). Contractions of the heart and mantle have
been found to be independent in previous studies on cuttlefish
(Chichery and Chanelet, 1972a),
octopus (Wells, 1979
) and
squid (Shadwick et al.,
1990
).
Contractions of vessels and gills
In all five cuttlefish kept at 15°C, we observed obvious contractions
of the anterior vena cava, the branch point, the lateral venae cavae, the
branchial hearts, the efferent branchial vessels, the auricles, the ventricle
and the mantle. The veins were considered to be actively constricting
(contracting) because they remained circular while their diameter decreased,
rather than being deformed. Furthermore, the vessels were not being passively
extended by a passing bolus of blood because there are no strongly pulsatile
organs upstream of the veins that could create such a bolus of blood. Informal
observations suggested that the auricles contracted approximately 180° out
of phase with the ventricle.
The gills changed shape and moved back and forth within the mantle (supplementary material, Movie 2). To determine whether gill movements were synchronized with mantle movements, we recorded the times when the gills were farthest from the mid-line of the cuttlefish (`maximum') and when they were closest (`minimum'). These were compared to the expansions and contractions of the mantle, respectively. The phase shift between the gill and mantle movements was consistent both within animals (4/4 cuttlefish where mantle movements could be accurately assessed, see Table 1) and between animals (N=4; maximum: r2=0.7168, P<0.05; minimum: r2=0.9708, P<0.01). Although the gills moved, we were unable to find evidence that they contracted. For each cuttlefish (N=5), we used the NIH Image program (version 1.62) to measure the transverse cross-sectional area of the gills at their `maxima' and `minima' for 21 consecutive oscillations. In 3/5 cuttlefish, the difference between the maximum and minimum areas was not greater than the measurement error (approx. 17 mm2 out of approx. 550 mm2). In the other two cuttlefish, one showed a maximum that was slightly larger than its minimum (G-statistic for goodness of fit: mean difference=14.9 mm2, G=11.9, d.f.=1, P<0.001) and in the other, the situation was reversed (G-statistic for goodness of fit: mean difference=-22.9 mm2, G=15.9, d.f.=1, P<0.001). It seems unlikely that the gills contracted. If they did, gill contractions were not obvious and not synchronized with their movements within the mantle cavity.
|
Of the vessels observed, only the anterior vena cava contracted at the same rate as the mantle (18/18 observations, five cuttlefish). The ventricle always shared the contraction rate of the branch point (9/9 observations, five cuttlefish), the lateral venae cavae (19/19 observations, 5 cuttlefish) and the branchial hearts (13/13 observations, four cuttlefish; for a fifth cuttlefish, no data was obtained on the branchial hearts). We were unable to reliably capture an efferent branchial vessel and the ventricle in the same image. Therefore, the efferent branchial vessel's contraction rate was compared with that of the branch point, which always contracted at the same rate as the ventricle (see above). The efferent branchial vessel contracted in 21/26 observations (five cuttlefish). We might not have seen all instances of efferent branchial vessel contraction. We were more likely to see contractions when image quality was good, when we held the transducer at certain angles and when we looked at the end of the efferent branchial vessel that joined the auricle. Using the criteria established in the methods, we selected 17 of the instances that the efferent branchial vessel contracted in five cuttlefish for analysis. In 13 of these, the efferent branchial vessel had the same rate as the branch point. In the 4 observations in which the rates differed, results were split between the efferent branchial vessel contracting faster (4.4% and 4.6% faster), and the branch point contracting faster (8.4% and 19.7% faster). The efferent branchial vessel never contracted at the same rate as the mantle.
Therefore, there appear to be two groups of organs actively contracting at two different rates. In one group were the mantle and the anterior vena cava. In the other were the ventricle, the branch point, the lateral venae cavae, the branchial hearts and, usually, the efferent branchial vessels.
Relative timing of vascular contractions
Vessels that contract at the same rate do not necessarily contract in an
order that will propel blood. We investigated the timing of contractions along
the anterior vena cava, and also between the branch point, lateral venae cavae
and the branchial heart, to test whether contractions could be propulsive.
Because these vessels and organs can only generate pressure gradients by
contracting (they do not actively expand;
Wells, 1978;
Schipp, 1987a
), only the
relative times of contraction were considered.
