Structure of the Shell and Tertiary Membranes of Eggs of
Softshell Turtles (Trionyx spin if e r us)
MARY J. PACKARD AND GARY C. PACKARD
Department of Zoology a n d Entomology, Colorado State Uniuersity,
Fort Collins, Colorado 80523
ABSTRACT
Eggs of the turtle Trionyx spiniferus are rigid, calcareous
spheres averaging 2.5 cm in diameter. The eggshell is morphologically very similar to avian eggshells. The outer crystalline layer is composed of roughly columnar aggregates, or shell units, of calcium carbonate in the aragonite form.
Each shell unit tapers to a somewhat conical tip a t its base. Interior to the
crystalline layer are two tertiary egg membranes: the outer shell membrane
and the inner shell membrane. The outer shell membrane is firmly attached to
the inner surface of the shell, and the two membranes are in contact except a t
the air cell, where the inner shell membrane separates from the outer shell
membrane. Both membranes are multi-layered, with the inner shell membrane
exhibiting a more fibrous structure than the outer shell membrane. Numerous
pores are found in the eggshell, and these generally occur a t the intersection of
four or more shell units.
Studies recently completed in our laboratory have revealed important physiological
similarities between avian eggs and eggs of
the softshell turtle Trionyx spinifems (Packard et al., '79). Eggs of Trionyx are rigid and
calcareous, as are avian eggs (Becking, '751,
and they apparently lose water vapor continuously to the nest environment (Packard et
al., '791, as do avian eggs (Rahn and Ar, '74).
Although incubating eggs of softshell turtles
may absorb liquid water from the substrate
(Packard et al., '791, neither avian embryos
nor the turtle embryos depend upon the absorption of liquid water to sustain embryogenesis (Packard et al., '79; Rahn and Ar,
'74). Given these functional similarities between avian eggs and eggs of Trionyx spiniferus, we felt it important to determine if
there also are structural similarities between
eggs of softshell turt,les and those of birds.
MATERIALS AND METHODS
Eggs of softshell turtles were collected from
natural nests on sand bars in the South Platte
River near Crook, Colorado, in June, 1977.
These eggs were used in an experiment assessing the effect of substrate water potential on
water exchanges between incubating eggs and
their environment (Packard et al., '79). Shells
J . MORPH. (1979)159: 131-144
of all eggs from which turtles hatched were
saved, and it is these eggshells that formed
the bulk of the material used in this study.
In addition, we examined eggshells from
unhatched eggs; some of these shells were
gathered from the vicinity of nests opened by
predators, whereas others came from eggs
that were incubated but from which no
hatchling emerged. Subsequent examination
of eggs which did not hatch following
incubation in the laboratory revealed t h a t
virtually no development had occurred, but it
was not possible to determine if these eggs
were infertile or whether the embryo had
simply died early in development. In any case,
shells of unhatched eggs did not differ
morphologically irrespective of their origin,
and observations on these eggshells are pooled
for comparison with shells from hatched eggs.
Observations were made on shell fragments
and/or shell membranes from a total of 35
eggs.
Most shell fragments were simply air-dried
and stored for future use. However, some fragments were boiled for five minutes in 5%
NaOH to remove the shell membranes and
make it possible to examine the inner surface
of the shell. The outer shell membrane, which
usually is bound to the calcium layer, was pre-
131
132
MARY J. PACKARD AND GARY C. PACKARD
pared for examination by decalcifying pieces
of shell in Decal” (Scientific Products) or in
concentrated HC1 (for 60 seconds). Both procedures gave similar results. All material
treated with NaOH, Decalo, or HCI was rinsed
in distilled water and air-dried. These preparations, and those receiving no chemical treatment, were placed in a desiccator over flakes
of KOH for a minimum of 24 hours prior to
preparation for scanning electron microscopy.
After drying, pieces of shell or membrane
were mounted flat on aluminum stubs using
reflective paint or Scotch@double-coated tape.
Radial fractures were prepared by breaking
off small fragments of shell and mounting
them on edge. In this manner, i t was possible
to examine the radial (cross sectional) face of
t h e shell. Mounted materials were then coated
with carbon and gold in a Technics@hummer
and examined with a n Hitachia scanning electron microscope (model HHS2-R) operated at
a n accelerating voltage of 20 kv. Pictures
were taken with Polaroid” 105 or 665 film,
and prints were made from negatives using
standard photographic techniques. Magnifications are not exact, but are within 20% of reported values.
The composition of t h e shell was analyzed
using techniques of X-ray diffraction (Cullity,
’56). Pieces of shell were ground to a powder
with a mortar and pestle, and the X-ray pattern was generated with a General Electric@
XRD-5 diffractometer using copper radiation
and a nickel filter (Cullity, ’56).
Although shells from eggs of softshell t u r tles are similar to those of birds, there are
some differences. For this reason, we have
used a rather simple terminology to describe
our observations (Erben, ’701, and have tried
to avoid extensive use of a terminology best
suited for descriptions pertaining specifically
to avian eggs (Tyler, ’69).
RESULTS
Eggs of softshell turtles are white, brittle,
and nearly spherical, averaging 2.5 cm in diameter (n = 65; s.d. = 0.13). Analysis by X-ray
diffraction revealed t h a t t h e eggshell consists
of calcium carbonate in the form of aragonite
crystals, as is the case with eggs of other
chelonians (Young, ’50; Erben, ’70; Solomon
and Baird, ’76). In contrast, avian eggshells
are composed of calcite (Erben, ’70). Interior
to the crystalline layer are two tertiary egg
membranes: the inner shell membrane, adja-
CALCAREOUS
/LAYER
OUTER
INNER
SHELL
MEMBRANE
AIR
CELL
Fig. 1 Schematic diagram of a n egg of Trionyx
spiniferus.
cent to t h e albumen, and t h e outer shell membrane, adjacent to the crystalline layer (fig.
1).The two membranes are closely apposed to
one another and cannot be distinguished
visually a s separate entities except a t the air
cell. At t h e air cell, the inner shell membrane
separates from the outer shell membrane, and
a space forms between them (fig. 1). Since
these eggs are nearly spherical, i t is not possible to identify a particular region of the egg a t
which t h e air cell forms. This is in contrast to
t h e situation in most avian eggs, where the air
cell forms at the so-called “blunt” pole of the
egg (Romijn and Roos, ’38).
Examination of t h e outer surface of shells of
softshell turtle eggs by scanning electron microscopy revealed t h a t the eggshell is formed
by t h e close association of individual crystalline aggregates or shell units. In surface
view, t h e borders of individual shell units are
most easily seen at points of intersection
adjacent to pores (fig. 3). The pores in these
eggshells a r e highly irregular in size and
shape, making i t difficult to characterize a
“typical” pore. (Pores can also be located
using a dissecting microscope, where they appear as irregularly-shaped holes in the shell.)
In radial or transverse section (fig. 4) i t is possible to see t h a t pores through a n eggshell provide for a direct connection between the external environment and t h e interior of the egg.
We found no evidence of a true cuticle
(Board et al., ’77) on t h e surface of these eggs.
A surface film and bits of debris were found to
be irregularly distributed on t h e surface of all
eggs examined, and this film occasionally oc-
STRUCTURE OF SOFTSHELL TURTLE EGGSHELLS
133
cluded pores (see fig. 4). However, most pores also observed on the surface of the membrane
seen in surface view (fig. 3) or in radial frac- from unhatched eggs which had been detures were free of occluding material. Al- calcified (fig. 9). Since decalcification rethough we are uncertain of the origin of this moved all visible traces of the calcium layer,
debris, we suspect that it results from ovidu- there are no crystallites and no crystalcal secretions or from the leaking of extraem- line matrix associated with these structures
bryonic fluids during hatching.
(fig. 9).
In addition to revealing that pores peneThe outer shell membrane can be gently
trate the thickness of the shell, radial frac- separated from the shell of unhatched eggs
tures also disclosed that each shell unit is after thorough desiccation, but this sort of
composed of fine, needle-like crystallites radi- separation generally leads to little or no disating out from a common center (figs. 4, 5). In ruption in shell structure (fig. 10). On the
some cases, the plane of fracture in transverse other hand, the separation of the outer shell
sections resulted in a near-median cross sec- membrane that occurs during incubation retion through the shell unit (figs. 4-61 and ex- sults in a more extensive disruption in strucposed the central or core area of a shell unit. ture (fig. 11).The only region of the shell to
The crystalline matrix of the central area is which the outer shell membrane remains a t smoother and more homogeneous than the ma- tached throughout incubation is a t the air
trix of the rest of the shell unit (fig. 7). Occa- cell. Removal of the membrane from the air
sionally, an amorphous mass of material cell (by treatment with NaOH) revealed a
adhered to the exposed center of a shell unit sharp transition in the appearance of the shell
(fig. 6, left), whereas in other shell units the units a t the margins of the air cell (fig. 12).
core area was free of adhering material (fig. 6, The shell units from beneath the air cell are
roughly columnar structures tapering to a
right).
In some radial fractures (usually those from somewhat conical tip at the base (fig. 12, botunhatched eggs), the outer shell membrane tom), whereas those shell units from the area
was visible adjacent to the calcium layer (fig. adjacent to the air cell appear to have had
5 ) , and the shell unit tapered to a somewhat their tips broken off (fig. 12, top), revealing
rounded tip a t its base (fig. 4). Note, too, that the central area of each shell unit.
fibers of the membrane penetrate the central
The structure of the inner and outer shell
area (fig. 5).
membranes of softshell turtle eggs is superfiIn general, the outer shell membrane is cially similar to that of avian eggs (Bellairs
tightly bound to the crystalline matrix of un- and Boyde, ’69; Becking, ’75). Both memhatched eggs, although this attachment can branes from eggs of softshell turtles appear to
be weakened if the eggshell is thoroughly be composed of several layers of fibers, aldried. However, the outer shell membrane though i t was not possible to determine the
does not remain bound to the crystalline layer exact number of layers present. The inner
of hatched eggs. Instead, the outer shell mem- shell membrane generally exhibits a more
brane separates from the shell sometime dur- fibrous structure than does the outer shell
ing incubation, carrying with it part of the membrane (fig. 13).The inner shell membrane
crystalline layer. As a result, the base of a was sheared during preparation in figure 13,
shell unit from a hatched egg appears flat (fig. revealing the presence of two or three layers.
61, rather than rounded (fig. 4). Moreover, a n On occasion, the outer shell membrane also
examination of the outer surface of the outer was sheared during preparation so that one of
shell membrane from a hatched egg revealed the inner layers of the membrane was exposed.
that the surface is obscured by spherical or When this occurred, tips of the shell units
amorphous masses from which crystallites could be seen protruding through the memradiate in all directions (fig. 8). In some cases, brane (fig. 14). Generally, the tips were obthese masses resembled those seen a t the base scured by an overlying layer(s) of the outer
of a shell unit from a hatched egg (fig. 61, shell membrane.
As in avian eggs, the inner surface of the inwhereas in others the spheres seemed to be
encased in a crystalline matrix similar to that ner shell membrane (i.e., that surface facing
of the core area of a shell unit (fig. 8). Masses the embryo and albumen) has an amorphous,
similar to those seen on the surface of the structureless appearance, similar to the “limouter shell membrane of hatched eggs were iting membrane” described by Bellairs and
134
MARY J. PACKARD AND GARY C. PACKARD
Boyde ('69) in their studies of avian eggshells.
This surface also is similar to the inner surface of the inner shell membrane from eggs of
the turtle Chelonia m y d a s (Solomon and
Baird, '76).
DISCUSSION
The process of shell formation in avian eggs
is relatively well understood (Tyler, '69) and
provides an excellent framework from which
to make inferences concerning shell formation
in eggs of Trionyx spiniferus.
After formation of the shell membranes in
avian eggs, the first evidence of calcification
is the appearance of small granules on the
outer surface of the outer shell membrane
(Fujii and Tamura, '70; Stemberger et al.,
'77). These small projections are not removed
by acid (Fujii and Tamura, '70), indicating
that their chemical composition is different
from that of the crystalline shell. Moreover,
the granules resemble projections remaining
on the outer surface of the outer shell membrane after decalcification of an intact egg
(Fujii and Tamura, '70). It is generally
assumed that these granules are the organic
cores which serve as nuclei for the growth of
crystalline aggregates (shell units) during
shell formation (Simkiss, '67; Tyler, '69).
The chemical composition of the granules
seen during the early stages of shell formation
in avian eggs is unknown (Stemberger et al.,
'77), and no one has yet demonstrated the
presence of an organic core on the outer shell
membrane of an egg removed from the oviduct
prior to the beginning of calcium deposition
(Tyler, '69). Nonetheless, the crystallites of an
intact shell unit do radiate out from a discrete
core which is organic in composition (Terepka,
'63b; Simkiss, '67; Tyler, '69). As pointed out
by Tyler ('691, the formation of the organic
core may be quite rapid, or it may occur simultaneously with the initiation of calcification,
making it unlikely that the naked cores could
be detected during studies of shell formation.
In avian eggs, the organic cores are anchored to the fibers of the outer shell membrane (Simons and Wiertz, '63; Simkiss, '67;
Bellairs and Boyde, '69) and are eventually
surrounded by the crystalline matrix of the
shell (Simons and Wiertz, '63; Simkiss, '67;
Bellairs and Boyde, '69). The crystallites of
calcium carbonate initially grow outward
from the core in all directions, growing into
the membrane and enclosing fibers of the
membrane (Terepka, '63a; Erben, '70) as well
as the organic core (Simons and Wiertz, '63;
Simkiss, '67; Tyler, '69). However, growth of
crystals toward the membrane is eventually
inhibited (Simkiss, '671, so that the bulk of
growth occurs laterally and outward (Simkiss,
'67; Tyler, '69). Ultimately, in an avian egg,
the borders of the individual shell units meet
at their lateral margins, and this contact between shell units limits growth in the lateral
direction. As the shell increases in thickness,
the individuality of the shell units is obscured,
and in radial (or transverse) section it is difficult to trace an individual shell unit through
the entire thickness of the shell (Tullett et al.,
' 7 5 ; Board et al., '77).
Based on our observations of the structure
of eggshells of Trionyx spiniferus, we infer
that a similar process of shell formation occurs in this species. Presumably, the small
masses remaining on the outer surface of the
outer shell membrane after decalcification of
an intact eggshell (fig. 9) served as the centers
of crystallization during calcification of the
shell and are analogous to the organic cores of
avian eggs (Tyler, '69). We assume that the
amorphous masses occasionally seen a t the
base of a shell unit in transverse section (fig.
6) and those seen on the surface of the outer
shell membrane when it detaches from the
crystalline layer (fig. 8 ) are comparable to the
cores seen after decalcification.
As in avian eggs, crystal growth apparently
occurs in all directions initially. Eventually,
however, the rate of growth away from the
outer shell membrane outstrips that occurring
toward the membrane, and the shell unit
tends to grow laterally and outward (fig. 2). In
contrast to the situation in avian eggs, however, each shell unit in an eggshell of a softshell turtle retains its individuality through
the thickness of the shell, making it possible
to identify an individual shell unit in transverse section (figs. 4-6). Thus, as pointed out
by Erben ('701, the entire shell of a turtle egg
corresponds to the so-called mammillary layer
of an avian egg (Tyler, '69; Erben, '70).
Fibers of the shell membrane penetrate the
core area of shell units in softshell turtle eggs
(fig. 51, and the tips of the shell units grow
into a layer (or layers) of the outer shell membrane (fig. 14). These observations suggest
that, as in avian eggs, the crystalline matrix
surrounds the core and some of the fibers of
the outer shell membrane. Since the core may
remain attached to the outer shell membrane
when it separates from the shell proper (fig.
STRUCTURE OF SOFTSHELL TURTLE EGGSHELLS
135
Fig. 2 Schematic diagram of a shell unit from an egg of Trionyz spiniferus. As we envision it, after formation of the shell membranes, the central core of a shell unit is secreted onto the outer surface of the outer
shell membrane. This core then serves as a nucleus for the initiation of crystal growth. The primary direction
of growth is here represented by the length of t h e arrows representing t h e crystallites of calcium carbonate.
However, no proportionality of size is intended.
81, we assume that. the core is anchored to the
fibers of the outer shell membrane, as it is in
avian eggs. Presumably, then, the cores seen
on the surface of the outer shell membrane of
hatched eggs (fig. 8 ) or a t the base of a shell
unit of hatched eggs (fig. 6) would occupy the
core area (figs. 4, 5, 7, 11) in an intact shell
unit. This interpretation is strengthened by
the observation that cores seen on the outer
shell membrane often are encased in a crystalline matrix similar in appearance to that of
the core area of a shell unit (figs. 7, 8 ) .
As shell formation proceeds to completion in
avian eggs, there may be an incomplete meeting of the borders of intersecting shell units,
resulting in the formation of a space or pore
between them (Becking, '75; Tullett, '75;
Board et al., '77). Pores generally occur a t the
intersection of four or more shell units in
avian eggs (Tullett, '75; Fox, '761, and a similar situation obtains in eggs of Trionyx
spiniferus. With respect to avian (and chelonian) eggs, i t is not known what causes the
discontinuities that lead to pore formation
(Tyler, '69). However, in both avian and chelonian eggs, i t is through these openings in the
shell that gas and water exchanges are effected.
The pores of an avian egg provide for a continuous loss of water from the egg throughout
incubation (Rahn and Ar, '74). Indeed, an
avian egg must lose a certain amount of water
during incubation to insure formation of a
large air cell (Romanoff and Romanoff, '49).
Avian embryos then pip the inner shell membrane over the air cell and use the air cell as a
source of air to inflate their lungs in the hours
prior to hatching (Romanoff and Romanoff,
'49). Eggs of Trionyx spiniferus, on the other
hand, tolerate a relatively wide range of water
loss during incubation (Packard et al., '79).
Although most eggs examined in this study
formed an air cell, some of the air cells were
extraordinarily small. We closely examined
the shell membranes of all eggs from which
hatchlings emerged and could find no evidence to suggest that embryos of Trionyx
spiniferus preferentially pip the inner shell
membrane over the air cell. These observations support the contention that embryonic
softshell turtles do not use the air cell as a
source of air to inflate the lungs prior to
hatching.
Sometime during development in avian
eggs, during the later stages of incubation, the
outer shell membrane separates from the
crystalline matrix except at the air cell, where
the outer shell membrane remains firmly a t tached to the shell (Tyler and Simkiss, '59;
Terepka, '63b). Since the tip of a shell unit
136
MARY J. PACKARD AND GARY C. PACKARD
grows into the outer shell membrane and
encloses fibers of t h e membrane (Bellairs and
Boyde, '69; Erben, '70; Fujii, '741, some of t h e
shell unit remains attached to t h e membrane
when i t separates from t h e shell (Erben, '70;
Fujii, '74). The shell units from which t h e tips
have been removed in this manner show a central depression (Tyler and Simkiss, '59; Erben,
'70; Fujii, '74). and adhering to t h e surface of
the outer shell membrane are roughened granules t h a t presumably were liberated from t h e
shell unit when the outer shell membrane separated from t h e shell proper (Tyler and
Simkiss, '59; Terepka, '63b; Erben, '70; Fujii,
'74). (Although i t is convenient for us to speak
of t h e outer shell membrane as separating
from the shell, this generality is only partially
correct. Actually, i t is the tip of a shell unit
t h a t separates from t h e rest of a shell unit,
and i t is unclear whether t h e outer shell membrane is a passive participant in this process
or whether shrinkage and drying of t h e
membrane during incubation facilitates this
separation.)
In any case, it is generally assumed t h a t resorption of calcium from t h e inner surface of
the shell by a n avian embryo weakens t h e attachment between the tip of a shell unit and
t h e rest of t h e shell unit, leading to a separation of the tip from the shell unit (Bellairs and
Boyde, '691. This interpretation is supported
by the observation t h a t t h e shell units remain
intact at t h e air cell (Tyler and Simkiss, '59),
which is the only region of the inner surface of
t h e shell where there is not a n intimate contact between the embryo, via the chorioallantoic membrane, and t h e shell (Simkiss, '671,
and thus, the only region from which calcium
is not removed. Moreover, a t about the time
t h e embryo begins to resorb calcium from t h e
shell (Johnston and Comar, '551, t h e ectoderma1 layer of the chorioallantois becomes highly differentiated (Coleman and Terepka, '72a)
and begins actively to transport calcium (Garrison and Terepka, '72). Presumably, dissolution of the shell is accomplished by carbonic
acid formed from water and carbon dioxide
(Dawes, '751, and t h e calcium liberated during
this process is actively transported across t h e
chorioallantoic membrane. The calcium may
be transported in certain specialized cells
(Coleman and Terepka, '72a,b) or in the extracellular space (Crooks e t al., '76; Saleuddin
et al., '76). Current evidence supports either
interpretation.
A similar separation of t h e outer shell mem-
brane from t h e shell apparently occurs in softshell turtle eggs during incubation, since, a t
hatching, the crystalline matrix falls away
from the outer shell membrane. Moreover, the
appearance of t h e shell units at t h e inner surface of t h e shell and t h e corresponding appearance of t h e outer surface of t h e outer shell
membrane strongly suggest t h a t the separation of t h e shell membrane from t h e shell is
quite similar to t h a t described for avian eggs.
Our interpretation is t h a t t h e spherical masses seen on t h e surface of the outer shell membrane are t h e central cores and t h a t they (and
part of the crystalline matrix of t h e shell unit)
are pulled from t h e shell unit when the membrane separates from the shell. Presumably,
t h e central depression seen in shell units from
hatched eggs (figs. 11, 12 top) once contained
t h e core, and it is this core plus t h e associated
crystals of t h e tip t h a t adhere to t h e surface of
t h e outer shell membrane (fig. 8 ) . We believe
t h a t t h e similarities between these eggs and
those of birds are too striking to warrant a n
entirely different interpretation.
Although we do not know if embryos of
Trionyx spiniferus remove calcium from the
inner surface of t h e shell for incorporation
into embryonic tissues, we infer from the
structural similarities with avian eggs t h a t
they must. The outer shell membrane can be
pulled away from t h e shell of a n unhatched
egg if the egg is stored over a desiccant first.
However, such mechanical separation of the
membrane from t h e shell is neither as complete nor as striking as t h a t occurring naturally during incubation (figs. 10, 111, suggesting t h a t some change takes place in t h e
shell during development of t h e embryo. Our
interpretation is strengthened by the observation t h a t embryos of other chelonians are
known to remove calcium from the shell during incubation (Bustard et al., '691. Whether
or not there also a r e changes in t h e chorioallantoic membrane t h a t are correlated with
t h e onset of calcium resorption in softshell
turtle eggs remains to be determined.
In conclusion, eggs of Trionyx spiniferus are
very similar morphologically to avian eggs.
Trionyx eggs are hard and rigid, as are avian
eggs; shells a r e comprised of a n outer,
calcareous layer bounded on t h e inner surface
by two shell membranes, as are avian eggs;
pores form at t h e intersection of four or more
shell units, as in avian eggs; and the process of
shell formation seems to be similar to t h a t occurring in avian eggs. Moreover, Trionyx eggs
STRUCTURE OF SOFTSHELL TURTLE EGGSHELLS
exhibit structural changes during incubation
similar to those occurring in avian eggs a s a
result of resorption of calcium from t h e shell.
These structural changes suggest t h a t embryos of Trionyx spiniferus also resorb calcium
from the shell, just as do avian embryos.
ACKNOWLEDGMENTS
We thank Marvin Gardner of t h e Tamarack
Ranch Wildlife Area, near Crook, Colorado, for
providing us with access to t h e collecting site.
D. Winder of t h e Department of Physics at
Colorado State University helped us to gather
and interpret data in t h e X-ray diffraction
analysis of eggshells. M. Stringer of t h e Department of Anatomy offered valuable advice
concerning scanning electron microscopy; and
we thank t h e Department of Anatomy for t h e
use of their facilities for electron microscopy.
G. Happ of t h e Department of Zoology and
Entomology provided access t o darkroom
facilities and equipment, and offered helpful
advice on t h e preparation of plates. S. Stack
(Department of Botany and Plant Pathology)
and C. R. Tracy (Department of Zoology and
Entomology) critically reviewed drafts of this
paper. The manuscript was expertly typed by
M. Wright. Our research has been supported,
in part, by grants from t h e National Science
Foundation (DEB 75-18179) and from t h e
BRSG Committee a t Colorado State University.
LITERA.TURE CITED
Becking, J. H. 1975 The ultrastructure of the avian eggshell. Ibis, 117: 143.151.
Bellairs, R., and A. Boyde 1969 Scanning electron microscopy of the shell membranes of the hen’s egg. Z.
Zellforsch., 96: 237-249.
Board, R. G., S. G. Tullett and H. R. Perrott 1977 An arbitrary classification of the pore systems in avian eggshells.
J. Zool., 182: 251-265.
Bustard, H. R., K. Simkiss and N. K. Jenkins 1969 Some
analyses of artificially incubated eggs and hatchlings of
green and loggerhead sea turtles. J. Zool., 158: 311-315.
Coleman, J. R., and A. R . Terepka 1972a Fine structural
changes associated with the onset of calcium, sodium and
water transport by the chick chorioallantoic membrane.
J. Membrane Biol., 7: 111.127.
- 1972b Electron probe analysis of the calcium
distribution in cells of the embryonic chick chorioallantoic membrane. 11. Demonstration of intracellular location during active transcellular transport. J. Histochem.
