Abstract
The viscera of bluefin tuna, Thunnus thynnus, are warm, and internal temperatures as
high as 25°C have been recorded in 5°C water (Block, pers. comm.). The source of this heat and
the visceral site of vascular heat conservation are not clear. Measurements taken after feeding of
bluefin implicate the caecum as the warmest visceral organ, however, little is known about the
general anatomy of the tuna caecum or its method of heat production. Specimens of yellowfin,
Thunnus albacares, and blackfin, Thunnus atlanticus, were used to study the anatomy, histology
and aerobic capacity of the tuna caecum. Anatomical comparisons were also made to the bonito,
Sarda chiliensis, an ectothermic relative of the tuna. Injections of silicon into the caecum
vasculature detected a parallel array of arteries and veins throughout the organ. Measurements of
citrate synthase activity, an enzymatic indicator of tissue metabolic capacity, show a high degree
of caecum metabolic activity, around 20 units/g tissue at 25°C. This value is 4-5 fold greater
than tuna white anaerobic muscle, but not as high as the aerobic red muscle. The high heat
contribution from aerobic activity, along with that contributed by digestive processes within the
caecum and stomach, may be able to account for much of the heat production within the viscera.
The endothermic nature of the organ and the parallel architecture of the vasculature suggest that
countercurrent heat exchange can occur. An increased visceral temp would lead to faster
digestion and provide a continuous source of fuel for the tuna's high metabolic needs.
Introduction
While performing visceral temperature measurements on bluefin tuna, Francis Carey
discovered the warmest recorded temperatures in the pyloric caecum, at an average of 15.7
above water temperature (Carey, 1984). He went on to implicate the caecum as the major site of
visceral heat production, suggesting that the heat originates from a combination of digestive and
metabolic processes within the caecum. Carey hypothesized that high heat production in
combination with heat conserving anatomy within the viscera would lead to faster digestion and
allow the migrating bluefin to take advantage of a sporadic food supply. Since Carey's study
archival satellite tag data has recorded visceral temperatures as high as 25°C above ambient
water temperature (Block, pers. comm.), however little has been done to uncover the role of the
caecum in heat production within the tuna. It may well be that research on the caecum has halted
due to a lack of information about the general anatomy of the organ, and an uncertainty as to its
specific function.
In most fish, the pyloric caeca are appendages of the anterior intestine and are not
considered to be a separate organ. The caeca are finger-like projections that arise from the
intestine and end blindly. The projections typically number between 1 and 100. Caeca appear to
function mainly in fat absorption (Greene, 1913), but in some fish have also been shown to
participate in water absorption (Boge et al., 1988). The caeca of most fish contain pancreatic
tissue between the pyloric fingers that produce many digestive enzymes such as trypsin and
chitinase (Jobling, 1995), however this has not been documented in tuna. In scombrid fish such
as the bluefin and the related species of the Thunnus genus, the caecum is classifiable as an
organ. The caecal mass dominates the viscera, often weighing in as the largest organ along the
digestive tract (Gibbs and Collette, 1967). While the role of the fish pyloric caeca in fat
absorption has been most recently supported by Jobling, no such studies could be found for the
tuna. Histological studies performed during this project show a high degree of lipids within the
tuna caecum suggesting that the general role of the caecum in fat absorption is the same.
Knowledge about the bluefin viscera is extensive despite the lack of information about
the caecum. The presence of countercurrent heat exchangers within the bluefin tuna has been a
subject of much interest because of the migratory nature of the fish. The bluefin is typically
found in temperate zones where water is cold and heat conserving strategies are important to its
survival. Studies on the visceral anatomy of tropical relatives of the bluefin, the yellowfin,
blackfin, and longtail have never been done. While all three tropical species employ
endothermic strategies, their visceral anatomy has never been of much interest. There is a
considerable dirth of information about the visceral anatomy of tropical species of tuna.
In the bluefin the viscera is thermally isolated from the rest of the fish body by five
countercurrent heat exchanging retia (Fudge and Stevens, 1996). The arterial portions of these
retia arise from the main artery that supplies the viscera, the coeliac artery. The coeliac artery
branches off of the dorsal aorta just below the posterior epibranchials (Gibbs and Collette, 1967).