Sonograms revealed that the anterior vena cava contracted in peristaltic waves that traveled posteriorly (see Movie 1 in supplementary material). The time of contraction of two points on the anterior vena cava (Fig. 1) were measured: Point A was close to the opening of the mantle, and Point B was at the opening of the anus. In 4/4 cuttlefish, there was a consistent phase shift between the contractions of Points A and B both within (for a fifth cuttlefish, there were only two data points, so significance was not calculated; see Table 1) and among animals (N=5, r2=0.88, P<0.01). Given that the period of the contractions of the anterior vena cava varied, it is perhaps not surprising that the speed of the peristaltic wave varied within and among cuttlefish. The lowest mean speed for a given cuttlefish was 0.05 m s-1 (range: 0.04-0.06 m s-1, 4 observations), and the highest was 0.1 m s-1 (4 observations). Cuttlefish #26 had the highest variability in speeds of peristaltic contraction along the anterior vena cava (0.05-0.08 m s-1, 4 observations). However, speed did not vary with the period of contraction (linear regression: 18 observations, 5 cuttlefish, r2=0.47, P=0.197, NS).
|
|
Anatomical separation between anterior vena cava and branch point
Since the branch point and the anterior vena cava contract at different
rates (BP and AVC of Fig. 1),
the branch point will occasionally contract when the anterior vena cava is
expanding. This might push blood anteriorly towards the head, instead of
posteriorly towards the hearts. We found a previously unidentified valve in
this location that prevented flow reversal
(Fig. 5A).
Tracing medium could be pushed from the anterior vena cava into the branch point. When we applied pressure to the lateral venae cavae or branch point, however, the tracing medium did not flow back into the anterior vena cava (Fig. 5B,C).
The presence of a valve was confirmed in histological section. Successive
serial longitudinal sections of the anterior and lateral venae cavae were
examined to reconstruct the structure of the valve
(Fig. 5A). The valve was
composed of a large flap of thin cellular valve tissue with a shallow,
off-center slit in it. The tissue was attached obliquely in the vessel along
the dorsal and one of the lateral walls, and part way along the ventral wall.
Where it attached to the vessel walls, the valve tissue was 2-3 cells thick,
and was supported by extensive extracellular fibres. These fibres stained
green with Masson's trichrome, indicating the presence of fibrous proteins,
which could be collagen (Flint et al.,
1975). The remainder of the tissue was usually only one cell thick
(Fig. 5D). Where it was split,
the edge of the larger portion of the valve was reinforced by
polysaccharide-rich extracellular matrix
(Fig. 5E). Polysaccharides were
selectively stained blue by the acidic Alcian Blue dye used in the tracing
medium (Klymkowsky and Hanken,
1991
). Muscle cells were interspersed within the fibrous matrix in
the smaller portion where the valve split
(Fig. 5F). Muscle cells stained
red. We did not stain specifically for nerves.
Role of mantle in circulation
Previous researchers have suggested that the mantle might drive venous
return, especially through the anterior vena cava
(Johansen and Martin, 1962;
Bourne, 1982
,
1987
). Indeed, the anterior
vena cava was the only vessel that contracted at the same rate as the mantle
in our study. Therefore we investigated the mantle's role in driving blood
through the anterior vena cava.
|
The phase shift between the anterior vena cava and the mantle, although consistent within cuttlefish, was not consistent among cuttlefish (Table 1: N=4; Point A expansion: r2=0.5183, NS; Point B expansion: r2=0.4767, NS). In other words, when the mantle was fully expanded, the anterior vena cava could be one quarter contracted in one cuttlefish, but entirely contracted in another (Fig. 6, two outermost circumferences). Therefore, none of the organs or vessels investigated were synchronized with the mantle in a way that was consistent between cuttlefish. Nevertheless, mantle dynamics may somehow interact with vascular function. Earlier, we noted that one group of organs contracted with the mantle's rate (anterior vena cava) and another group contracted with the ventricle's rate (lateral venae cavae, branchial hearts and efferent branchial vessel). The ratio of contraction rates between the mantle and the ventricle groups was highly correlated to the phase shift between mantle and anterior vena cava expansion (Fig. 7; N=4, r2=0.9837, P=0.0082). The reason for this is unclear.
|
Discussion
The large veins (the anterior vena cava, the lateral venae cavae and the
efferent branchial vessels) have been observed to contract in vitro
(Williams, 1909;
Tompsett, 1939
;
Schipp, 1987a
), in dissected
octopods (Smith and Boyle,
1983
), and in anaesthetized octopods whose mantles were turned
inside out (Wells and Smith,
1987
). Wells and Smith
(1987
) proposed that all
veins, except the anterior vena cava, contract actively and contribute to
venous return. However, venous function in intact coleoid cephalopods has been
difficult to study using implanted pressure transducers
(Chichery and Chanelet, 1972b
;
Bourne, 1982
,
1987
;
O'Dor et al., 1990
;
Shadwick et al., 1990
;
Pörtner et al., 1991
)
and, until now, non-invasive techniques have not been applied to this system.
