DIGESTIVE MORPHOLOGY AND LIPID CONTENT IN THE INTERTIDAL
ISOPOD
HARFORDI
175H Hopki
Stanfor
Introduction
The intertidal isopod Ci
rolana harfordi (Lockington,1877)
is knowi to feed heavily at discrete intervals and to appar-
ently "fast" in the interim period of up to one month. (John¬
son,1973). Cirolana has been observed by the author to prey
upon small annelids, although it is perhaps best described
as an opportunistic carnivore.
Some preliminary dissections revealed the presence of
large amounts of fat in Cirolana, particularly in the form
of droplets or globules in the digestive diverticula of the
midgut region. This accumulation of lipids, in an area of
endodermal origin, suggested a mechanism whereby the animal
could deplete special metabolic reserves to survive long
periods of time with no feeding. To explore this, I decided
to examine the morphology and contents of the digestive tract
in Cirolana during a period of food deprivation, and to coup¬
le this with a study of the animal's overall levels of li¬
pid content during the same period.
Sequential changes in the amounts of food and fåv in the
gut, as well as a series of extracted lipids were measured,
with the hope of elucidating some of the processes which oc¬
fordi.
cur between feedings in C.
There are several alternative survival strategies which
an animal might employ over periods of food deprivation. It
is possible for food to remain in an undigested state in some
area of the gut and to be recalled at intervals to a site
where digestion and absorption occur.
The carnivorous isopod Eurydice may store large quan¬
tities of food in its hindgut, where processes of digestion
may be begun, and digest and absorb food in the midgut gland.
(Jones,1968)
It is also possible that the animal survives a period
without feeding by using a dynamic supply of metabolic re¬
serves. Several forms of metabolic reserves might be pos¬
sible storage substrates in such a case.
Steeves((1963) monitored the changes in glycogen con¬
tent during starvation in the isopod Lirceus brachyurus, and
found that it was being used as a metabolic storage product.
The crustacean hepatopancreas (midgut gland) is a site
of both lipid biosynthesis and storage; O'Connor and Gil¬
bert (1968, 1968) have correlated fluctuations in this lip¬
id content with cycles of ecdysis.
Vonk (1960) and others have shown that the dissappear-
ance of proteins from the hepatopancreas during molting is
coupled with tissue growth, and that proteins accumulate
in the hepatopancreas between molts.
These alternative stategies are not mutually exclusive
and any of them might be coupled with a long process of slow
digestion and low energy requirements. I have observed that
in the laboratorycCirolana are markedly less active after
feeding. I felt that any overall analysis of lipid con¬
tents should be paralleled by a morphological study that
would attempt over time to trace the fate of ingested food
within the digestive system.
lual focus allows the experimenter to
Such
measured changes in body lipids with observed, serie
in the states of the gut and its contents.
Materials and Methods
A. Collection: Cirolana harfordi were obtained from three
sources: Macrocystis holdfasts, under rocks in the intertidal,
and from baited traps. The collection area was the Great Tide
Pool in Point Pinos, Pacific Grove, California.
Animals frommthe holdfasts were chosen as the original
population. However, the age structure and sex ratio of this
population limited its usefulness. Brooding females do not
generally eat.(Johnson,1973) and were not desired for the
experiment. I have, however observed them to eat in the lab¬
oratory. The holdfast population had too great a concentration
of brooding females and specimens under five millimeters in
length to provide enough large males for the experiment. Ani¬
mals overrseven point five millimeteres long were selected to
facillitate dissections. Traps set were baited with squid
according to the method of Johnson (1973) and were success¬
ful in attracting about three-fourths large, and hungry males.
This was important because eighty males from the hold¬
fast refused food even after twelve days in a holding tank
with no access to food, except through possible cannabilism.
Many of these males had bright red pieces of annelid persist-
ing in their guts during this twelve day period.
B Methods: Since the red color produced a rough assay for
food in the hindgut, it was decided to feed the animals dyed
food. Squid was soaked for twelve hours and macerated slight-
ly in the vital dye Methylene blue. Obviously Ovigerous fe¬
males were discarded and the remainder of the animals from
all three sources were allowed to feed in the dark for twen¬
ty-four hours. Cirolana is a nocturnally active species.
The fed organisms were subsequently placed in individual
sequestration chambers which allowed the passage of aerated
sea water but prevented the Cirolana from swimming freely.
The chambers were screened to prevent them from eating each
other's molts and to discourage any possible cannabilism.
Molts were removed as they occured. Live births and their
mothers were also removed, as were dead animals.
The sampling regime carried out over the twenty-three
day period of food deprivation included the removal of ani¬
mals for dissections and for composition analysis. At a stage
where the dissections showed great asynchrony more than the
minimal number of five organisms were examined.
+ DISSECTED
4 ANALYSED
ME
14
control
10
immed.
post feed
4 hour
10
12 hour
16 hour
11
24 hour
36 hour
11
48 hour
72 hour
1 week
11
10 day
13
2 week
23 day
Cirolana to be used in the lipid extraction were quick
frozen in plastic bags immersed in a dry ice and ethanol bath.
After one to two minutes the frozen animals were transferred
to a zero?C freezer, and maintained there no more than two weeks
untilanalysis. They were then defrosted at room temperature
and dried to a constant weight in a 50°0 oven.
Dissections were performed by two methods. If the gut
diverticula were to be squashed and examined microscopic¬
ally to determine the presence and condition of food and fat,
the technique was as follows. The animal was placed ventral
side down and its head was slit in a downward motion by slicing
from the fusion of the cephalothorax to just below the eyes.
This piece of exoskeleton wasrremoved, and a small horizontal
cut at the midline was made through the second thoracic seg¬
ment to loosen the layer of connective tissue. The entire
gut was then drawn out with a pair of fine forceps. This
method is ideal for removing whole guts because it allows
the maintainence of relative proportions and positions of
different areas of the gut, and avoids bringing away the lay-
ers of invaginated exoskeleton surrounding the hind and fore-
guts. Squashes were made with distilled water, but when a
whole section of a gut, as, for example, one digestive
lobe ,was to be examined whole it was bathed in fluid from
the isopod's own gut.
The remaining two specimens in each sample were dissect-
ed by placing the animal on its ventral side, cutting through
the midline of the uropod and slitting the dorsal surface to
one side of the midline with a scalpel inserted through the
cut in the uropod and moved anteriorly through the cut.
The two halves of the exoskeleton were then removed, along
with the underlying layers of connective tissue, exposing
the digestive tract.
The procedure for extracting lipids was suggested to
me by Dr. Phillips (1973). It consisted of soaking intact,
irofana twenty-four hours in 10milliliters acetone
dried C
per animal in sample, grinding samples in fresh acetone in
a tissue grinder, centrifuging in fresh acetone, and resus¬
pending and centrifuging in fresh acetone. Each change used
10 milliliters acetone per animal. The pooled acetone for
each sample was evaporated to dryness over a steambath and
washed four times with 5 milliliters chloroform per animal
to extract the residues. The chloroform was evaporated
over a steambath to a volume of approximately 20 milliliters.
This was poured off into preweighed test tubes and evap
ated to dryness.
Results
Figure 1 presents the digestive tract of Cirolana har-
fordi (legend explained in figure captions). The gut ex¬
tends from the esophagus to the anus located on the second
pleopod of the uropod. Except for the short midgut with its
six long diverticula, the entire digestive tract is of ecto¬
dermal origin and is lined with a chitinized invagination
of the exoskeleton (Jones, 1968). The lumen of the sac-like
diverticula empty into the foregut and contain only liquid
or fine particulate matter.(Jones, 1968). The diverticula
are visibly muscularized. The hindgut is quite extensible
but always remains narrowed at its most posterior end. Along
the length of one hindgut there may be considerable variation
in diameter, with the widest area 4-6 mm. anterior to the
anus. The jointure of the midgut and the hindgut is also
sibly narrowed, at least in a distended gut. In an empt
gut the diameter is more constant, because of the collapsed
appearance of the hindgut, the lumen of which is folded in
on itself. The third, posterior pair of caceum are the shortest.
Feeding visibly distends the hindgut;
A. Feeding Behavior:
the animals appear bloated when viewed dorsally. When filled
with dyed material the digestive tract shows blue; however.
there was no clear outline of the diverticula. Ten Cirolana
placed in an aqueous solution of methylene blue did not stain
in the gut region. Observed feeding in five large males
(over 13mm.) involved boring into the meat with the mouth¬
parts until the entire cephalon was up to 4mm. deep and the
telson protruded vertically. The animals gorged for from
2-5 minutes. Cirolana with guts that did not appear trans-
parent were not observed to feed; it is assumed that these
isopods do not feed if their hindgut is fairly full.

The results presented here
B. Dissections and Squashes:
represent the averages of the two dissections and three to
six whole gut removals for each time interval. The most
prominent morphological landmarks for the stages of gut ap-
pearance are generalized. Thus the changes observed over a
period of time do not represent strict and discrete changes,
but rather a continuum ofechanges in the means of whole groups
of animals. The drawings in Fig.2 represent this series of
changes for two parameters: the diameters of the hindgut and
the positions of the dyed food within the hindgut (legend
explained in figure captions).
1. Control dissections; these animals were not necessarily
starved, but six of them had empty hindguts. The hindgut
tissue was translucent and colorless, there was no fluid in
the lumen. Three specimens contained recognizable pieces of
annelid worms or setae in the hindgut, which was filled with
clear fluid. The diverticula of all specimens, including a
brooding female, contained greenish brown fat droplets that
appeared to spiral around the lumen.
2. Immediately post feed to 48 hours: there are marked sim¬
ilarities in many of the characteristics ofthese time stages.
The hindgut is filled with a clear fluid, especially after
36 hours. By 48 hours the amount of fluid has increased so
that the food mass no longer appears to occupy more than 80%
of the body cavity. By 48 hours the food mass no longer con¬
forms in outline to the outline of the gut. Although it is
still regurgitated as one mass, it has narrowed considerably
in some areas. The diverticula contain fat droplets at all
these stages. Changes in gut diameter and in the position
of the dyed food in the hindgut are recorded in figure 2.
3. Between 48 and 72 hours; the amount of clear fluid in
the hindgut steadily increases and the foregut contains a
blue liquid. The food mass in the hindgut has continued to
narrow in places and may have an hourglass appearance. Re¬
gurgitation was induced in three individuals;the connections
between some areas of the food mass were quite tenuous. Two
of the Cirolana regurgitated pieces of squid with intact
chromatores. As in the earlier dissections, the food in the
hindgut was still recognizable as squid. See figure2.
Allthe diverticula contain fat droplets around the lumen.
4. Between 72 hours and 1 week; the gut is less swollen,
anterior and posterior regions of the hindgut are easily dis¬
tinguishable. There is proportionately more of the clear
fluid in the hindgut, which is smaller. The foregut still
contains blue liquid. The food mass in the gut is now in
discrete pieces, and the posterior portion of the gut has
regained its transparent, prefeed appearance. The digestive
tract is still stained a deep blue. The diverticula all
contain fat droplets. See figure 2.
5. Between week 1 and week 2; Viewed morphologically, the
rate of change for several parameters has begun to accelerate.
The gut is narrower than in 4 (above) and external swelling
is less visible. The posterior portion of the hindgut is
transparent, and the food masses at the anterior end of the
hindgut are smaller than at the previous stages. The lumen
of the posterior portion of the hindgut contains very little
fluid; the whole hindgut region only occupies about 50% of
the body cavity. The amount of blue liquid in the foregut
is still increasing. Diverticula still contained fat drop¬
lets, and the lumen were seen to contain a blue liquid in
the blind end. For two days at some stage between week 1
and week 2 animals began to acquire a light blue tinge that
was not confined to the gut region, after this tinge dissap¬
peared the dye in the gut remained light. See figure 2.
6. Between week 2 and 23 days; the rate of change is not as
5% of
rapid as in 5 (above) but the processes continue. In
the animals examined the hindguts were emptied of food and
fluid. The remaining 66% of the C. harfordi examined had
small food masses at the anterior extremes of the hindgut,
and within the short midgut region. The foregut still con-
tained blue fluid in these 66% of the sample, and small a¬
mounts of blue color were visible in the mid and foregut re¬
gions. All the diverticula contained fat droplets. See figure 2.
7. The diverticula: As noted above Cirolana has three pairs
of sac-like diverticula, the third pair of which I have ob¬
served to be one half to two thirds the length of the first
two pairs. Various observations were made on the changes
in position relative to the hindgut, the other diverticula
and the foregut of the animal. This variance did not appear
to be a function of time; it would seem that the diverticula
often move actively. These, changes are schematically shown
in figure 3. (legend explained in figure captions).
Squashes made of diverticula during the course of the
experiment revealed patches of blue cells scattered in one
70
or all of the diverticula of approximately 5% of the speci¬
mens examined. Evidently, some of the squid, at least,
made its way into the lumen of the hepatopancreatic lobes.
