The Connection Between the Hemal and Digestive Systems
of the Holothuroid, Parastichopus Californicus.
Heather Vandeweghe
Hopkins Marine Station
Pacific Grove, CA 93950
Running Title: The Hemal System
Spring, 1983
The Hemal Syste
Abstract
The hemal system and its association with the
intestine was investigated in P. californicus. Two
main vessels run along the length of the intestine;
the dorsal hemal vessel is attached to the intestine
by small dorsal connections and the ventral vessel
lies nearly on top of the intestine. The dorsal
vessel beats about every eighteen to twenty seconds,
creating a flow pattern of the hemal fluid. The
association of the hemal fluid with the intestine was
determined by the use of cobalt and latex injections.
It was concluded that the hemal fluid flows through
concentric folds that face the lumen of the intestine.
The transport or absorbtion of small molecules from
the lumen of the intestine into the hemal vessels
occurs within a fairly short time-fifteen minutes--
but the passage of larger molecules is blocked.
deweghe
Vandeweghe
The Hemal System
Introduction
While holothurians are widespread in all seas and
at all depths, our knowledge of their ecology and
biology is rather incomplete. (Pawson, 1966 as
stated by Filimonova, 1980.) The hemal system of
some echinoderms has been compared to the circula¬
tory system of higher vertebrates. (Burton, 1964.)
But in no echinoderm is the comparison more apt than
in the holothuroid P. californicus. The structure
and function of the holothurian hemal system has been
studied both histologically (Fish, 1967) and histo¬
chemically. The hemal system of this animal beats
with a distinctive rhythm, has an associated capil¬
lary system, and a close relationship to the digestive
tract. However, the function of these clear vessels
is virtually unknown and not well investigated. This
work proposes a structure for the fine connection
between the hemal vessels and the intestine, shows
that there is passage of some small molecules from the
intestine to the hemal fluid, and describes the
beating of the dorsal hemal vessels.
Vandeweghe
The Hemal System
Techniques
Some of the experiments were done on animals
with empty guts, accomplished by starving the animals
for about thirty hours. Before incisions were made,
the cucumber was relaxed in magnesium chloride (739/1,
lal seawater) for about fifteen to thirty minutes,
until the body wall no longer contracted when handled.
The animals were cut longitudinally on the ven¬
tral side. Pinning out the body wall exposed the
intestines clearly. The animals would remain alive
in this state for at least twenty-four hours if kept
in running seawater most of the time. It was found
to be important to have the animal alive with the
hemal system pumping strongly.
The initial experiments entailed injecting a
fluorescent dye, carboxy fluorescein, into the intes¬
tine and later the hemal vessels. The dye was injected
with a 33-gauge needle at different points along these
organs. A hand-held fluorescent lamp was held over
the animal, making the location of the dye visible.
The clear, thin membranes of these organs made it
possible to see the fluprescence as it flowed within
these organs.
-4-
Vandeweghe
The Hemal System
Cobalt experiments were done by injecting (with
a hypodermic and a 33-gauge needle) a two molar co¬
baltous-chloride solution in seawater into the hemal
system, waiting a few seconds, rinsing with running
seawater for ten or twenty seconds, then topically
applying straight ammonium sulfide (light, Baker's
analyed.) This created a black precipitate wherever
the cobalt solution had flowed in the hemal system.
For the microtome sections, an FD-3 plastic em¬
bedding kit (Poly Sciences) was used after the tissue
was fixed. Fixation was done as follows:
1. Cut intestine into small pieces (1 inch)
2. Perfuse for 2 hours in fixative solution of:
1% gluteraldehyde
1% formaldehyde
85% seawater
5Omm HEPES solution
3. Rinse two times in distilled water.
4. Dehydrate in alcohol series.
The latex experiments were done by injecting
approximately 5 cc of the blue solution (latex in¬
jection medium, Caroline Biological Supply) into the
hemal vessels using 27-gauge neddle on a hypodermic.
After thirty minutes, the latex became sufficiently
hard so that dissecting could be done.
Vandeweghe
The Hemal System
Results
In preliminary dissections, the gross structure
of the internal anatomy was studied with emphasis
on the hemal system and the intestine. The digestive
tract runs the length of the animal three times, as
seen in Figure 1. It is held in place by a mesentery
that attaches the dorsal side of the intestine to the
body wall.
The two main hemal vessels, the dorsal and ven-
tral, appear as clear vessels that run the length of
the intestine. Both are present along the intestine
as it exits the calcarous ring of the mouth. They
are said to originate from the hemal ring which lies
beneath the water vascular ring (Hyman, 1955.) Their
ending is obscure but they seem to fade into the in¬
testine about seven centimeters above the anus.
The ventral vessel lies very close to the in¬
testine along the entire length, and has a transverse
connection running from the bottom third of the de¬
scending part of the intestine to the middle position
of the ascending part of the intestine. This vessel
has the largest diameter of all hemal vessels seen.
-6
Vendeweghe
The Hemal System
The dorsal vessel lies some distance from the
intestine, attached to it by small dorsal connections
which also carry hemal fluid. These connections
begin at the constriction point, and get longer as
they move posteriorly along the intestine and are
sometimes seen to bifurcate after leaving the main
dorsal vessels. At the first bend of the intestine,
the connections become very thin and dense, beginning
what is known as the rete mirable. (Hyman, 1955;
Stutt, 1957.) To the naked eye, this looks like an
orange mass, but under a dissecting scope they appear
as fine capillaries filled with an orange material.
They intertwine among each other and the respiratory
tree, but do not open into the respiratory tree.
(Prosser and Judsen, 1952.)
Approaching the second bend, the rete begins to
concentrate back into the small dorsal connections,
with the bright orange fading out. About midway down
the large intestine, the main dorsal vessel moves
closer to the intestine, soon to blend into it.
The Hemal System
Vandeweghe
Beating and Hemal Flow
A number of investigators have reported a contrac¬
tile nature for parts of the hemal system. (Kawamoto,
1927; Prosser and Judson, 1952; see also Hyman, 1955.)
The dorsal vessel and its small connections beat
rhythmically every eighteen to twenty seconds. (Pros¬
ser and Judsen, in 1952, wrote that the vessels beat
four to five-point-four times per minute.) The con¬
traction moves along the dorsal vessel like a wave,
beginning just posterior to where the rete tapers off,
moving anteriorly and fading out just below the con¬
striction point. The dorsal connections are also seen
to beat with the waves, moving from the main dorsal
vessel towards the intestine. The capillaries of
the rete are not seen to beat, but the hemal fluid
does flow within them. (Figure 2A and 2B.)
Under a compound scope focused on a point along
the dorsal vessel, the flow pattern of the hemal fluid
can be detected by watching small particles that are
suspended in the fluid. The beating creates a mixing
of the hemal fluid with a general flow anteriorly and
towards the intestine. In the dorsal vessel, the
fluid movement is as follows:
The Hemal System
Vandeweghe
1. Fast thrust anteriorly and towards intestine
at the time of contraction,
2. Brief pause,
3. Quick back-wash at the time of vessel relax-
ation, posteriorly and away from intestine,
4. About five seconds of no movement,
5. Slow anterior flow for about five seconds,
slowing to a stop until the next contraction.
In the ventral vessel the flow is more erratic,
depending on the peristaltic movements of the intes¬
tine, but net movement seems to be posteriorly. The
ventral vessel doesn't contract naturally, but with
physical stimulation it will contract at the site of
the stimulation. Physical stimulation also causes the
dorsal vessel to contract. But in this case, the con¬
traction spreads as waves in both directions from the
point of stimulation. The vessel has a latent period
of at least seven seconds before it will contract
again.
-9-
Vandeweghe
The Hemal System
Fine Anatomy:
Watching the flow of the hemal fluid where the
dorsal connections enter the intestine, it can be seen
that the vessels bifurcate beneath the outer epithelium
of the intestine, but where they go from there was the
next question. In previous papers on other holothur¬
oids, authors have described a layer of the intestinal
wall beneath the muscle layer called the "tissue-fluid
complex." (Fish, 1967) The fluid in this layer is
said to be continuous with the hemal fluid. Yet, how
the connection is made was never described, so it seemed
to deserve some attention.
In p. californicus, this layer was not seen, but
rather, an interesting system of folds face the lumen
of the intestine. Under a good dissecting scope, the
digestive tract seems to be comprised of two layers:
a smooth outer epithelium and the inner layer, which
is convoluted into ridges or folds projecting into
the lumen of the intestine. (Figure 3.) These folds
are not solid masses, but have a cavity within them.
(Figure 4.) The best analogy is that of an accordion,
with pleats, or ridges, circling the intestine,
-10-
The Hemal System
Vandeweghe
A single fold is not uniform around the lumen of
the intestine. At the dorsal side, there is a small
groove opposite where the dorsal connections enter
the intestine. Along the sides, the folds are large
and prominent, but they taper down near the ventral
side to tiny, irregular folds, giving a ruffly appear-
ance. This network of tiny ridges is about four
centimeters wide, with the ventral hemal vessel lieing
down the center on the outside of the intestine. It
was also noticed that on one side of the intestine,
the folds are larger, but no functional difference was
seen.
Along the length of the intestine, the folds are
not constant, either. They are absent above the con¬
striction point, and begin just after it. Opposite
the rete mirable, the large folds are not present,
although the intestinal lining appears superficially
to be ruffled all the way around, very much like what
was just described for the ventral part of the intes¬
tine. The folds are distinct where the dorsal con¬
nections are fairly long. The folds are of various
sizes and follow the general pattern of small, large,
small, medium, although there were fluctuations in
that; never were two small or two large folds seen
to be adjacent. (Figure 4.)
The Hemal System
Vandeweghe
The folds can be scraped from the outer epithelium
with ease at the dorsal side, and with a little more
difficulty near the ventral side. At the dorsal side
the folds seemed to be attached at a number of discrete
points, whereas at the ventral side the attachment
seemed continuous. Figure 3 is a drawing from a slide
of a microtome section, which revealed that there is
connective tissue which is responsible for holding the
folds to the outer epithelium.
Could these folds carry the hemal fluid? To
investigate this, two techniques were used: cobalt
precipitation and latex injection. With both of
these techniques, the gut appeared striated from the
outside. When the intestine was cut open, the black
cobalt and blue latex revealed where the hemal fluid
flowed.
It was found that hemal fluid flows within these
folds, thus creating the striated appearance from
the outside. When it came to the ventral area, the
interpretation was more difficult, though it appeared
that the lumen of the folds led into an intricate net¬
work of tiny tubules that lay close to the outer epi¬
thelium. This complex system is schematically pieced
together in Figure 5.
-12
Vandeweghe
The Hemal System
Absorption:
The structure of the hemal system certainly
indicates a possible role as a nutrient transporter.
(Fish, 1967; Filimonova and I. V. Tokin, 1980.) To
examine this, a fluorescent dye, carboxy fluorescein,
was injected into the intestine at different points.
In a full gut, the dye is seen in the hemal vessels
after about fifteen minutes, and after about thirty
minutes in an empty gut. Transport is not seen above
the constriction point, nor opposite the rete mirable
in any measurable quantities. (The orange material
could have made it difficult to see.) Thus, there
is a way for molecules to pass from the lumen of the
intestine into both the dorsal and ventral hemal
vessels.
If the dye is injected into either of the main
hemal vessels, it diffuses quickly along the vessel
and the gut appears striated with fluorescence, as
was seen with the cobalt and latex. This was thought
to represent the dye in the folds. Samples of intes¬
tinal fluid were taken at intervals, and after two
hours, no sign of fluorescence was seen in the lumen.
Vandeweghe
The Hemal System
The gut never took on the appearance of solid
fluorescence either, so it appears as though trans¬
port from hemal to intestine is under different regula¬
tion.
When injected opposite the rete in the main dor-
sal vessel, fluorescence is seen to travel along the
vessel and partly into the tiny capillaries, only if
the vessel is beating strongly. But still, the in-
testine opposite the rete does not become fluorescent
in any way. Either there is a filtering system, or
the dye is moving through in quantities too small to
see.
A large fluorescent protein (75,000 MW, FITC dex¬
tran) was used to see if there is a size limit to
what is transported. When injected into the intes¬
tine, it moved along the gut as the other dye did,
yet after at least two hours, was not seen in any of
the hemal vessels. This indicates that the digestive¬
hemal association allows the transport of substances
smaller than 75,000 MW.
The Hemal System
Vandeweghe
Discussion
A detailed physiological description of the
intestine and the hemal vessels was given. The
hemal fluid is propelled, as described, through
the large vessels and compartments within the men¬
branes of the intestine. This close association
allows the transfer of small molecules from the lumen
of the intestine to the hemal vessels. (Oomen, 1926;
Schreiber, 1932, as reported by Fish, 1952.) Sub¬
stances of small molecular weight (carboxy fluorescien)
were seen to transfer from the lumen of the intestine
to the hemal vessels. The hemal fluid is propelled
through the large vessels and compartments within the
membranes of the intestine. The beating of the dor¬
sal vessel produces a mixing motion with slow move¬
ment anteriorly and across the intestine to the
ventral vessel. The beating was found to be important
for moving hemal fluid into the smaller vessels such as
the dorsal connection and rete capillaries. The pro¬
posed anatomical connection is complex, but its descrip¬
tion will be of great value to further studies in this
area.
Vandeweghe
The Hemal System
For future studies with absorption, the use of
materials that are more sensitive to measurement would
be useful. With the dye, a certain concentration had
to be reached before it was detectable, which could
have given some erroneous results. Also, the use of
more natural substrates (such as sugars or fats) would
be more relevant, in that it would be something the
animals would normally ingest and need for energy.
Past investigators have suggested that transport
across the gut is directly into the coilom and is ac¬
complished by active transport (D'Agostine and Farman¬
furmaian, 1960; Kawaguti, 1964) and have concluded that
the hemal sinuses are not significantly involved in
nutrient transport in either echinoids or hologhurians.
(Farmanfurmaian, 1963) G. F. Filimonova and I. B. Tokin
(1980) mention "The Coelomocyte Theory" (first introduced
by Enriques, 1902) where transport of enzymes and digested
material is by amoebocytes in the hemal fluid. The work
reported in this paper shows that some small molecules,
such as caboxyfluorescein, can be transferred from intes¬
tine to the hemal system. To ascertain whether nutrients
Vandeweghe
The Hemal System
can be transferred, further examination of the types
of molecules that pass across the intestinal-hemal
barrier would be necessary. (Radio isotopes of dif¬
ferent substrates would be useful.) Also, does the
hemal system have vessels that flow along the body
wall? These areas should be investigated to determine
if this system carries nutrients to the body wall of
the P. californicus.
Vandeweghe