Using ultrasound, we have made the first non-invasive measurements of
cardiovascular function in unanaesthetized, free-moving, intact cephalopods,
and the first measurements of any kind on vascular function in cuttlefish. All
the large veins, including the anterior vena cava, contracted actively in
resting cuttlefish.
As in octopods (Smith,
1962), the cuttlefish anterior vena cava pulsed at the same rate
as the mantle. In our cuttlefish, the anterior end of the anterior vena cava
usually contracted when the mantle was expanding, and vice versa.
Therefore the anterior vena cava does not appear to be compressed by the
mantle in resting cuttlefish. Furthermore, contractions of the anterior vena
cava were peristaltic, traveling posteriorly towards the branch point. We
conclude that the anterior vena cava contracts actively and peristaltically to
propel blood towards the branch point. The peristaltic waves of the anterior
vena cava of S. officinalis traveled at speeds of 0.04-0.1 m
s-1, slightly slower on average than the 0.1 m s-1
reported previously for the arm veins of the octopus Enteroctopus
dofleini Wülker (Smith,
1962
). The variable speed of peristalsis along the anterior vena
cava suggests that its speed might be influenced by nervous or hormonal input.
Variable speeds might help synchronize venous contraction with ventilatory
activity.
The anterior vena cava contracts at a different rate than the branch point.
Consequently, contractions of the branch point could occasionally push blood
back towards the anterior vena cava. We discovered a valve that prevents
backflow from the branch point to the anterior vena cava, thereby ensuring
that blood flows only in the proper direction. We suggest this valve be called
the Wells valve in recognition of his influential pioneering work on
cephalopod circulation. Many vascular valves in crustaceans
(Wilkens, 1997;
Davidson et al., 1998
) and
octopods (Smith and Boyle,
1983
) are both muscular and innervated. The Wells valve was
muscular, but we do not know whether it was innervated. If innervated, it
could regulate cardiac output by controlling blood flow into the lateral venae
cavae and therefore into all three hearts; like other molluscan hearts, the
systemic heart of coleoids adjusts its cardiac output according to venous
return (Wells and Smith,
1987
). Furthermore, by regulating flow into the lateral venae
cavae, the Wells valve could regulate the pressure in the renal appendages,
which are involved with solute exchange between the blood and the forming
urine (Martin and Harrison,
1966
).
Once past the Wells valve, blood was propelled by a second peristaltic wave. This wave started at the branch point, traveled along the lateral venae cavae, and ended with the contraction of the branchial hearts. Because there is a valve at both ends of the lateral venae cavae (the Wells valve and one at the entrance to the branchial heart), the lateral venae cavae could act as auricles to the branchial hearts.
The gills moved within the mantle, probably in response to mantle-driven
water movement. However, unlike previous authors
(Johansen and Martin, 1962;
Bourne, 1982
), we found no
evidence that the gills contracted in intact cuttlefish. We saw no
contractions and could not detect contractions when we measured the
cross-sectional area of the gills at their closest and furthest points from
the cuttlefish's midline. If the gills did contract, contractions were either
less than 3% of the gill's transverse area (measurement error), or were not
synchronized with their movements within the mantle. The gills might still
contribute to circulation; preliminary evidence suggests that, in
vitro, individual gill lamellae or vessels within the lamellae contract
to propel blood through the branchial capillaries
(Wells and Smith, 1987
).
After passing through the gills, blood appeared to be forced into the
auricles by contractions of the efferent branchial vessels. These contractions
were especially evident in the section of efferent branchial vessel connected
to the auricles. Contractions usually had the same frequency as ventricular
contractions, but always a different frequency than mantle contractions.
Unlike octopods (Johansen and Martin,
1962), the efferent branchial vessel is probably not an extension
of the auricle in cuttlefish because there is a valve separating the efferent
branchial vessel and auricle in cuttlefish
(Versen et al., 1997
).
