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Embryonic Development within the Marsupium
The brood pouch or marsupium in isopods, located
ventrally on the animal, is formed of five pairs of
brood plates or oostegites which arise from the inner
regions of the basis of the first five pairs of pareao-
pods. The edges of the plates overlap to a large
degree, but there is no evidence of a structural inter-
locking mechanism of plates. Embryos within the
marsupium in both marine and terrestial species are
bathed in a "marsupial fluid" which fills the brood
chamber.
The mechanical protection of the developing brood
by the marsupium in part explains the biological
significance of the structure. However, if this were
its only function, the brood might more appropriately
remain in the body cavity. Verhoeff (1920) suggests
other advantages provided by the marsupium:
a) The marsupium is capable of a much greater
expansion than the body cavity and therefore
can harbor a more numerous brood.
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page 2
Embryonic Development within the Marsupium
b) The brood, despite lying outside the mother,
is still within a cavity, and is protected
from strong evaporation and dessication.
In addition to these advantages, it can be suggested
that the brood pouch is an important structure mediating
mineral, nutrient, and metabolic exchange between mother
and embryo. Verhoeff discounts this final function,
but Saudray and Lemercier (1960), in noting a consider-
able increase in embryo size before hatching (in Ligia
oceanica) and an even more rapid increase in weight after
hatching, in great part due to mineral uptake, suggest
that such increases were of maternal origin.
I will attempt to determine whether indeed the mother
contributes to embryonic development by supplying organic
nutrients and/or minerals within the brood fluid in
Porcellio scaber (Latreille 1804) and Idothea resecata
(Stimpson 1857).
For my purposes, the brood within P. scaber and I.
resecata were assigned three developmental stages:
stage 1: early egg; only yolk visible; roughly
spherical.
stage 2: middle larvae; eye spots visible; larvae
elongate within membrane; body curved
away from developing appendages.
nbryonic Development within the Marsupium
page 3
stage 3: late larvae; exoskeleton and appendages
well formed; pleopod beat visible in marine
species; larvae motile; completely free
of membrane.
I studied these stages to determine if the brood
receives any organic and/or inorganic nutrients di
during the brood period. Relative wet and dry weights
(corrected for salt content of surface seawater on embryo)
of the three developmental stages were compared for both
P. scaber and I. resecata. Fifteen mothers of each
species were collected for this study, including five
mothers containing stage 1 embryos, five containing stage 2
and five containing stage 3 embryos. Wet weights were
obtained by placing embryos on glass fiber filter paper,
removing surface water with gentle vacuum, and immediately
weighing. Dry weight was obtained by weighing these same
embryos dessicated for 48 hours to constant dryness.
Five embryos were weighed in each trial, on a Cahn electro-
gram balance with an accuracy of + 2.5 ug. Results for
I. resecata and P. scaber are plotted in graphs 1 and 2.
In graphs 1A and 2A, the embryos from the five
mothers of one stage were combined to form one large
embryo pool of a particular stage (graph designation:
mothers pooled). Five embryos of each stage were removed
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page 4
Embryonic Development within the Marsupium
from the embryo pool and weighed in each of five trials.
Bar graph total height indicates mean wet weight of
one embryo. Each bar is divided into two sections; the
lower section represents mean dry weight of one embryo.
In graphs 1B and 2B (mothers individuated) each mother's
brood was sampled independently. Graphical representation
is identical to the previous graph.
There is a significant decrease in dry weight
in development from stage 1 to stage 2 in two of four
trials (p=0.01), and a decrease in dry weight between
stage 1 and stage 2 with probability less than 0.1 and
0.4 in the other two trials. However, as development
proceeds from stage 2 to stage 3, dry weight increases
significantly again in three of four trials (p = 0.001),
and increases with probability less than 0.4 in the other.
The apparent constant increase in size of the developing
embryo can in part be attributed to the constant increase
of wet weight. Mean embryo weight never varied more than
2 ug between samples at each stage (sample size: three
embryos each stage), which is within the limits of accuracy
of the balance.
Seasonal variation of nutrient availability, temper-
ature, etc., probably cannot account for any observed
differences in weight between stages, since the studies
page 5
Embryonic Development within the Marsupium
portrayed in graphs 1B and 2B were done two weeks after
the studies in 1A and 24. The fact that these second
studies show similiar significant trends suggests that
seasonal variation is not a factor. Further, in the
course of following experiments, embryo weights for the
three stages were obtained for other purposes and were
similiar to those weights observed in graphs 1 and 2.
The dry weight increase between stage 2 and stage?3
can be attributed to inorganic uptake alone. (primarily
minerals), organic uptake alone, or a combination of both.
Individual broods corresponding to the three develop-
mental stages in both P. scaber and I. resecata were
divided in the following ways: partt of the brood was
removed from the mother (gentle pipetting of embryos
after brood plates slightly lifted) and utilized for
ex-marsupial cultures; part of the brood was immediately
weighed; remainder of the brood was allowed to develop
normally within the mother. Ex-marsupial culture dishes
consisted of 50X12 mm tight-lid Petri dishes (holes in
lid for oxygen exchange) filled with five milliliters
of either:
filtered seawater and penicillin (50 parts/million)
a)
plus tris buffer pH 8.3
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Embryonic Development within the Marsupium
b) filtered isotonic to seawater NACl solution
plus penicillin and tris buffer
c) seawater plus penicillin minus buffer
d) NACl solution plus penicillin minus buffer
e) seawater plus buffer minus penicillin
f) NACI solution plus buffer minus penicillin
After one week, at 14°0. embryo dry and ash weights
were obtained (ashed at 290°C, 12 hours) and compared
to dry and ash weights of original embryos and original
embryos allowed to develop normally within the brood
pouches of the marked original mothers. Results are
plotted in graphs 3 to 8. Animals that died before the
end of the week period were not included in the results.
Each separate graph 3 to 8 charts the developmant
of a particular brood. Each bar charts mean actual
weight change per animal during the experimental period.
Total height of the bar represents mean dry weight of
one animal; each bar is divided into two sections; the
lower section represents ash weight (a measure of inorganic
weight) whilethe upper portion represents ash-free weight
(organic weight). Numbers above the bar represent the
stage of the embryo at the end of the experiment.
The changes in dry weights during development in
graphs 1 and 2 were confirmed within a particular brood.
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page 7
Embryonic Development within the Marsupium
As expected, development within the brood pouch was more
advanced than in cultures. Highlighted bars of graphs
3B, 4A, and 5A show a decrease in dry weight of embryos
going from stage 1 to stage 2 (which cannot be entirely
accounted for by normal embryo weight variation within
a brood) compared to the original weight of the brood;
this decrease can be accounted for by an absolute decrease
in organic weight perhaps as vitelline reserves of yolk
are utilized. Apparent anomalies such as 3AN, 3CS, 4AS2,
in which an increase in organic weight was observed despite
no increase in dry weight cannot be readily explained.
In bars 4BST, 4BS2, 5AN, 7AR, 7AS, 7CR, the increase in
dry weight can be entirely accounted for by increases
in inorganic weight. However, in graphs 5BR, 5CR, 7BR,
8A and 8B (charting development from stage 2 to stage 3),
increases in dry weight cannot be entirely accounted for
by increases in inorganic weight. There appears to besa
definite increase in both absolute inorganic weight and
absolute organic weight. While the noted anomalies in
graphs of development from stage 1 to stage 2 can perhaps
suggest that observed weight changes could be due to normal
variation within embryos of a particular brood, the
magnitude of the weight changes from stage 2 to stage 3
far exceed the maximum limits of these possible intra-brood
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page 8
Embryonic Development within the Marsupium
weight variations. Thus a significant increase in both
organic and inorganic weight in some embryos (particularly
from stage 2 to stage 3) occurred.
Of special interest is the apparent differences in
increase of absolute organic weight in graphs 8A and 8B.
At the termination of the experiment, the larvae represented
in bars 8AS, 8A82, 8B83, had cannabalized several other
embryos. Perhaps this was the source of the additional
increase in inorganic and organic weight.
Tests for total nitrogen content in the various
embryonic stages, which involved first digesting ten
embryos of each stage by the Folin-Wu method, then assaying
for ammonia content by the phenol-hypochlorite method,
suggest that the ratio nitrogen/drybody weight is constant:
that is, as dry weight increases, total nitrogen increases.
Such a result would support the hypothesis that organic
weight increases during development, but the results
of the tests were not statistically significant perhaps
due to a small sample size. However, during development,
the embryo is apparently utilizing an additional source
of organidand inorganic compounds, aside from those
originally contained within the egg itself.
The most obvious source of additional nutrients
would be the brood fluid itself. Refractometer studies
0
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page 9
Embryonic Development within the Marsupium
of the brood fluid of P. scaber and I. resecata suggest
that the mother is actively concentrating solutes within
the brood fluid. I. resecata, despite observation of
active ventilåtion of the marsupium (approximately 22 times
per minute) through mechanical contraction of the brood
cavity and movement of brood plates, maintains a marsupium
of brood fluid 150 to 200% normal seawater osmotic solute
concentration. Brood fluid of P. scaber is between 8 to 14%
or two to three times that of seawater (fresh water rated
at 0.2%). Blood of P. scaber is approximately 3.5%.
SUMMARY
Evidence suggests that developing embryos increase
in both inorganic and organic weight within the brood
pouch. Brood fluid appears to contain a higher concen¬
tration of solute than other fluids found in the animals'
environment. Several hypotheses can be developed to
describe these results:
the mother is actively concentrating minerals
and/or nutrients within the brood fluid
b) a decrease in brood size is noted through
development. Perhaps decaying embryos provide
an important source for other developing embryos.
More work is needed to concretely say if the mother
is actively supplying minerals and/or nutrients to
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Embryonic Development within the Marsupium
developing embryos. The evidence I have presented is
circumstantial but highly suggestive that indeed
the mother is active in embryonic development.
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mbryonic Development within the Marsupium
page 1
Icknowledgements:
I would like to thank Dr. Robin Burnett, Dr. Donald
Abbott, and Dr. John Phillips for their advice and
assistance in this research project. Special thanks
to Connie Whiteside for aid in preparation of final
draft and illustrations. Finally, thanks to the
aculty and staff of Hopkins Marine Station for many
many things.
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Embryonic Development within the Marsupium
REFERENCES
1. Davis, Charles C. 1964. A Study of the Hatching
Process in Acquatic Invertebrates. IX. Hatching
Within the Brood Sac of the Ovoviviparous Isopod
Cirolana (sp. Isopoda, Cirolanidae). Pacific
Science. 18: 378-384
Folin, Otto, H. Wu. 1919. The System of Blood
2.
Analysis. J. Biol. Chem. 38: 81-110
Naylor, E. 1955. The Comparative External Morphology
and Revised Taxonomy of the British Species of
Idothea. J. Mar. Biol. Ass. U.K. 31: 467-493
Solorzano, L. 1969 Determination of Ammonia in
Neutral Waters by the Phenol-Hypochlorite Method.
Limnol. Oceanogr. 14: 799-801
Verhoeff, K.W. 1920. Zur Kenntnis der Larven,
des Brutsaches und der Bruten der Oniscoidea Isopoda.
Aufsatz 28 Zool. Auz. 51: 169-189
5.
page 12
I
8
470
450
250
200
150
100
50
10
SRAPH1A
(MOTHERS
POOLED)