It is unclear where the venous branches originate, however it is known that they travel through
the retia, into the liver and out two efferent vessels, collecting the venous blood in the sinus
venosus.
The visceral retia thermally isolate the viscera by facilitating countercurrent heat
exchange. Heat being produced in the viscera, through digestive and metabolic processes, is
carried in the blood through the veins. The heat is transferred to the adjacent arteries bringing in
cool blood from the gills, allowing the venous blood to be cooled before it enters the liver and
heart and retaining the heat within the viscera (Block, pers. comm.). The liver, does not receive
a significant amount of the conserved heat (except perhaps by conduction), as it is on the cold
side of the heat exchanger (Carey, 1984). The bluefin retia are well diagrammed by Eschricht
and Müller, and have been studied in an effort to better understand the endothermic nature of the
fish (Fudge and Stevens, 1996). Despite the knowledge of the bluefin visceral retia, little is
known about the blood supply to the caecum or its role in heat production.
Because of the difficulty and expense of obtaining bluefin specimens for study,
two congeneric species, the yellowfin, Thunnus albacares, and the blackfin, Thunnus atlanticus,
were used to study the caecum. The yellowfin and blackfin tuna are tropical species which,
while considered endothermic, do not display the same degree of endothermy as the bluefin.
While it is known that both possess well developed countercurrent heat exchangers associated
with their swimming muscle, an in depth anatomical study of their viscera, like that of Eschricht
and Müller (1835), has never been done.
Ihypothesized that the yellowfin and blackfin would share similar anatomical features
with the bluefin in the form of retia leading from the coeliac artery to each of the visceral organs.
After studying the gross anatomy of the yellowfin and blackfin, I then injected silicon rubber
into the vessels leading to and from the viscera to determine the perfusion of the caecum and the
surrounding organs. Histological studies were also performed to better understand the caecum
on a cellular basis. The study also included a dissection and silicon injection of a bonito, Sarda
chiliensis, an ectothermic species in the same family as the tuna, for comparison.
Once the caecum blood supply was mapped out and the presence of a countercurrent heat
exchangers was determined in yellowfin and blackfin tuna, the extent of heat production was
assessed by enzymatic study. To determine the tissue metabolic capacity of the caecum, citrate
synthase (CS) activity was assayed. Citrate synthase, the rate limiting factor for the citric acid
cycle, is considered an enzymatic marker for aerobic metabolism. Heater tissues of scombroid
fish typically exhibit high levels aerobic activity (Tullis et al., 1991). Since metabolic activity
produces heat, it may be possible to gauge relative heat production according to CS activity. For
comparison, CS activity in another visceral organ, the liver, was measured. What resulted was a
picture of the caecum as a site of countercurrent heat exchange within the yellowfin
Materials and Methods
Four yellowfin tuna, referred to as YFTI, YFT2, YFT3, and YFT4, and two bonito, were
obtained from the Tuna Research and Conservation Center in Monterey, California. Deaths
occurred either from anesthesia or by pithing. Yellowfin and bonito used for dissection/injection
were either fresh or previously frozen.
Two blackfin tuna, were caught on longline in the Gulf of Mexico and frozen before
dissection.
Silicon Injections
Injections were performed using Microfil injection compounds MV-120 (blue), MV-130
(red) and MV-117 (orange). Components were mixed in a 5 (color): 4 (diluent): 1 (catalyst)
ratio. Curing time was approximately twenty minutes depending on the core temperature of each
fish and the ambient temperature in the lab.
The desired vessels were first found through dissection. A catheter attached to a Cole-
Pamer three-way stopcock was inserted as far as possible into the vessel of choice and the
system was flushed with sea water in order to rinse out blood and test for leaks or blockages.
Leaks were clamped with hemostats or blotted with gauze. Silicon compound was then pushed
through using gentle hand pressure on a 10 ml syringe. Injection continued until applied
pressure became too great or until all the injectant was used. Sites of injection and amounts of
injectant varied as techniques were developed. Table 1 outlines the sites and amounts of
injection in each fish.