Cephalopods have maximized the numbers of contractile veins, likely to ensure
ample venous return to feed the elevated cardiac output of the coleoid
heart.
The lateral venae cavae, branchial heart, efferent branchial vessels and
ventricle all contracted with the same frequency, and in a specific order.
These contractions were not simply driven by contractions of the mantle, which
had a different frequency. Furthermore, the lateral venae cavae and the
branchial hearts are on one side of the branchial capillary bed, whereas the
efferent branchial vessels and the ventricle are on the other side. Therefore,
the rate and order of contractions was not driven by simple serial peristalsis
between organs, as was suggested by early authors [Bert (1867), Fredericq
(1914) and Skramlik (1929), as cited in
Johansen and Martin, 1962;
Wells and Smith, 1987
]. The
nervous system probably plays a role in coordinating contractions; an
investigation into the innervation of this area revealed that the lateral
venae cavae, the branchial heart, the efferent branchial vessels, the auricles
and the ventricle are all connected by nerves in octopods
(Smith, 1981
;
Smith and Boyle, 1983
). The
auricles may set the rate of ventricle
(Versen et al., 1997
). It has
been suggested that an element in the ventricle is responsible for
establishing the contraction rate of all these interconnected organs
(Wells and Smith, 1987
). Our
results raise the possibility that such a region might instead be in the
branch point (Fig. 4).
Both heart rate and ventilation rate decreased with decreasing temperature.
Adapted Q10 values for heart and ventilation rate were,
respectively, 2.50 and 1.97. These values might be confounded by uncontrolled
age and size effects, but they are similar to Q10 values for heart
rate and ventilation rate in Octopus vulgaris Cuvier
(Wells, 1979) and the squid
Lolliguncula brevis Blainville
(Wells et al., 1988
).
We have assumed that the mantle does not compress venous vessels directly
in resting cuttlefish in part because none of the vessels' contractions were
timed to the mantle's movements in a way that was consistent between
cuttlefish. However, new evidence suggests that although mantle cavity
pressure changes have the same frequency as mantle movements, they are not
timed to the ventilatory period in a way that is consistent between cuttlefish
(F. Melzner, personal communication). If this is true, the anterior vena cava,
but none of the other veins observed in our experiments, may contract when the
mantle pressure is high. Even if contractions of the anterior vena cava are
timed to pressure increases within the mantle cavity, however, it does not
mean that mantle pressure is directly compressing the anterior vena cava. The
anterior vena cava contracts peristaltically towards the posterior of the
cuttlefish, and the pressures created during resting ventilation likely travel
anteriorly above the anterior vena cava. The fact remains that none of the
veins contracted in a way that was consistent with the pressures produced by
the mantle. Instead, the coordination between the contractions of the anterior
vena cava and the pressures in the mantle may facilitate venous return from
the head and arms during the low venous and mantle pressures that coincide
with anterior vena cava and mantle expansion
(Smith, 1962;
Wells et al., 1987
).
It is not to say that mantle contractions have no possible role in circulation. Contractions of the mantle and anterior vena cava might be linked to contractions of the rest of the vasculature. The mantle and anterior vena cava contracted at the same rate, and all other investigated organs contracted at the same rate as the ventricle. The ratio of contractions between mantle and ventricle groups is strongly correlated with the phase shift between the onset of mantle contraction and the onset of anterior vena cava contraction. However, the nature of the connection between these organ groups is obscure.
The interaction between the heart and the mantle is modified during
cephalopod jetting. During octopus jetting, the heart stops
(Wells et al., 1987), and
during squid jetting, heart rate increases
(Shadwick et al., 1990
). It is
unclear how jetting will affect cuttlefish circulation. Further investigations
into mantle vasculature dynamics, peripheral vascular resistance, vessel
pliability and regional pressure changes within the mantle cavity (especially
if they are synchronized with sonograms) might clarify the mantle's role in
circulation in both resting and jetting cuttlefish.
Like vertebrate cardiovascular systems, coleoid cephalopod cardiovascular
systems are proving to be complicated, certainly much more complicated than a
series of vessels that propel blood simply by serial peristalsis, or a system
driven solely by a systemic heart (Wells,
1978; Schipp,
1987b
). In some regards, coleoid circulation is strikingly
mammalian. This convergence has been shaped by similar factors
(Packard, 1972
;
O'Dor and Webber, 1986
);
however, the original Bauplan of each group has resulted in important
and interesting differences between these groups. Non-invasive technologies
such as ultrasound provide us with tools to further the investigation of this
sophisticated but poorly understood invertebrate circulatory system.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
Present address: School of Medicine, Department of Orthopedics and School
of Veterinary Medicine, Department of Anatomy, Physiology and Cell Biology,
University of California, Davis, CA 95616, USA
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berne, R. M. and Levy, M. N. (1997). Cardiovascular Physiology. St Louis: Mosby.