The Hemal System
BIBLIOGRAPHY
Burton, M.P.M., 1964. Hemal System of Regular Echinoids.
Nature, 204: 1218
Choe, S., 1962. Biology of the Japanese Common Sea
Cucumber, "Stichopus japonicus" Selenka. 226pp.
Pusan, Korea. (In Japanese with English summary.)
D'Agostino, A.S., and A. Farmanfarmaian, 1960. Trans¬
port of Nutrients in the Holothurian Leptosynapta
inhaerens. Biol. Bull., 119 : 301
Farmanfarmaian, A., 1963. Transport of Nutrients in
Echinoderms. Proc. XVI. Int. Cong. Zool., 1 : 118
Filimonova, G.F., and I. B. Tokin, 1980. Structural
and Functional Peculiarities of the Digestive System
of Cucumaria frondosa. Mar Biol (Berl) 60(1):9-16.
(recd. 1981)
Fish, J.P. The Digestive System of the Holothurian,
Cucumaria elorgata. I. Structure of the gut and
hemal system. Biol. Bull. Mar. Biol. Lab., Woods
Hole 132 : 337-353.
-18-
Vandeweghe
The Hemal System
BIBLIOGRAPHY, Cont'd
Fish, J.P. The Digestive System of the Holothurian,
Cucumona elongata. II. Distribution of the Diges-
tive enzymes. Biol. Bull. Mar. Biol. Lab.,
Woods Hold 132 : 354 - 361.
Hyman, L.H., 1955. The Invertebrates: Echinodermata.
The Coelomate Vilateria. McGraw-Hill Book Co.,
New York.
Kawaguti, S. Electron Microscopy of the Intestinal
Wall of the Sea Cucumber with Special Attention
to its Muscle and Nerve Plexus. Biol. J. Okayama
Univ. 10, 39-50. (1964).
Kawamoto, N., 1927. Anatomy of Cardina Chilersis.
Tohoku Univ. Sci. Repts. Ser 4 Biol., 2 : 239-265.
Krishnan, S. and S. Krishnaswamy: Studies on the Trans¬
port of Sugars in the Holothurian Holothuriascabra.
Mar. Biol. 5, 303-306 (1970).
Lawrence, D.C., Lawrence, A.L., Greer, M.L. and Mailman,
D., 1967. Intestinal Absorption in the Sea Cucumber,
Stichopus Parvinenensis. Comp. Biochem. & Physiol.
20 : 619-627.
-19
Vandeweghe
The Hemal System
BIBLIOGRAPHY, Cont'd
Millot, N., 1966. A Possible Function for the Axial
Organ of Echinoids. Nature, 209 : 594-596.
Oomen, H.A.P.C., 1926. Verdanungsphysiologische
Studien an Holothurien. Pubbl. Staz. Zool.
Napoly 7 : 215-297.
Prosser, C.L., & C. L. Judson, 1952. Pharmacology of
the Hemal Vessels of Stichopus. Biol. Bull.,
102 : 249-251.
Stott, F.C., 1957. Observations on the Food Canal and
Associated Structures in the Holothurian Holothuria
Forskali Delle Chiaje. Proc. Zool. Soc. London,
129 : 129-136.
Vandeweghe
The Hemal System
FIGURES
Fig 1. A diagram of the intestine, shaped as it is
in the opened animal. A full description is given
in the text. The two numbered arrows indicate
the areas where beating begins (1) and ends (2).
Fig 2. These two photographs show the considerable
reduction of the dorsal hemal vessel when it
contracts. (Arrows)
Fig 3. This is a piece of intestine that has part of
it cut and layed open flat. The folds can clearly
be seen along the cut edge (a magnified picture
of this appears in Figure 4.) Notice how the
folds taper out into a flat network of capillaries
near the ventral hemal vessel.
Fig 4. A sketch taken from a slide of one of the
sections from the plastic embedding. It is a
side-on view of the edge of the intestine cut
lengthwise, showing the hollowness of the folds.
Hemal fluid flows into and out of the paper,
through these folds.
Vandeweghe
The Hemal System
FIGURES, Cont
Fig 5. A diagram of the proposed structure of the
hemal system with the intestine removed for
clarity. The dorsal connections appear to bi¬
furcate after entering the intestine. The hemal
fluid then flows within the folds circulating
the intestine. At the ventral side the lumen
of the folds lead into an intricate network of
tiny tubules that have connections to the ventral
hemal vessel.
1 Pharyng
— Stomach
Constriction Point
-Ventral Hemal Vess.
—





Ascendir

Small intestine



Descending



Small Intestiné




---

—

Ventral



Transverse

Connection
TENT



Fig.




Dorsal Connections
Dorsal Hemal Vessel
Large htestine,
lete Mirable
Fig. 2
Fig. 3
Tops of folds
Dorsal Hemal Vessel




Folds


Ventral Hemal Vessel
Outer Epithelium
Fig. 4
Lumen of intestine (location of food)
— Folds
Collogen Materia!



—

ml
—Outer Intestinal Epithelium
Fig. 5







ap;



— Dorsal Hemal Vessel
Dorsal connections
Tubule within a fold


Ventral Hemal Vessel
C
O