Lipid and Glycogen Levels in the Tissues
of Two West Coast Chitons;
Mopalia muscosa and Nuttallina californica
ABSTRACT
Analysis was done on the foot, gut, digestive gland, and
gonad using the methanol-ethyl ether method for lipids
and the phenol-sulfuric acid method for glycogen.
Species differences were found only in the foot tissues.
Female Mopalia muscosa (Gould, 1846) had a lower percentage
of lipid in the foot tissue than did female Nuttallina-
californica (Reeve, 1847). Comparing both sexes, N.
muscosa was found to have a higher level of glycogen in
the foot tissuethan N. californica. In the tissues of
both species studied lipids are quantitatively the more
important storage materials. Original paper containing
full findings is in library of Hopkins Marine Station.
Robert Tenold
Hopkins Marine Station
Pacific Grove, Calif. 93950
R. Tenold
Storage Reserves In Two Chitons
Page 1
Introduction
Of the several species of Polyplacophora found in
the intertidal area of Monterey County California,
Nuttallina californica (Reeve, 1847) and Mopalia muscosa
(Gould, 1846) are two of the most common. The habitat,
behavior, and food of these two species differ greatly.
Nuttallina californica is a small chiton, usually
no longer than 3.7 cm which is usually restricted to
the middle intertidal of fairly exposed rocky shores
(Ricketts & Calvin, 1962). They are often found in small
furrows and crevices in the rocks and appear to lead a
rather sedentary life. From personal observation some
have been found not to move at all during a 48 hour period.
They have been found to feed mostly on red algae in their
immediate vicinity. These include Endocladia muricata,
Gigartina papillata, and Rhodoglossum spp. They are also
known to feed on the green alga Cladophora trichotoma
(Nishi, 1974).
Mopalia muscosa is the larger of the two species
and is in the size range of 7.6-10.2 cm. They are found
in more protected areas such as; tidepools, attached to
the underside of rocks, or on top of rocks under a covering of
Pelvetia fastigiata or Fucus furcatus (Legard & Andrus,
1974). Their activity is more pronounced than that of
R. Tenold
Storage Reserves In Two Chitons
Page 2
Nuttallina californica. They have been found to show
much activity during the hours of high tide, occurring
during the night (Smith, 1974). Boolootian (1951)
found them to feed on various red and green algae such
as; Gelidium spp., Ulva spp., and Ectocarpus spp.
The impetus for this comparative study of lipid
and carbohydrate stores was a test of the following
hypothesis; differences in habit and habitat are reflected
in the levels of tissue lipid and glycogen. Using
movement as an indication of active feeding it was hypoth-
esized that the more sedentary Nuttallina californica,
which has prolonged periods of inactivity, may have higher or
lower lipid and carbohydrate stores than those of
Mopalia muscosa, which seems to move and feed more fre-
quently.
Materials & Methods
Nuttallina californica of relatively the same size
were collected from similar habitats at Mussel Point
near the Hopkins Marine Station, in Pacific Grove and
at Point Pinos on the Monterey Peninsula. Mopalia
muscosa were collected from Mission Point in Carmel Bay.
Animals were collected during the last week of April
and the month of May, 1974.
Immediately after collection the animals were
R. Tenold
Storage Reserves In Two Chitons
Page 3
brought into the laboratory, weighed and then dissected
to obtain the foot, gut, digestive gland, and gonad.
The gut was extended in a dissecting pan and a bent
dissecting probe was run the length of it squeezing out
the contents to insure that only the gut tissue was
analyzed. The tissues were then placed in separate
crucibles and dried in an oven at 75 degrees centigrade
for 24 hours. Upon completion of drying the tissues were
kept in a desiccator until the analysis could be made.
Dried foot tissue was ground into a fine powder using
a mortar and pestle and the other tissues were broken
into fine pieces using a dissecting probe and the end
of a small test tube. Lipids were extracted using the
methanol-ethyl ether method of Freeman, Lindgren, Ng,
and Nichols (1957).
After the lipids had been extracted from the tissue
it was digested in 10 ml of hot 10% trichloroacetic acid
for 30 minutes. The supernatant was then analyzed for
carbohydrate content using the phenol-sulfuric acid
method of Dubois, Giles, Hamilton, Rebers and Smith
(1956) with reference to a glucose standard curve.
Therefore, all carbohydrate levels are expressed in terms
of glucose.
The entire organ of each animal was used for
analysis. Therefore, percent of tissue dry weight is
also percent of organ dry weight.
c
6
Storage Reserves In Two Chitons
Page 4
R. Tenold
Results & Discussion
Ten organisms from each species, five of each sex,
were analyzed. The results of lipid and carbohydrate
levels for female Mopalia muscosa are shown in Table 1,
male M. muscosa are shown in Table 2, female Nuttallina
californica are shown in Table 3, and male N. californ¬
ica are shown in Table 4. The mean level of carbohydrates
for both species, standard error for each tissue and
the range between individuals are shown in Figure 1.
Figure 2 shows the same information for lipid levels
in both species.
The findings show a great variability in lipid and
carbohydrate levels between individual organisms within
each species. It has been shown by Giese and Araki (1962)
that the chemical composition of tissues does change
depending on what stage of the reproductive cycle the
animal is in. This study was only conducted over a five
week period and individuals analyzed were of differing
gonadal developement. Dry weights of the gonad from
each individual were recorded but there was no apparent
relationship between gonadal size and tissue storage levels.
In answering the original hypothesis the only
significant difference found between the two species in
terms of lipid stores was in that of the foot tissue of
the females of both species. Using the t-test this
R. Tenold
Storage Reserves In Two Chitons
Page 5
difference was found to be significant to the 97.5%
confidence limit. Mean lipid content of the foot of
female Nuttallina california was 8.7% of the dry weight
of the tissue. This is to be compared to a value of
4.5% for the same tissue in the female Mopalia muscosa.
This finding would tend to confirm the original hypothesis
however no significant difference could be found between
any of the other tissues.
Looking at the carbohydrate levels the only
significant difference between the two species was found
in the foot tissue of both sexes. These findings were
subjected to a two-tailed t-test and were found to surpass
the 99% confidence limit. The mean value for carbohydrates
in the foot of female and male Mopalia muscosa was 7.0%
and 4.3% respectively. These levels are to be compared
to 1.6% and 1.7% for female and male Nuttallina californica.
These findings show the foot tissue of M.muscosa as a
prominent storage site for carbohydrates as compared to
the other tissues in both species. It is to be remembered
that M. muscosa is a far more active animal than N.
californica.
The findings also showed intraspecies differences
between females and males concerning lipid and carbohy¬
drate content of the gonads. Lipid content of the ovary
was much higher than that of the testis, probably reflecting
R. Tenold Storage Reserves In Two Chitons
Page 6
the large amount of storage reserves deposited in the
eggs to be used in developement. In Mopalia muscosa
the level of carbohydrates is significantly higher at
the 95% confidence limit level in the testis than it is
in the ovary. The possible reason for this difference
is not apparent. However when the tissues are corrected
for their difference in lipid content there is not a
significant difference between the carbohydrate levels
of the ovary and testis. The results of the "t-test
for significant difference between mean values is
presented in Tables 5 and 6.
As in other species of Polyplacophora studied;
Katharina tunicata, Cryptochiton stelleri (Giese, 1969)
and Mopalia hindsii (Giese & Araki, 1962) Mopalia
muscosa and Nuttallina californica seem to have most
of their storage reserves in the form of lipids, and
their carbohydrate stores are much lower. This trend
probably indicates a fast overturning carbohydrate reserve
used for immediate energy and a more stable lipid reserve
drawn on for the production of gametes and in periods
of starvation.
Summary
1. Lipid and carbohydrate levels were determined in
the foot, gut, gonad, and digestive gland of two
Page 7
R. Tenold Storage Reserves In Two Chitons
Monterey Bay chitons, Nuttallina californica
(Reeve, 1847) and Mopalia muscosa (Gould, 1846).
A difference between species in storage reserves of
2.
lipids and carbohydrates due to an observed
difference in activity and feeding frequency was
investigated.
The foot tissue was found to be different between
species in terms of lipids and carbohydrates.
Other tissues were not significantly different.
4.
Lipids are quantitatively the more important storage
materials.
The levels of these two storage substances was
5.
found to be extremely variable from organism to
organism in both species studied.
Storage Reserves In Two Chitons
Page 8
R. Tenold
Acknowledgments
I would like to thank Dr. John H. Phillips for his
timely advice concerning this project and for his
assistance in writing this paper. I also wish to thank
Dr. Robin Burnett for his help with the statistical
analysis of data. My sincere thanks also go to
Rich Williams for the use of his chiton restrainer.
I also would like to thank all of the faculty and
personnel of Hopkins Marine Station and especially
my fellow students for making Biology 175H an unforgettable
experience.
R. Tenold
Storage Reserves In Two Chitons
Page 9
Literature Cited
Boolootian, R. A.
1964 On growth, feeding and reproduction in the
chiton Mopalia muscosa of Santa Monica Bay.
Wessenschaften Meeresuntersuchen 11: 186-199
Dubois, Michel; Giles, K.A.; Hamilton, J.K.; Rebers,
P.A.; Smith, Fred
1956 Colorimetric method for determination of
sugars and related substances. Anal. Chem.
28: 350
Freeman, N.K.; Lindgren, F.T.; Ng, Y.C.; Nichols,
A.V.
1957
Serum lipid analyses by chromatography and
infrared spectrophotometry. J. Biol. Chem.
227: 449-464
Giese, Arthur Charles
1969 A new approach to the biochemical composition
of the Mollusc body. Oceanogr. Mar. Biol. Ann.
Rev. 7: 175-225
Giese, Arthur Charles & Araki, George
1962 Chemical changes with reproductive activity
of the chitons, Katharina tunicata and Mopalia
Storage Reserves In Two Chitons
R. Tenold
Literature Cited (cont.)
hindsii. J. exp. Zool. 151: 259-267
Legard, William & Andrus, Jon
1974
Personal Communication.
Nishi, Rae
1974
Personal communication.
Ricketts, Edward F. & Calvin, Jack
1962 Between Pacific Tides.
Stanford Univ.
Press; xii + 516 pp.
Smith, Suanne
1974 Personal communication.
Page 10
R. Tenold Storage Reserves In Two Chitons
Figure Captions
Figure 1
Carbohydrate levels in the digestive gland, gut,
gonad, and foot of both sexes of Nuttallina californica
and Mopalia muscosa are shown. Horizontal lines show
mean carbohydrate levels expressed in per cent of dry
tissue weight. Shaded areas show standard error for
each sample analyzed and arrows show the range of levels
found within each sample analyzed. Sample size was five
in all cases.
Figure 2
Lipid levels in the digestive gland, gut, gonad,
and foot of both sexes of Nuttallina californica and
Mopalia muscosa are shown. Horizontal lines show mean
lipid levels expressed in per cent of dry tissue
weight. Shaded areas show standard error for each
sample analyzed and arrows show the range of levels
found within each sample analyzed. Sample size was
five in all cases.
2
8 10
3 8
2
8
Digestive
Gland
Mopalia
raad
Nattallina
Gonad
Gut
muscosa
Figare 1
californica
+
+
8
Foot
8
30
20
/0
O
50
10
S 30
20
10
+
Digestive
Gland
Digestie
Gland

Gat
Gut
Figure 2
califorica
Vattallina
Gonad
Foot
Mopalia muscosa
+
8
Foot
Gonad
R. Tenold Storage Reserves In Two Chitons
Table Captions
Table 1
Lipid and carbohydrate levels, expressed in per-
cent of dry tissue weight are shown for the digestive
gland, gut, gonad, and foot of five female Mopalia
muscosa analyzed. Mean values are also shown. Wet body
weight and dried gonad weight for each animal are also
given; body weight is expressed in grams and gonad
weight is expressed in milligrams.
Table 2
Lipid and carbohydrate levels, expressed in
percent of dry tissue weight are shown for the digestve
gland, gut, gonad, and foot of five male Mopalia
muscosa analyzed. Mean values are also shown. Wet body
weight and dried gonad weight for each animal are
also given; body weight is expressed in grams and
gonad weight is expressed in milligrams.
Table 3
Lipid and carbohydrate levels, expressed in
percent of dry tissue weight are shown for the digestive
gland, gut, gonad, and foot of five female Nuttallina
california analyzed. Mean values are also shown. Wet
body weight and dried gonad weight for each animal are
R. Tenold Storage reserves In Two Chitons
Table Captions (cont.)
also given; body weight is expressed in grams and
gonad weight is expressed in milligrams. N.R. means
weights were not recorded.
Table 4
Lipid and carbohydrate levels, expressed in
percent of dry tissue weight are shown for the digest-
ive gland, gut, gonad, and foot of five male Nuttallina
californica analyzed. Mean values are also shown.