Bourne, G. B. (1982). Blood pressure in the Squid, Loligo pealei. Comp. Biochem. Physiol. 72A, 23-27.
Bourne, G. B. (1984). Pressure-flow relationships in the perfused post-systemic circulation of the squid, Loligo pealei. Comp. Biochem. Physiol. 78A,307 -313.
Bourne, G. B. (1987). Hemodynamics in squid. Experientia 43,500 -502.
Brusca, R. C. and Brusca, G. J. (1990). Invertebrates. Sunderland: Sinauer Associates.
Chichery, R. (1980). Etude du comportement moteur de la seiche Sepia officinalis L. (Mollusque céphalopode): Approches neurophysiologique et neuropharmacologique.PhD thesis , L'Université de Caen, France.
Chichery, R. and Chanelet, J. (1972a). Enregistrement et étude de l'électrocardiogramme de la Seiche (Sepia officinalis). C.R. Soc. Biol. 166,1421 -1425.
Chichery, R. and Chanelet, J. (1972b). Action de l'acétylcholine et de diverse substances curarisantes sur le système nerveux de la Seiche. C.R. Soc. Biol. 166,273 -276.
Davidson, G. W., Wilkens, J. L. and Lovell, P.
(1998). Neural control of the lateral abdominal arterial valves
in the lobster, Homarus americanus. Biol.
Bull. 194,72
-82.
Farrell, A. P. and Jones, D. R. (1992). The Heart. In Fish Physiology, vol.XII , Part A (ed. W. S. Hoar, D. J. Randall and A. P. Farrell), pp. 1-88. Toronto: Academic Press.
Flint, M., Lyons, M., Meaney, M. F. and Williams, D. E. (1975). The Masson staining of collagen - an explanation of an apparent paradox. Histochem. J. 7, 529-546.[CrossRef]
Greenwood, J. A. and Durand, D. (1955). The distribution of length and components of the sum of n random unit vectors. Ann. Math. Statist. 26,233 -246.
Hanlon, R. T. and Messenger, J. B. (1996). Cephalopod Behaviour. Cambridge: Cambridge University Press.
Hill, R. B. and Welsh, J. H. (1966). Heart, Circulation and Blood Cells. In Physiology of Mollusca, vol. II (ed. K. M. Wilbur and C. M. Yonge), pp. 125-174. New York: Academic Press.
Johansen, K. and Martin, A. W. (1962). Circulation in the cephalopod, Octopus dofleini. Comp. Biochem. Physiol. 5,161 -176.[CrossRef][Medline]
Klymkowsky, M. W. and Hanken, J. (1991). Whole-mount staining of Xenopus and other vertebrates. Methods Cell Biol. 36,419 -441.[Medline]
Mangum, C. P. (1990). Gas Transport in the Blood. In Squid as Experimental Animals (ed. D. L. Gilbert, W. J. Adelman and J. M. Arnold), pp.443 -468. New York: Plenum Press.
Martin, A. W. and Harrison, F. M. (1966). Excretion. In Physiology of Mollusca (ed. K. M. Wilbur and C. M. Yonge), pp. 353-386. New York: Academic Press.
Mislin, H. (1966). Über Beziehungen zwischen Atmung und Kreislauf bei Cephalopoden (Sepia officinalis L.). Synchronregistrierung von Elektrocardiogramm (Ekg) und Atembewegung am schwimmenden Tier. Verh. dt. zool. Ges.; Zool. Anz. Suppl. 30,175 -181.
O'Dor, R. K. and Webber, D. M. (1986). The constraints on cephalopods: why squid aren't fish. Can. J. Zool. 64,1591 -1605.
O'Dor, R. K. and Webber, D. M. (1991). Invertebrate athletes: trade-offs between transport efficiency and power density in cephalopod evolution. J. Exp. Biol. 160,93 -112.
O'Dor, R. K., Pörtner, H. O. and Shadwick, R. E. (1990). Squid as elite athletes: locomotory, respiratory and circulatory integration. In Squid as Experimental Animals (ed. D. L. Gilbert, W. J. Adelman and J. M. Arnold), pp.481 -503. New York: Plenum.