Table 1: Sites and Amounts of Silicon Injections
Injection Amount
Injection Site (5)
Fish
Venous
Arterial
——
20 ml
ventral stomach vein
YFTI
(blue)
(Fig. 1a)
60 m
50 ml
lateral cutaneous
YFT2
(blue)
(orange)
artery and vein
45 ml
30 m
lateral cutaneous vein
YFT3
(blue)
anterior epibranchial
(red)
YHI4*
40 ml
10 ml
anterior epibranchial
(red
(blue)
ventral stomach vein
(Fig. 1b)
lateral cutaneous
25 ml
15 ml
BLKI
(orange)
(blue)
artery and vein
(Fig. 1c)
——
40 ml
lateral cutaneous artery
BLKZ
(blue)
—
30 ml
Dorsal Aorta
bonito
(orange)
* Only successful double injection
The double injection of YFTI began with the venous injection. For YFT2, the arterial
side was injected first, followed by the venous side. YFT3 began with the venous injection
followed by the arterial injection. For YFT4, the arterial side was injected, followed by the
venous side. Finally, for BLKI the arterial side was injected first, followed by the venous side.
Following injection, viscera were excised from each fish and fixed in 10% phosphate
buffered formalin. Viscera were studied intact first, following the path of the coeliac artery, then
caeca were separated from the remainder of the viscera, sectioned, and viewed under a Leica
dissecting scope.
Histology
Caecal tissues from yellowfin and blackfin were removed from fish shortly after death,
fixed in 10% phosphate buffered formalin, sectioned, stained with Masson's Trichrome, and
prepared on a slide for light microscopy.
CS Assays
Assays were performed on one caecum and liver sample from each of seven yellowfin, as
well as two caecum samples from blackfin tuna (they were not the same fish that were used for
injection). All tissues were stored in a-80°C freezer prior to use in assays. Tissue specimens
were weighed in 0.2-0.3 g wet weight portions. Removal of fatty tissue from caecal tissues was
not attempted due to the high fat content of the caecum. Upon thawing, tissues generally lost
0.1g of water weight. The tissue was then minced on an ice cold stage, and extraction buffer
added to achieve a 1:10 dilution. Extraction medium consisted of 40 mM Hepes, ImM EDTA,
and 2mM MgCl2, pH 7.98 020°C. The mixture was then homogenized in a ground glass
homogenizer. The homogenate was sonicated in two 15 second bursts with a 30 second break in
between, and the final dilution was performed by adding more extraction buffer. Dilutions were
1:50 for caecum samples and 1:33 for liver.
Assays were performed at 25°C using a Perkin-Elmer Lambda 6 spectrophotometer at
412nm. Activity was measured as micro moles of DTNB reduced per minute (Tullis, et al.
1991). Each homogenate was assayed 6 replicate times.
Statistics
A single factor ANOVA was run for caecum and liver citrate synthase activities between
the seven sampled fish. The same was done for the blackfin caecum CS activities. A pe.05 was
considered to be significant. A regression plot of CS activity as a function of animal mass was
also run to test for a correlation.
Results
Yellowfin, Thunnus albacares
The caecum takes up a large portion of the visceral cavity, lying ventral to the stomach
and dorsal to the liver (Fig. 2-3). It is composed of hundreds of pyloric fingers, blind tubules
lying in a parallel array (Fig. 3b). The pyloric fingers are grouped in bundles with open spaces
between each bundle. The entire organ is surrounded by a thin transparent mesentery that may
prevent it from moving around within the visceral cavity. The liver envelops the ventral anterior
portion of the caecum, covering a large surface area. It is difficult to separate the two due to this
close anatomical relationship. The caecum receives partially digested material from the pylorus
of the stomach (Fig. 2a) which divides into two smaller tubules as it enters the caecum. These
two tubules lead into the caecum and travel about halfway down its length before anastomosing
in the pyloric fingers which make up the bulk of the organ.
The caecum receives arterial blood from two of three branches of the coeliac artery (Fig.
2a). The first and largest branch runs along the center of the middle lobe of the liver, beneath the
stomach supplying both the middle and right liver lobes and the main caecal artery. The second
branch travels along the left lobe of the liver, first feeding the left gastric artery and then sending
smaller branches to the liver, caecum, gall bladder, spleen, and intestine. The third and final
branch of the coeliac feeds the right gastric artery and the right liver lobe. The caecum also
receives multiple arterial branches from each lobe of the liver. These branches divide into many
small arterioles upon entering the caecum. The arterial blood traveling through the caecum alsc
travels into the ventral stomach artery. This artery is part of a rete connecting the stomach to the
caecum (Fig. la). This rete is made up of many parallel arteries and veins and is the only
connection between the stomach and the caecum.
Venous blood from the stomach travels into the caecum through the ventral rête.
Immediately upon entering the caecum, the veins split into the multiple small parallel vessels
which make up the countercurrent array. From the caecum, the venous blood continues into
each of the three lobes of the liver and then out two efferent vessels, one on the right liver lobe
and one on the middle liver lobe. The arterial vessels leading from the liver to the caecum lie
adjacent to the venous vessels from the caecum to the liver, but no well developed retia, like the
one between the stomach and caecum, are present. A general diagram of yellowfin circulation
based on dissections of the four injections in shown in figure 4.
Extensive vasculature is seen in the center of the dorsal surface of the caecum (Fig. 5).
These blood vessels appear to branch out into smaller vessels which continue throughout the
caecum. The first injection was performed in order to achieve a better understanding of the
vasculature within the caecum. Injection of YFTI led to a complete venous injection of the
caecum. This was confirmed by dissecting into the sinus venosus and noting that the silicon had
entered the atrium of the heart. Dissection of the caecum revealed a high degree of venous
vascularization. Many small venules run throughout the caecum down to the tip of the organ
(Fig. 6a). These vessels often appear in bundles, but no clear pattern is seen in most of the
caecum. Where the ventral stomach vein enters the caecum, there is extensive branching of the
vessels and an array of tightly packed venules can be seen running parallel to each other (Fig. 6b
and c). A closer inspection identified a number of empty arterioles amongst the venules. In
order to see the relationship between the arterioles and venules, dual injections were attempted.
The second and third injections were successful arterial injections permitting a clear
picture of the arterial arrangement. A similar setup of the arteries was discovered, with the
arterioles branching throughout the individual pyloric fingers of the caecum. As observed in the
venous injection, the same parallel architecture of arterioles can be seen where the ventral
stomach vein enters the caecum (Fig. 6b and c).
The injection of YFT4 was a successful double injection of arteries and veins. The
injection resulted in the conclusive identification of a countercurrent array of vessels. As the
arteries approach from the top of the caecum, forking off the first branch of the coeliac artery,
the venules from the bottom of the caecum meet up to form a parallel network surrounding the
pyloric tubules (Fig. 7a). For the first time, I could identifity the two main arterial vessels, both
extensions of the coeliac artery, feeding the countercurrent region of the caecum (Fig. 7b).
These vessels both have venous blood surrounding them. The injection was not entirely
complete and the venous / arterial relationship could not be determined for the entire caecum. For
those regions that were doubly injected, however, it was observed that the venules and arterioles
continued together into the farthest regions of the caecum.
Histology also reveals that parallel venules and arterioles are present between each
pyloric finger and are found in varying numbers throughout the caecum. The histological
sections also depict the countercurrent vasculature within the caecum. Slides of the
countercurrent area show the clear parallel nature of the venules and arterioles (Fig. 8a and b).
Arterioles can be distinguished by their larger proportion of smooth muscle and their small
diameter relative to the amount of that muscle. The venules appear to be more flexible than the
arterioles, often conforming to match the local arrangement of arterioles. Each pyloric finger has
a surface area made larger by epithelial loops projecting into the lumen of the finger (Fig. 8c).
Mucous secreting goblet cells are present along the epithelial lining of each pyloric finger (Fig.
8d).
Blackfin, Thunnus atlanticus
The caecum of the blackfin, and its relationship to the other organs in the viscera, is very
similar to vellowfin (Fig. 9). The blood supply is the same as that diagrammed for the yellowfin
(Fig. 4). The only two apparent differences are in the caecum's relationship with the liver and
the organization of its arterioles.
The liver of the blackfin is tightly bound by tissue connections to the caecum, making it
more difficult to view the arterial and venous connections between the two. The vessels in the
countercurrent region are less densely packed than those of yellowfin and appear to be more
delicate (Fig. 10a-c). The arterioles in the countercurrent region show less organization than in
the vellowfin although they are laid out in the same fashion; with the largest population at the
center where the ventral stomach vein feeds the venous blood to the caecum.
The blackfin caecum is histologically similar to the yellowfin. The pyloric fingers of the
blackfin are smaller and many more can be seen at the same magnification in the same amount of
space as the vellowfin (Fig. 11a), however, they display a similar organization of the epithelial
tissue. At higher magnification, the goblet cells are easily recognizable as are the different type
of cells that make up the epithelial lining (Fig. 11b). They appear to share the same
countercurrent array of arterioles and venules that could not be positively identified without a
dual injection (Fig. 11c). There appear to be no significant anatomical or histological differences
between the yellowfin and the blackfin.
BLKI vielded a successful arterial injection, but the venous injection failed to get past
the heart. At this point, I realized that an injection into the lateral cutaneous vein after injection
into the lateral cutaneous artery would never be successful because it would always get blocked
in the heart. In order to solve the problem, BLK2 was injected first into the lateral cutaneous
vein in an attempt to fill the venous side. Unfortunately, the catheter was inserted into the lateral
cutaneous artery rather than the vein. In an effort to salvage the injection, the fish was opened
viscerally. The injection could not be saved as the viscera was already too decomposed. Only
BLKI was used for anatomical study.
Bonito, Sarda chiliensis
The bonito viscera is laid out much like the tuna with slight differences in the size and
structure of the organs (Fig. 12a). The liver of the bonito is almost inseparable from the caecum.
The bonito caecum lacks any of the fine structured parallel vasculature of the yellowfin and
blackfin although it is highly vascularized (Fig. 12b).
The blood supply to the bonito viscera follows the same model as that laid out for the
vellowfin (Fig. 4). The coeliac artery has three main branches which send blood to all three
lobes of the liver, the stomach, caecum, and all the other visceral organs. It is not clear whether
any retia are present in those connections.
11
The one bonito injection performed was not successful because the caecum was too
decomposed. Most anatomical observations were performed on freshly dead bonito that were
dissected without injection in order to learn about the general visceral makeup.
Enzymes
Assays were performed on caecum and liver tissue from seven yellowfin. The average
value recorded for caecum tissue is 21.4 units/g tissue + 1.9. The range of values for the caeca
of these fish is between 11.88 units and 30.06 units (Table 2). There is significant variation
between all seven of the caecum tissues sampled. Liver samples resulted in an average of 2.36 +
18 units and a range between 1.69 units and 2.85 units (Table 2). Liver samples also show
significant differences amongst fish, but these differences were much less than those recorded
for the caecum. Liver assays were used both for comparison and as a control. Caecum values
are approximately 9-fold larger than liver values for each of the seven fish (Fig. 13a).
Assays run on two blackfin show an average value of 20.27 +0.1 activity/g tissue (Table
2) with no significant difference between the two samples.
To account for the differences seen amongst caecum CS activity of fish, a table of
historical information about each fish was compiled (Table 4). The regression plot of fish mass
ys. caecal CS activity (Fig. 13b) shows a trend toward increasing activity with increasing body
size, however there is no significant correlation. A regression plot performed on days in
captivity vs. caecal CS activity (Data not shown) also shows no significant correlation.
Discussion
My observations of the gross anatomical organization of the circulatory anatomy
indicates an extensive countercurrent heat exchange system throughout the tuna caecum. The
presence of a rete combined with the high degree of aerobic metabolism within the caecum both
support this conclusion.
There is no doubt that the anatomical requirements for countercurrent heat exchange exist
in the tuna caecum. Both silicon injections and histological studies reveal the presence of a rête
within the caecum. Injections and histology also showed that both the yellowfin and the blackfin
possess this rete in their caeca, suggesting that this characteristic may be true for all tuna. The
fact that the bonito lacks such a rete may mean that this caecum anatomy is limited to
endothermic fish. The documented parallel network of vessels running all throughout the organ
is well developed and highly organized. Similar countercurrent architecture was observed in the
red muscle of the injected tuna. The caecum is perfused with cool oxygenated arterial blood
arriving from the gills. As it meets up with the warm venous blood carrying metabolic and
digestive heat produced within the caecum, heat exchange can occur. The countercurrent nature
of the vessels allows the heat to be transferred with the highest efficiency.
The rete in the caecum is receiving cool arterial blood from both the gills and the liver.
The countercurrent flow of blood in the vessels leading from the caecum to the liver and vice
versa suggests that little heat transfer is occurring between the caecum and liver. The venous
blood the caecum receives from the stomach passes first through the ventral stomach rete which,
depending on the temperature gradient between the two organs, may or may not be conserving
heat in the stomach. Regardless, heat remains where it is produced. What results is the complete
isolation of the caecum from the surrounding organs. Any heat increase in the caecum can only
come from the processes taking place within the tissue. The only exception would be conductive
heat processes between the organs. The liver probably receives the largest portion of this
conductive heat due to its close relationship to the caecum, which would account for the warm
liver temperatures observed by Carey (1984).
The isolation of the caecum from the rest of the viscera means that any heat produced by
the caecum will remain in the caecum, but it also means that the high caecum temperatures
recorded by Carey in his bluefin study must be accounted for by heat produced mainly in the
caecum (although some conductive heat transfer may be occurring between the caecum and other
visceral organs). Citrate synthase assays have shown that the caecum is a highly aerobic tissue
The average value of 21.4 units/g tissue is comparable to values around 27 units/g tissue
measured for swordfish red muscle (Tullis et al., 1991). Values that showed the most variation
amongst vellowfin caeca were most likely the result of variations in enzyme concentration along
the tissue. Fish number seven (Fig. 14), showed low activity in both the caecum and the liver,
suggesting faulty sampling procedure or damaged tissue. Even with the variation amongst
samples, when compared to liver values obtained for the same fish, the aerobic capacity of the
caecum seems even greater. Why is it that two visceral organs should be so different in their
metabolic activities? How does the tuna benefit from an aerobically active caecum?
The histological slides revealed a high degree of lipid within the caecal tissues of
vellowfin and blackfin. If indeed the caecum is the main site of lipid digestion, the tuna would
reap an evolutionary benefit by maintaining an aerobically active caecum. The tuna requires an
immense amount of fuel to power their most aerobically active tissue, the red muscle. The
muscle receives this fuel in the form of ATP produced aerobically. The production of ATP
requires 2-carbon substrates supplied by lipid metabolism (Tullis et al., 1991). The caecum
would be responsible for supplying this lipid fuel. The lipid would be absorbed into the venules
amongst the pyloric fingers of the caecum and sent to the liver where it would be stored for
future mobilization. The liver is a well known site of lipid storage and it is logical that this lipid
would come from the caecum (Jobling, 1995).
Assuming that the caecum is a site of lipid absorption, it may be possible to predict the
major sources of heat within the organ. While heat is a known by-product of metabolic activity,
this activity alone cannot account for all the heat produced in the caecum. The remaining heat
must be supplied by the enzymatic processes involved in fat digestion. Carey predicted that in a
20kg portion of food the hydrolysis of fat and protein would release approximately 13Okcal of
heat and account for about 1/3 of the heat increase within the visceral organs (Carey, 1984). He
does not predict anything for the heat produced in the caecum, however, a similar proportion of
heat production is probable due to fat hydrolysis alone.
What remains to be answered is how the tuna would benefit from countercurrent heat
exchange within the caecum. By conserving heat produced within the caecum, the tuna would
benefit from elevated enzymatic activity. The faster enzymes can work, the faster food can be
digested. For migrating fish like the yellowfin and bluefin, faster digestion could mean more
14
efficient utilization of an ephemeral food supply as Carey suggests. Faster digestion also means
faster fat absorption. This speed in absorption will allow more frequent feeding, increasing the
supply of lipid to the liver. These lipids can either be stored for future use or mobilized in order
to fuel the powerful swimming muscles that allow the tuna to be great predators.
Assays of lipid oxidation within the caecum will prove useful for further study. Now
that it is clear that heat is being produced metabolically, the source of the remaining heat cannot
be elusive for long. Further injections performed on yellowfin and blackfin may lead to a more
complete injection, and injections on other species of tuna will determine whether countercurrent
heat exchange is possible throughout the genus.
Conclusion
The presence of countercurrent heat exchanging architecture within the caecum of the
vellowfin tuna along with a high degree of aerobic activity contributing to heat production
indicates a model for heat conservation within the tuna caecum. Similar anatomy within the
blackfin tuna suggests that this model is not confined to the yellowfin and may be present in all
species of tuna.
Acknowledgments
Many thanks to my advisor, Dr. Barbara Block, who always demonstrated enormous
enthusiasm for my project. She spent countless hours assisting me on injections and giving me
advice when things didn’t work out quite the way I had expected. Without her support, the
mysteries of the caecum would still lie uncovered to this day. Thanks to the entire Block lab,
whose willingness to help with any problem made life as an undergraduate that much easier.
Thanks to Dr. Tom Williams for helping me to ask all the right questions about the caecum.
Thanks to Dr. Heidi Dewar, for her constant encouragement and interest in my progress, and for
her help with my paper. Special thanks to Ellen Freund, for lending me an extra set of hands
when they were needed and for being the first person who dared to read my paper. Thanks to
Daniel Lau for helping me with those pesky computers and for being my chauffeur. Thanks to
David Marcinek for allowing me to impede his progress toward his thesis by answering all my
questions all the time. Thanks also to Alex Nelson for always being the smiling face that made
me look forward to doing enzyme assays. Thanks to Simon Fletcher for leading me through
those first few weeks. Thanks to Meghan for keeping me company in the Carey lab and for
letting me listen to the same awful music over and over again. Thanks also to Jim Watanabe for
his statistical insights. Finally, enormous thanks to my housemates at 801 Terry St. who have
made this one of the best quarters of my Stanford career.
Literature Cited
Papers
Boge, G., Lopez, L., and Peres, G. 1988. An in vivo study of the role of pyloric caeca in water
absorption in rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 91A(1): 9-13.
Carey, F.G., Kanwisher, J.W., and Stevens, E.D. 1984. Bluefin tuna warm their viscera during
digestion. J. Exp. Biol. 109:1-20.
Gibbs. R.H., and Collete, B.B. 1967. Comparative anatomy and systematics of the tunas genus
Thunnus. Fish. Bull. 66(1): 81-90.
Greene, C.W. 1913. The fat-absorbing function of the alimentary tract of the king salmon.
Fish. Bull. Fish Wildl. Serv. U.S. 33: 153-175.
Fudge, D.S., and Stevens, E.D. 1996. The visceral retia mirabilia of tuna and sharks: an
annotated translation and discussion of the Eschricht and Muller 1835 paper and related
papers. Guelph Icthyol. Rev. 4: 1-92.
Tullis, A., Block, B.A., and Sidell, B.D. 1991. Activities of key metabolic enzymes in the
heater organs of the scombroid fishes. J. Exp. Biol. 161:383-403.
Books
Jobling, Malcolm. 1995. Environmental Biology of Fishes. Chapman and Hall; London: 1995.
Table 2: Average CS Activities of Caeca and Liver of 7 Yellowfin Tuna
and for Caeca of 2 Blackfin Tuna
Average Liver CS
Average Caecum
Fishf
activity/g tissue
CS activity/g tissue
(n=6)
(n=6
30.06 H2.87
1.99 10.22
2.64 +0.09
21.71 -3.54
2.56 +0.07
18.40 +0.29
2.34 +0.09
18.09 +2.24
23.98 +0.88
2.85 +0.28
2.44 +0.26
25.71 -3.28
1.69 1021
11.88 +0.03
20.36 10.45
Blackfin 1
20.17 40.11
Blackfin 2
*Values are means -SE
Table 3: Autopsy Data for Seven Yellowfin used in Caecum CS Assays
Cause of
Days in
Weight of
Weight of
Weight of
Weight of
Fish*
Death
Captivity
Stomach
Liver
Caecum
Fish
(9).
(Kg
Euthanasia
516
65
482
5.326
55.7
Euthanasia
503
65.9
113.2
123.1
8.57
Euthanasia
123
66.9
60.2
34.7
4.642
Euthanasia
36.1
120
70.7
74.8
4.757
Euthanasia
120
79.9
103.2
6.002
464
Euthanasia
75.7
72.
38.3
7.96
Euthanasia
473
145.2
12.94
164.1
Figure Legends
Fig. 1. (A) Arrow indicates the ventral stomach rete connecting the caecum and the stomach.
Venous injection site for YFTI and YFT4. (B) Arterial injection of YFT4. Left operculum is
removed to reveal the gills, and catheter is inserted into the posterior epibranchial of the gills.
(C) Arterial injection of BLK1. Catheter is inserted into the lateral cutaneous artery via a small
window in the muscle.