C Lipid content: The percentage values, (from obtained val¬
ues of mg. lipid per mg. dry body weight) are expressed in
figure 4. The values never returned to the value obtained
for unfed animals. After a twenty-three day period of food
deprivation, fed animals were still not starved.
Discussion
During a period of food deprivation in previously gorged
Cirolana harfordi, a slow process of digestion occurs in which
food held in the hindgut is eventually digested. Within the
hindgut large pieces of food are gradually broken into smaller
food masses.in a fluid medium. Movements observed in the di¬
gestive diverticula may represent a means by which fluid pro¬
duced in their lumen is passed into the hindgut. It is prob¬
able that this fluid is related to the observed breakdown of
large masses of food in the hindgut; no fluid was observed
in the hindgut lumen when the hindgut was empty of food par¬
ticles. As digestion progresses the hindgut empties and its
diameter gradually decreases. The most posterior portions
of the hindgut are seen to empty first, and the tissue of
the emptied areas is clear with no blue color.
The diverticula always contain fat droplets. Some food
particles were found in the anterior hindguts of animals de¬
prived of food for twenty-three days. Overall lipid composition
never returned to unfed levels. This shows that Cirolana de¬
prived of food for twenty-three days are not in a state of
tarvation.
A temporal co-incidence of food decrease in the hindgut
with an increase in overall lipid content may be an indication
that lipids are stored and/or synthesized either over their
rate of use as a metabolic substrate or that other products
of digestion are preferentially utilised on a short term basis.
The regurgitation observed shows the existance of a mechan¬
ism by which partially digested food could be moved anteri¬
orly to the hepatopancreatic gland, a site of digestion
and absorption. The presence of small patches of blue cells
in an irregular arrangement within diverticulum, and the ob¬
served small pieces of food within the short midgut tend to
support this scheme.
A period of slow digestion would seem to be an adaptative
advantage for Cirolana in that a decreased feeding frequency
is paralleled by a decreased need for exposure to predators.
Summary
During a 23 day period after feeding with dyed food,
gut contents and morphology of C. harfordi were monitored.
Food in the hindgut,in changing volumes of fluid, was broken
down into small pieces and eventually digested. The midgut
gland showed a continualspresence of fat droplets and fluids
and contained patches of the dyed food cells. The dye used
did not stain the tissue of the hindgut which became clear as
the hindgut emptied. The gut diameter increased after feed¬
ing and gradually narrowed. Changes in the relative positions
of the diverticula were noted.
Overall lipid contents increased sharply with feeding and
remained at higher levels than those in non-fed animals through¬
out the period, indicating that starvation was not occuring.
0
Literature Cited
Herreld, C. (1973) Hopkins Marine Station of Stanford Univ.
Johnson, W. (1973) PhD. Seminar, Hopkins Marine Station of
Stanford niversity
Jones, (1968) Journal Zoology 156,363
O'Connor, J. andGGilbert,L. (1968) American Zoologist 8,529
(1969) Comp. Biochem. Ph
siol.
26,311
Steeves, H. (1963) J. Exptl. Zool 154,21
Vonk, H. (1960) The Physiology of Crustaceans (Waterman, ed.)
Acknowledgments
I would like to thank Nat Howe, Dr. Lee, and Dr. Phillij
for their patient advice.
e
Figure Captions
Figure 1: The Digestive Tract of Cirolana harfordi
A= foregut
B = midgut
cdiverticula (one pair)
D= hindgut
icula are cut away to expose one joined pair.
Four diver
Thetop of the page corresponds to anterior, the bottom
to posterior.
Figure 2: Changes in Hindgut Diameters and Food Contents
in Fed, then Food Deprived,Cirolana har
fordi
at selected times.
Spackled areas represent food distribution within the hindgut.
Four Arrangements of the Midgut D,verticula in
Figure 3:
rolana harfordi
A- waving motion, away from hindgut, separating
air of diverticula
B- each pair overlapped and wrapped around
the anterior portion of the midgut.
C- all six diverticula elongnate, exposed and
hanging near hindgut.
D- each pair overlapped and wrapped around the
foregut.
Figure 4: % Lipid Composition in Fed, then Food Deprived
rolana harfordi
numbers in parentheses represent numbærs in
samplesused.
unfed control group
- fed animals
FIG 1


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