Packard, A. (1972). Cephalopods and fish: the limits of convergence. Biol. Rev. 47,241 -307.
Pörtner, H. O. (1994). Coordination of metabolism, acid-base regulation and haemocyanin function in cephalopods. In Physiology of Cephalopod Molluscs: Lifestyle and Performance Adaptations (ed. H. O. Pörtner, R. K. O'Dor and D. L. Macmillan), pp. 131-148. Basel: Gordon and Breach.
Pörtner, H. O., Webber, D. M., Boutilier, R. G. and O'Dor, R. K. (1991). Acid-base regulation in exercising squid (Illex illecebrosus, Loligo pealei). Am. J. Physiol. 261,R239 -R246.[Medline]
Priestley, M. B. (1981). Spectral Analysis and Time Series. New York: Academic Press.
Schipp, R. (1987a). The blood vessels of cephalopods. A comparative morphological and functional survey. Experientia 43,525 -537.
Schipp, R. (1987b). General morphological and functional characteristics of the cephalopod circulatory system. An introduction. Experientia 43,474 -477.
Shadwick, R. E., O'Dor, R. K. and Gosline, J. M. (1990). Respiratory and cardiac function during exercise in squid. Can. J. Zool. 68,792 -798.
Smith, L. S. (1962). The role of venous peristalsis in the arm circulation of Octopus dofleini. Comp. Biochem. Physiol. 7, 269-275.[CrossRef][Medline]
Smith, P. J. S. (1981). The role of venous pressure in the regulation of output from the heart of the octopus, Eledone cirrhosa (Lam.). J. Exp. Biol. 93,243 -255.
Smith, P. J. S. and Boyle, P. R. (1983). The cardiac innervation of Eledone cirrhosa (Lamarck) (Mollusca: Cephalopoda). Phil. Trans. R. Soc. Lond. B 300,493 -511.[Medline]
Tateno, S. (1993). Non-invasive analysis of mantle movements in Octopus vulgaris. In Recent Advances in Fisheries Biology (ed. T. Okutani, R. K. O'Dor and T. Kubodera), pp. 559-569. Tokyo: Tokai University Press.
Tompsett, D. H. (1939). Sepia. Liverpool: University Press of Liverpool.
Versen, B., Gokorsch, S., Lücke, J., Fiedler, A. and Schipp, R. (1997). Auricular-ventricular interacting mechanisms in the systemic heart of the cuttlefish Sepia officinalis L. (Cephalopoda). Vie Milieu 47,123 -130.
Wells, M. J. (1978). Octopus: Physiology and Behaviour of an Advanced Invertebrate. London: Chapman and Hall.
Wells, M. J. (1979). The heartbeat of Octopus vulgaris. J. Exp. Biol. 78, 87-104.
Wells, M. J. (1994). The evolution of a racing snail. In Physiology of Cephalopod Molluscs: Lifestyle and Performance Adaptations (ed. H. O. Pörtner, R. K. O'Dor and D. L. Macmillan), pp. 1-12. Basel: Gordon and Breach.
Wells, M. J. and Wells, J. (1983). The circulatory responses to acute hypoxia in Octopus. J. Exp. Biol. 104,59 -71.
Wells, M. J. and Smith, P. J. S. (1987). The performance of the octopus circulatory system: A triumph of engineering over design. Experientia 43,487 -499.
Wells, M. J., Duthie, G. G., Houlihan, D. F., Smith, P. J. S. and Wells, J. (1987). Blood flow and pressure changes in exercising octopuses (Octopus vulgaris). J. Exp. Biol. 131,175 -187.
Wells, M. J., Hanlon, R. T., Lee, P. G. and DiMarco, F. P. (1988). Respiratory and cardiac performance in Lolliguncula brevis (Cephalopoda, myopsida): the effects of activity, temperature and hypoxia. J. Exp. Biol. 138, 17-36.
Wilkens, J. L. (1997). Possible mechanisms of
control of vascular resistance in the lobster, Homarus americanus.
J. Exp. Biol. 200,487
-493.
Williams, L. W. (1909). The Anatomy of the Common Squid, Loligo pealii, Lesueur. London: E. J. Brill.
Zar, J. H. (1999). Biostatistical Analysis. Upper Saddle River: Prentice Hall.
Related articles in JEB: