Cotylodons in isopods. pa
Introduction
The marsupium of the brooding females of Porcellio
scaber Latreille contains four soft-walled processes
which emerge from the midventral body wall on thoracic
segmonts 3 to 6 inclusive. The processes have been
called cotyledons, and their morphology and position
have been well documented by Akahira (1956). The
existance of the structures was noted by earlier workers
(eg. Schöbl, 1880; Verhoeff, 1920; Heeley, 1911),
and some have speculated on their possible function.
The present studies were directed toward extending
present knowledge of the distribution of these struc-
tures in the Oniscoidea, and gaining further insight
on their function.
All isopods studied were collected in Monterey
County, California. P. scaber americanus Arcangeli
were collected from under and within a rotting log in
a shady area on Mussel Point, Pacific Grove and from
moist leaf mulch in upper Carmel Valley. Identifi-
cation to subspecies was checked by Dr. M. A. Miller,
University of California, Davis. The animals were
maintained in the laboratory in covered plastic buckets
containing moist Monterey pine leaf litter and wood
chunks at temperatures of 20-25°0 and a relative hum-
idity of 65-75%. Porcellio dilatatus Brandt and
Cotylodons in isopods. page 3
Ratzonburg and Armadillidium vulgare (Latreille) were
collected in upper Carmel Valley from under moist leaf
litter. Ligia occidentalis Dana was collected from
a protected beach on Mussel Point, Pacific Grove.
All three species were examined on the day of collec¬
tion. Philoscia richardsonae Holmes and Gay, Arma-
dilloniscus lindahle (Richardson), Alloniscus percon-
vexus Dana, and Alloniscus cornutus Budd and Lund
were collected by James Moore from the west beach on
Mussel Point, Pacific Grove. These four species were
preserved in 70% alcohol prior to examination; all
other animals were studied in a living state.
Animals in which cotyledons were to be examined
were placed ventral side up in a plasticine clay trough
moulded to match the width of the animal. The animal
was secured by placing insect pins across the body
anterior and posterior to the marsupium and embedding
both pin ends in the sides of the trough. The cotyl-
edons were exposed by first removing the legs and
oostogites with forceps, then either washing the em-
bryos out with fresh water or picking them out with
forceps. Measurement of cotyledons were taken using
an ocular micrometer on a dissecting microscope.
All drawings except figure 5 were made with a camera
lucida.
Cotyledons in isopods. page 1
Occurence and appearance of cotylodons
Specimens of Porcellio scabor americanus repre-
sonting all stages of the life history were examined.
Cotyledons were found to occur only in brooding females.
No traces aro present in males or in any non-brooding
females except those from which the brood has just
departed. Four cotyledons are present (figure 1).
They arise from the centers of ridges which run trans-
versly across the centers of thoracic segments three
through six. They are hollow, finger-like evaginations
of the body wall which are soft and pliable. Inter-
nally they are filled with loose connective tissue
which allows fluid circulation; their surfaces bear
no observable pore openings (Akahira, 1956).
Cotyledons were examined in brooding females in
eight species of Oniscoidea (figures 1 through 5).
Porcellio scaber americanus, P. dilatatus, and Arma-
dilloniscus lindahli all have similar cotyledons;
four are present, one per segment. Cotyledons on P.
dilatatus seem larger than those on P. scaber whereas
those on A. lindahli seem shorter. Philoscia richard-
sonae has nine cotyledons, one on the third thoracic
segment, two on the sixth thoracic segment, and three
each for thoracic segments four and five. The cotyl-
edons appear smaller in diameter yet somewhat longer
Cotylodons in isopods. pago 5
than those of P. scaber. Alloniscus porconvexus and
A. cornutus both have 12 cotyledons regularly arranged
with three por segment. They are more similar to the
cotylodons of Philoscia than to those of P. scaber.
Armadillidium vulgare has ten structures, two per
segment for the end segments of the marsupium, and
three per segment for the two middle segments. These
structures are extremely different from the cotyledons
of the previously mentioned species in that they are
balloon-like rather than finger-like projections.
Their surface occupies a large part of the surface
of the roof of the brood pouch, yot they seem to arise
from the same points of attachment as those of the
preceeding species. Ligia occidentalis appears to
lack cotyledons completely.
Cotyledon size in relation to body size and stage of
developement of the brood in Porcellio scaber
Sixty brooding females of P. scaber americanus
ranging in length from 7 mm to 11.5 mm were examined.
Length of each cotyledon was measured to the nearest
O.5 mm. The results (figure 6) show there is a direct
relationship between body length and cotyledon length.
Variability between individuals was such that adjacent points
on the same curves are not significantly different
at the .05 level, but most points one or more places
Cotylodons in isopods. page 6
romoved are significantly difforont at the.05 level.
Verhooff (1920) romoved all the embryos from the
brood pouch of female Porcellio scabor and maintained the fe-
males in the laboratory. He recorded no change in
with time
cotyledon length,and so concluded that the cotylodons
do not change size during brooding as was suggested
by Schöbl (1880). Verhoeff also presented an outline
for recognizing stages in developement for the young
in the marsupium. All embryos still within the embry-
onic membrane are placed in one class, and the period
between emergance from the extraembryonic membrane
and release from the brood pouch is divided into four
larval stages. Also the characteristics defining
his stages are a set of continuous variables, eg.
relative length of the first segment of the antennae.
In the present study I found a larger number of females
with young still within the embryonic membrane than
with hatched young in the larval stages. I have there-
fore made use of the scale shown in figure 7. The
stages used here are more exactly defined than Verhoeff's
since I used the first appearence of a characteristic
to mark the beginings of a new stage.
Information on the cotyledon size in relation to
brood stage, for the 60 brooding females of P. scaber
americanus mentioned earlier, is presented in figures
Cotylodons in isopods. page 7
6 and 9. Cotyledons 1 and  in females 10 to 11.5
m long show no significant differences from one an¬
other, and no significant changes in size during brood
developement. In contrast cotyledons 2 and 3, while
they do not differ significantly from one another,
show a clear increase in size during developement
(significant at the.01 level).
Observation on 18 individuals showed that the
cotyledons shrink to a length of 0.2 to O.1 mm upon
release of the brood. They become nipple-like, and
appear somewhat retracted into the basal ridge though
they cannot be extracted when pulled with forceps and
do not extend when distilled water is applied. They
persist in this shrunken condition until the next
molt.
Properties and possible function of the cotyledons
in P. scaber
Schobl (1880) proposed that the cotyledons served
a nutritive function. Verhoeff (1920) disagreed and
suggested a respiratory function instead. He further
suggested an antibacterial action of the brood fluid.
Personal observation leads me to suggest the cotyledons
may play a part in anchoring the young, preventing
them from being lost from the brood pouch. They may
also function in maintaining the proper amount of
Cotylodons in isopods. page
fluid in the brood pouch.
There are several properties of the cotyledons
and tho rolated brood fluid which are pertainent to a
discussion of possible function. Permeability of the
cotylodons to water and small molecules was tested
with solutions of Nacl ranging from 0% to 10%, at
2.5% intervals. Several drops of each solution were
placed in the brood pouches of females from which oosteg-
ites and young had been removed such that the fluid
just covered the cotyledons. The cotyledons show an
increase in turgidity when exposed to tap water (0%).
There was a noticable reduction in turgidity when the
saline solutions were applied. There is not as great
a difference between any of the saline solutions as
there was between tap water and the 2.5% saline.
The increase in turgidity with tap water and the loss
of turgidity with exposure to the saline solutions
demonstrates that the ventral side of the
female is permeable to water. The thin walled
cotyledons are the most likely permeable structures.
In another experiment an aqueous solution of
Fast Green dye was injected into the body cavity of
an otherwise undisturbed female. A very small amount
of the dye was introduced using a glass micro-syringe
at the base of the last peraeoped, and care was taken
to prevent any leakage. After 20 minutes the female
Cotylodons in isopods. page
was oxaminod in the normal way and green colored mucus
was found in the brood puuch and around the cotyledons.
This tost demonstrated the outward permeability of
moloculos other than water.
Drops of marsupial fluid removed from tho brood
sacks of females with young in all stages were examined
by placing the dropson glass slides. They showed a
cloudy appearance and upon evaporation left white spots
which contained some crystals but represented predom-
inately a white film. Young taken from the brood
pouch show a great deal of adhesion to each other
and to other things. These observations suggest the
fluid is somewhat more complex than a simple salt
solution. Verhoeff (1920) and others have mentioned
'albuminous' materials in the marsupial fluid.
Nourishment
Verhoeff (1920) grew brooding females on a sand
substratum without feeding them anything during the
brood period. Several females were able to produce
normal broods. From this evidance he claimed to have
refuted Schobl's (1880) suggestion that the cotyledons
function in nutrition of the young. Yim (1973) has
shown an increase in both organic and inorganic content
of the brood in P. scaber americanus during develope-
ment. That the young increase in organic and inorganic
Cotylodons in
isopods. pago 10
content suggosts that thore must bo a source of mater¬
ials oxtornal to tho embryo. Possibly death and decom¬
position of somo of tho young originally presont in a
givon brood could both reduce crowding and provide
a source of nutrition already within the brood pouch.
However, I have seen very few decomposing young in
examinations of the brood sacks of more than 60 Por-
cellio females. Two other possible sources of nutri-
ith
ents are,the external environment or materials diffusing
through the roof of the brood pouch. Active uptake
of nutrition from the environment in a way other than
through the mother seems doubtful especially in terres-
trial species. Diffusion across the cotyledon mem-
brane can occur as is shown by the dye and the water
experiments. Also a mucdus-like substance is found
within the brood pouch and along the cotyledons which
may be of some use in nourishment. It seems likely
that whereas the female may not actively nurse the
young, she may suppliment their yolk supply. This
would yield more room in the brood sack to allow for
a larger number of eggs at the begining of developement.
Anchoring larvae in place
The states of the cotyledons in brooding females
held in the laboratory under more or less constant
conditions varies a great deal; cotyledons ranged
Cotylodons in isopods. page 11
from turgid and oroct to flacid, lying flat against
tho roof of tho brood pouch. This variability in the
sizo and shape of tho structuros makos it doubtful
that thoy are of major importance in directly retaining
the young. The author has never soen any regular ori-
entation of the young around the cotyledons or regular
orientation of the cotyledons within the brood sack.
However any mucus secreted into the brood sack, for
what ever purpose, may be an indirect aid in retaining
the young due to its natural viscosity and adhesive-
noss.
Water balance
Ten animals were maintained in small jars for
two to three days, with their normal laboratory sub¬
stratum but also with standing water in the jars making
a 100% relative humidity environment. These animals
showed normal variability in cotyledon turgidity when
compared with the population in the normal laboratory
environment. Desiccated animals exposed to between
35 and 15 % relative himidity show a shriveling of
the cotyledons without a noticable shortening. The
cotyledons shrink and presumably lose their function
after release of the young. The shrinking corresponds
to the release of the young, and the end of the re¬
quirements for the marsupial fluid. The high degree
Cotyledons in isopods. pago 12
of pormeability to water of the cotyledons indicates
those may play a part in water balance in the brood
pouch.
Bacteriostatic activity of marsupial fluid
A fow experiments were run to test Verhooff's
(1920) hypothesis that the marsupial fluid may have
an inhibitory effect on bacterial or fungal growth.
100 ml of a nutriant medium was made up of the following
ingredients: 0.5 % Difco yeast extract, .05 % Mgso,
7H,0, .02 % Na,HPo,, .01% kcl, .006 % Cacl, (added
in solution), .003% Fecl,-6H,O (also added in solution),
and 2 Difco agar in tap water brought to a pH of 6.9
with 10 ml 2M Tris HCl and.5 ml Tris base yielding
a final 100 ml solution. An incubating broth was also
made using the above formula without the agar. Sub-
rate from the Mussel Point habitat of P. scaber
was finely ground, then diluted with tap water to one
tenth concentration. Iml of this solution was added
to 5 ml to the agar medium and plated on a petri dish.
This was allowed to incubate at 35°0 for 24 hours.
Colonies from this plate were used to innoculate five
agar-less nutriant baths which were also allowed to
incubate for 24 hours at 35°0 until there was a percep-
table cloudy appearance to the fluid. These cultures
were used to innoculate a set of plates using ! mi
of broth with 5 ml of agar medium. Two plates were
Cotylodons
in isopods. page 13
made por nutrient bath and 20 embryos wet with mar-
supial fluid were placed on each dish using a different
female for each plate. The plates were incubated at
35°0 for 21 hours in 100% humidity at which time a
good growth of bacteria was evident. There was no
observable bacteriostatic effect by the fluid or embryos
for the substrate sampled or the medium tested.
Respiration
The cotyledon membrane is highly permeable to
water, and as a general rule membranes permeable to
water are permeable to gasses. For this reason it
is quite possible the cotyledons function as a site
for oxygen exchange. However, no experiments were
performed to test Verhoeff's (1920) hypothesis.
Summary
Brooding females in 7 out of 8 species of onis¬
coidean isopods examined exhibited cotyledons or re¬
lated structures. Diagrams are presented showing their
form and position. Porcellio scaber americanus, P.
dilatatus, and Armadilloniscus lindahli all have four
cotyledons, one on each of four segments making up the
brood pouch. Philoscia richardsonae has nine coty-
ledons, while Alloniscus perconvexus and A. cornutus
both have twelve. Cotyledons are arranged in single,
in double or in triple on the brood pouch segments.
tyledons in isopods. page 1!.
rmadillidium vulgare has ten structures markodly dif-
ferent from cotyledons. Ligia occidentalis appears
to lack cotylodons complotely.
rcellio scaber americanus body size is di¬
For Po
rectly correlated with cotyledon length. There is
also a correlation between stage of developement of
the brood and cotyledon length for the two middle
cotyledons. The cotyledons shrink upon release of the
brood. The cotyledons appear permeable to water and
aqueous dye. The marsupium on the brooding female
contains a mucus-like fluid of uncertain origin.
Neither the marsupial fluid nor the brooded embryos
xhibited any bacteriostatic action with the experi-
mental methods used.
0
Cotyledons in isopods
cknowledgemer
I would like to thank Dr. Donald P. abbott for his
tremendous amount of help and encouragement on this
project.
Cotyledons in isopods
Literaturo citod
Akahira, Yukio. 1956. The Function of Thoracic Processes
Found in Females of the Common Wood-louse, Porcollio
scabor. Jour. Fac. Sci. Hokkaido Univ. Ser. VI
Zool. 12 (4): 493-198.
Hoeley, Wm. 1941. Observations on the Life-Histories
of some Terrestrial Isopods. Proc. Zool. Soc.
London. 111 (B) : 79-149.
Schöbl, J. 1880. Die Fortpflanzung isopoder Crustacean.
Arch. mikr. Anat. Bonn. 17 : 125-110.
Verhoeff, K.W. 1920. Zur Kenntnis der Larven, des
Brutsaches und der Bruten der Oniscoidea-Isopoden
Aufsatz 28. Zool. Anz. 51 : 169-189.
im, Randall. 1973. Embryonic Developement and Develope-
mental Conditions Within the Marsupium of Por-
cellio scaber and Idothea resecata. Unpublished
manuscript on file at Hopkins Marine Station
Library.
Cotylodons in isopods.
Figures
Figs. 1-4. Figuros 1- show position, number, and
appearance of cotyledons in A - Porcollio scaber amer¬
icanus, B - Porcellio dilatatus, C - Armadilloniscus
lindahli, D - Philoscia richardsonae, E - Alloniscus
perconvexus, F - Alloniscus cornutus, G - Armadillidium
vulgare, H - Ligia occidentalis.
Fig. 5. Ventral and lateral views of the brood pouch.
Letters A-H refer to species as in figs. 1-4.
Fig. 6. Relation of cotyledon size to body length in
brooding females of Porcellio scaber americanus.
Vertical bars show 95% confidence limits of the means.
Cotyledons are numbered 1-1 in order from anterior to
posterior. Black triangles on the horizontal axis
show actual mean body length for the population sample
of each size class; verticle lines show the limits
for each size class. Means ranges and standard devi-
ations etc. appear in Appendi A.
Fig, 7. Distinguishing characteristics of the five
stages of brood developement recognized in the present
study.
Fig. 8. The relation between lengths of cotyledons
1 and Lys. stage of developement of the young in
Cotylodons
nisopods.
brooding femalos of Porcollio scabor
americanus 9.75
to 11.75 mm in body longth.
Fig. 9. The relation betweon mean length of cotyledons
2 and 3 and stago of developement of the young, in
americanus 9.75
brooding females of Porcellio scaber
to 11.75 mm in body length. A regression line is
drawn through the Y axis intercept and the last point.
Gotyledons in isopods.
FIGURE
C
C