Fig. 2, Illustrations of the yellowfin, Thunnus albacares, viscera. (A) Organs are shown as they
relate to each other in the peritoneal cavity and major stomach vessels are drawn in purple. (B)
Illustration of the branches of the coeliac artery and their destinations in the viscera.
Fig. 3. (A) View of the yellowfin viscera as it lies in the peritoneal cavity. Muscle has been
removed ventrally to reveal the organs. Caecum is located in the lower left of the cavity. (B)
Digital photo of the yellowfin caecum depicting the arrangement of pyloric fingers within the
organ.
Fig. 4. Block diagram of the arterial (red) and venous (blue) blood supply to the visceral organs
of yellowfin, and blackfin, Thunnus atlanticus.
Fig. 5. Uninjected yellowfin caecum rete. Dark red area shows rete on dorsal surface of the
caecum.
Fig. 6. Venous blood flow within the yellowfin caecum. (A) Injection of venules (blue) between
the pyloric fingers (white) of the caecum. 4.Ox magnification. (B) Cross-section of the caecum
rete. The two white tubes in the top left corner are the pyloric tubules bringing in food from the
stomach. There is no caecal tissue within the rete, only blood vessels. 2.5x magnification. (C)
Close-up of caecum rete. Same location as 6b. I.Ox magnification.
Fig. 7. Double injection of YFT4. Red denotes arteries and blue denotes veins. (A) Cross¬
section of caecum rete. 2.5x magnification. (B) View of the two main arteries supplying the
caecum. Both are surrounded by blue venous blood. 0.63x magnification.
18
Fig. 8. Cross-sectional slides of yellowfin caecum tissue stained with Masson's Trichrome. (A
and B) Caecum rete at lOx magnification. (C) 40x close-up of epithelial extension in the lumen
of a caecum pyloric finger. (D) Caecum pyloric finger viewed at lOx magnification.
Fig. 9. Illustration of the blackfin, Thunnus atlanticus, viscera with the vasculature of the
stomach and caecum.
Fig. 10. Arterial injection (red) of the blackfin. (A) Entire viscera removed from peritoneal
cavity. (B) Close-up of caecum rete and arterial connections to the stomach. (C) Arterioles of
the caecum rete at 1.25x magnification.
Fig. 11, Cross-sectional slides of blackfin caecum tissue stained with Masson’s Trichrome. (A)
Caecum pyloric fingers viewed at lOx magnification. (B) 40x close-up of epithelial extension in
the lumen of a pyloric finger. (C) Caecum rete at lOx magnification.
Fig, 12. Illustrations of bonito viscera and caecum. (A) Layout of viscera and relationship
between the organs. (B) Caecum with extensive vasculature.
Fig. 13. (A) Citrate synthase activities for caecum and liver tissues of seven yellowfin tuna.
Caecum is in gray and liver is in yellow. A single factor ANOVA shows significant differences
between the value for each fish. (B) Fish mass plotted against caecum CS activity. A regression
plot shows no significant correlation.
Figure 1
A
(AECUN
Figure 2
A
SPEEEN
TATESTE
LL GAR
BS
Figure 3
0
Figure 4
Dogsd

JEART
LEFT
LIVE
o61
GINU2
AIDDLE
VERS
LVER
LOBE

RIOAT
LIVER
LOGE

CAS
BLADDER

—
—
(AE

—


S
—

—  -

—
MUSCLE

SPLEE
Figure 5
Figure 6
Figure 7
Figure 8
A
B
Figure 9
LVER (RT 1286)
CAECUA
ESOPH AOU S
SOMACH
MER (UT 1oeë)
-GALUBLADOEE
-ENTESTINE
GPUEEN
Figure 10
Figure 11
Hoaus
LIVEE
(RT. LOBE)
CGECUN
Figure 12
DORSALVEW
To ESoPHA6US
A



APLEEN
LIVER
(1 86
- GALL GLRDDER
— INTESTINE
Figure 13a:
45
40
30
Figure 13b:
45 -
30
25
15
Citrate Synthase Activities
For Seven Yellowfin Tuna
Ecaecum
Eliver





Individual Fish
Mass of Fish vs. Caecal CS Activity
y = 0.0016x + 12.456
R2=0.4348
1000 2000 3000 4000 5000 6000 7000 8000 9000
Mass (9)