Wet body weight and dried gonad weight for each animal
are also given; body weight is expressed in grams and
gonad weight is expressed in milligrams.
Table 5
Data from comparison of lipid content of tissues
using the t-test is shown. The table shows tissues
from the two types of chitons being compared, also
the sample size n, mean value,X, of each tissue;
standard deviation, S.D., of each tissue; the t value
arrived at and the probability, p, of the two tissues
being significantly different.
Table 6
Data from comparison of carbohydrate content of
tissues using the t-test is shown. The table shows
R. Tenold
Storage Reserves In Two Chitons
tissues from the two types of chitons being compared;
also the sample size, n; mean value, X, of each tissue;
value
standard deviation, S.D., of each tissue; the t
arrived at and the probability, p, of the two tissues
being significantly different.
Table 1
Chiton
17.30 18.92 18.98 24.09 32.27 grams
Weight
Gonad
210.3 242.0 152.6 198.8 223.6 milligrams
Weight
Lipid Content
Mean
Digestive
50.28 9.0 9.2% 7.4% 5.0%
16.2%
Gland
15.8
Gut
24.0 13.3 12.4 8.6 20.6
Gonad
20.8 19.9 20.7 20.6 15.6
19.5
Foot
6.2 4.0 5.2 4.4
4.5
2.7
Carbohydrate Content
Digestive
2.6 %
4.8% 1.1% 1.0%
3.5% 2.8%
Gland
Gut
4.0
4.2 1.6
2.7
1.8
2.9
Gonad
2.7
3.4 2.3 1.2 2.8
2.5
Foot
8.4
9.6 8.8 4.7 3.4
7.0
Table 2
Chiton
26.16 22.92 36.40 31.16 24.13 grams
Weight
Gonad
271.5 245.9 252.1 277.8 247.8 milligrams
Weight
Lipid Content
Mean
Digestive
7.0% 8.3% 9.8% 11.9% 8.1%
9.0%
Gland
Gut
13.0 12.0 20.2 13.7 14.6 14.7
Gonad
7.8 8.8
9.6 9.1 9.7
9.0
Foot
4.1 5.2 4.6 9.8 4.5
5.6
Carbohydrate Content
Digestive
3.7% 3.2% 4.3% 2.1%
3.2%
2.5%
Gland
4.0
4.0
Gut
3.5 3.0 2.3
3.4
4.8
4.4
Gonad
4.4 1.8
4.1
5.3
Foot
4.4 3.6
4.3
6.0 2.2
5.2
0
Table 3
Chiton
N.R. N.R. 8.15 4.72 6.32 grams
Weight
Gonad
N.R. N.R. 41.04 65.75 25.16 milligrams
Weight
Lipid Content
Mean
Digestive
12.8% 3.8% 6.6% 12.5% 31.49
13.4%
Gland
Gut
18.8 16.2 13.1 16.7 32.3
19.4
Gonad
15.0 21.8 15.3 22.4 23.0
19.5
Foot
7.4 12.6 6.4 5.6 11.6
8.7
Carbohydrate Content
Digestive
2.3% 3.1% 3.1% 2.1%
2.6 %
2.5%
Gland
4.5 3.2
Gut
2.4
2.6
2.5
3.0
Gonad
3.9
2.0 2.8 2.0
2.6
2.6
Foot
1.4
1.8
1.6
1.9 1.7
1.4
Table 4
Chiton
4.94 4.45 6.95 9.40 4.16 grams
Weight
Gonad
22.02 28.89 3.43 119.16 34.33 milligrams
Weight
Mean
Lipid Content
Digestive
7.3%
6.6% 4.4% 7.9% 7.6% 10.1%
Gland
13.4
Gut
7.2 7.5 13.3 21.8 17.2
11.9 15.3 9.3 8.7
9.8
11.0
Gonad
Foot
8.3
5.1 11.8 9.9 9.1
5.7
Carbohydrate Content
Digestive
2.1%
1.4%
2.6 %
1.3% 3.7%
1.6%
Gland
4.9
Gut
3.8
2.7 5.3
2.9
3.9
Gonad
3.2
3.1 3.3 2.4 3.8
3.2
Foot
1.6
1.7
2.0 1.6 1.6 1.8
Table 5
Chitons
issue
Compared
A. Digestive
Gland
A. Female
B. Digestive
Gland
Nuttallina
californica
A. Gut
B. Gut
Female
A. Gonad
Mopalia
muscosa
B. Gonad
A. Foot
B. Foot
A. Digestive
Gland
B. Digestive
A. Male
Gland
Nuttallina
californice
A. Gut
B. Gut
B. Male
Mopalia
A. Gonad
muscosa
B. Gonad
A. Foot
B. Foot
A. Digestive
Gland
B. Digestive
A. Female
Gland
Nuttallina
A. Gut
californica
B. Gut
B. Male
Nuttallina
Gonad
californica B. Gonad
A. Foot
B. Foot
X S.D.
P
ts
5 13.4 10.76
.279 —-
16.2 19.10
7.44
19.4
32 -830 —
15.8
3.99
19.5
2.22 010 --
19.5
8.
3.17
2.76 97.5
4.5
1.72
7.3
2.07
1.36 ---
9.0
1.89
13.4
6.29
.411 —-
14.7
3.22
2.69
11.0
1.60 ---
0.76
9.0
2.85
8.3
2.34 1.62 —-
5.6
5 13.4 10.76
1.24 ---
2.07
7.3
19.4
7.44
6.29 1.37 --
5 13.4
3.90
19.5
11.0 2.69 3.95 99.5
3.17
8.7
8.3 2.85 0.21 --
Chitons
Compared
A. Female
M. muscosa
B. Male
M. muscosa
Table 5 (cont.)
Tissue
S.D.
t P
A. Digestive
16.2
19.10
Gland
O.83 --
B. Digestive
9.0
1.89
Gland
A. Gut
15.8
6.3
O.34 --
B. Gut
14.7
3.22
19.5
A. Gonad
2.22 10.11 99.9
B. Gonad
9.0 0.76
4.5
A. Foot
1.72 0.95 ---
5.6 2.34
B. Foot
Table 6
Chitons
issue
Compared
A. Digestive
Gland
B. Digestive
A. Female
Gland
Nuttallina
california
Gut
Gut
Female
B.
Mopalia
Gonad
muscosa
Gonad
B.
A. Foot
B. Foot
Digestive
Gland
Digestive
A. Male
Gland
californica
A. Gut
B. Gut
B. Male
A. Gonad
muscosa
B. Gonad
A. Foot
B. Foot
A. Digestive
Gland
B. Digestive
A. Female
Gland
californica A. Gut
B. Gut
B. Male
A. Gonad
californica B. Gonad
A. Foot
B. Foot
2.6
2.6
3.0
2.9
2.7
2.5
1.6
7.0
2.1
3.2
3.9
3.4
3.2
4.1
1.7
4.3
2.6
2.1
3.0
3.9
2.7
3.2
1.6
1.7
S.D.
S
.460
.026 —
1.61
.873
1.20 :240 —
.779
.357
———
.816
O.23
4.33 99.5
2.74
1.02
———
1.72
O.89
1.16
———
1.73
0.72
O.50
1.36 1.61 --
0.18 3.87 99.5
1.46
O.46
1.00 --
1.02
O.87
———
1.16 1.35
0.78
1.40 --
O.50
0.23
0.62 --
O.18
Chitons
Compared
A. Female
muscosa
B. Male
muscosa
Table 6 (cont.)
Tissue
A. Digestive g
Gland
B. Digestive
Gland
A. Gut
B. Gut
A. Gonad
B. Gonad
A. Foot
B. Foot
S.D.
2.6
1.61
0.89
3.2
2.9
1.20
3.4
0.72
2.5
0.82
4.1
1.36
2.74
7.0
4.3 1.46
t P
O.63 ---
O.61 --
2.34 95.0
1.94 ---
+
Tened
1
++
—
Tenolt
0
Figare
dattalling caldernca
*
4
++
3 0
6
Cond Pak
Oigestrie 6ue
Gland
Mopala masosa
/0
3


21
P
a-
Digestwe
Gut Gonad Foot
Gland
—.
10 -
Terdt
.
30
15
/0
5
50
45
10
§ 35
30
25
20
0
are 2.
6
Diaestwe
laad
9 8
Ogestwe at
Gland
Nattallina caltorce
0
23
9 d
Gut Gonad Foot
Popala musese
P S
P
foet
Gonad
Terde