4

S
E
11
o o0




- -- .--





O o



D

1


O
O
S


—
- —
—
Cotylodons in isopods.
FIGURE 2
o d

ka-

)
S

S
2
e e i

O

X


oo
oooo
kaaaa-

6

o
O

ua
—
Cotylodons in isopods.
FIGURE 3
C
o o
a-




S









oo
ka-



S




.
Cotylodons in isopods.
FIGURE 4
0
—11.


kaa-
2
N






aa

0
—


.

.
/

—
e




a
639
Cotylodons in isopods.

ddd
178
FIGURE 5
B
X

J



C

2

2
A
S


S.

J
A

OO
0

O C
+5
Cotylodons in isopods.
FIGURE 6
25
I
2.0
L
O 15


—

O 10
O6



(O
O
O
SIZE CLASSES
Gotylodons in isopods.
FIGURE 7
No definable
appendagos

irst appearane of
limb buds
Obvious appendages
No eye pigment
11
First appearance of
— black eye pigment
Obvious appendages
Eye pigmentation
L
Within embryonic
membrane
Emergence from
embryonic membrane
Obvious appendages
Eye pigmentation
Free from embryonic
membrane
Released from
Brood sac
Free living
Cotyledons in isopods.
O)

—
0
HIONA NOGZ1o5
NVZN
383


2.

O
5


IL
1
1)
Q
Q
HIONT NOdX1OS

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(
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Cotylodons ir
Sizo
Class Cot.
isopods.
Appendix
NC
NC
NC
NC
NC
NC
1.125
.2165
12 1.667
.1219
.3200
12 1.512
.1188
12 1.083
20 1.325
.1518
20 1.875
.3191
18 1.69
.330
.2958
20 1.25
1.361
.5262
2.111
.5152
.3685
1.911
1.3
.3685
1.11
.1285
2.313
.3307
2.1
1.61
.1581
NC
NC
NC
NC
NC
NC
NC
NC
NC = not computod
957
range
1.0
NC
O.5-1.5
NC
O.5-1.0
NC
O.5-1.0
NC
1.0-1.5
.1114
1.0-2.5
.282
1.0-2.0
.212
0.5-2.0
298
.218
O.5-2.0
.168
1.0-2.5
1.0-2.0
.171
O.5-1.5
.112
1.0-2.5
.376
1.5-3.0
.12
1.5-2.5
.3
0.5-2.0
.302
1.0-2.0
1.5-3.0
.186
375
1.5-2.5
1.0-2.5
.373
0.5-2.0
NC
O.5-2.0
NC
O.5-2.0
NC
NC
O.5-1.5