L
RESECATA
GRAPH
1.
(MOTHERS INDIVIDUATED)
DRY WT.
HAR HILLT
STAGE
21
22
390
380
330
5.
.
250
200
150
100
5C
10
GRAPH 2A
(MOTHERS
POOLED)
LIHHE
ul
DORCELLIO
GRAPH 28
(MOTHERS INDIVIDUATED)
HHHDRRY WT
HAU
STAGE
1
H
S1
2
S.
N
2
N.

(3-8)
GRAPH LEGEND
BROOD AT BEGINNING OF- EXPERIMENT
BROOD ALLOWED TO DEVELOE
NORMALLY WITHIN MOTHER- UNTIL
END OF EXPERIMENT
BROOD GROWN IN SEAWATER PLUS
PENICILLIN AND TRIS BUFFER
S SOLUTION MINUS PENICILLIN
S SOLUTION MINUS BUFFER
BROOD GROWN IN NACL SOLUTION
PLUS PENICILLIN AND TRIS BUFFER
Na SOLUTION MINUS PENICILLIN
BUFFER
N SOLUTION MINUS
WEIGHT
ASH
WEICHT
ASH-FREE
100-
50
10
DI
)(
——
11
—
Os
2
100
50
10
GRAPH 34
R
S1
GRAPH 30
PORCELLIO
STACE
No
50
10
GRAPH
HHH
R
S
38
dead ?
8
S(
10
50

50

7
R
2
Ihn
S1
RESECATA
STAGE
44
GRAPH
Nr
48
GRAPH
STAGE
dead ?
N.
23
N.
100
250
O
100
50
10
GRAPH
GRAP

5A

9
50
ORCELLIO
STAGE
2
GRAPH
N
50
10
3
58
N
8
10
50
10
U
1
10

10
O
R
R
early
S
RESECATA
STAGE
GRAPH
GA
So
N.
STAGE
6B
GRAPH
early
8
2
N

early
early
Na
50
100
50
10

L
150
OO
50
1C
O
SRAPH
12
GRAPH
7A
3
S1
70
PRCELUO
STAGE
3
GRAPH
50
100
N1
50
10
Hhili HAAA
8
78
HH
9 9
O
2
50
100
50
SRAP
STAGE
84
3
RESECATA