Cytochem., 20: 414-424.
Crooks, R. J., C. P. M. Kyriakides and K. Simkiss 1976
Routes of calcium movement across t h e chick chorioallantois. Quart. J. Exp. Physiol., 61: 265-274.
Cullity, €3. D. 1956 Elements of X-ray Diffraction.
Addison-Wesley Publishing Co., Inc., Reading, Massachusetts.
137
Dawes, C. M. 1975 Acid-base relationships within the
avian egg. Biol. Rev., 50; 351-371.
Erben, H. K. 1970 Ultrastrukturen und Mineralisation
rezenter und fossiler Eischalen bei Vogeln und Reptilien.
Biomineralisation, 1: 1-66.
Fox, G. A. 1976 Eggshell quality: its ecological and physiological significance in a DDE-contaminated common
tern population. Wilson Bull., 88: 459-477.
Fujii, S. 1974 Further morphological studies on the formation and structure of hen’s eggshell by scanning electron microscopy. J. Fac. Fish. Anim. Husb. Hiroshima
Univ., 13: 29-56.
Fujii, S., and T. Tamura 1970 Scanning electron microscopy of shell formation in hen’s eggs. J. Fac. Fish. Anim.
Husb. Hiroshima Univ., 9: 65-80.
Garrison, J. C., and A. R. Terepka 1972 Calcium-stimulated
respiration and active calcium transport in the isolated
chick chorioallantoic membrane. J. Membrane Biol., 7.
128.145.
Johnston, P. M., and C. L. Comar 1955 Distribution and
contribution of calcium from the albumen, yolk and shell
to the developing chick embryo. Amer. J. Physiol., 183:
365-370.
Packard, G. C . . T. L. Taigen, T. J. Boardman, M. J. Packard
and C. R. Tracy 1979 Changes in mass of softshell turtle
(Trionyx spiniferus) eggs incubated on substrates differing in water potential. Herpetologwa, in press.
Rahn, H., and A. Ar 1974 The avian egg: incubation time
and water loss. Condor, 76: 147-152.
Romanoff, A. L., and A. J. Romanoff 1949 The Avian Egg.
John Wiley & Sons, New York.
Romijn, C., and J. Roos 1938 The air space of the hen’s egg
and its changes during the period of incubation. J.
Physiol., 94: 365-379.
Saleuddin, A. S. M., C. P. M. Kyriakides, A. Peacock and K.
Simkiss 1976 Physiological and ultrastructural aspects
of ion movements across t h e chorioallantois. Comp.
Biochem. Physiol., 54A: 7-12.
Simkiss, K. 1967 Calcium in Reproductive Physiology. A
Comparative Study of Vertebrates. Reinhold Publishing
Corp., New York.
Simons, P. C. M., and G. Wiertz 1963 Notes on the structure of membranes and shell in the hen’s egg. An electron
microscopical study. Z. Zellforsch., 59: 555-567.
Solomon, S. E., and T. Baird 1976 Studies on the egg shell
(oviducal and oviposited) of Chelonia rnydas L. J. Exp.
Mar. Biol. Ecol., 22: 145-160.
Stemberger, B. H., W. J. Mueller and R. M. Leach, J r . 1977
Microscopic study of the initial stages of eggshell calcification. Poultry Sci., 56: 537-543.
Terepka, A. R. 1963a Structure and calcification in
avian egg shell. Exp. Cell Res., 30: 171-182.
19631, Organic-inorganic interrelationships in
avian egg shell. Exp. Cell Res., 30: 183.192.
Tullett, S. G. 1975 Regulation of avian eggshell porosity.
J. Zool., 177: 339-348.
Tullett, S. G., P. L. Lutz and R. G. Board 1975 The fine
structure of the pores in the shell of the hen’s egg. Brit.
Poultry Sci., 16: 93-95.
Tyler, C. 1969 Avian egg shells: their structure and
characteristics. Int. Rev. Gen. Exp. Zool., 4: 81-130.
Tyler, C., and K. Simkiss 1959 Studies on egg shells. XII.
Some changes in the shell during incubation. J. Sci. Food
Agric., 10: 611-615.
Young, J. D. 1950 The structure and some physical properties of the testudinian eggshell. Proc. Zool. SOC.London,
120: 455-469.
Y
720
Near-median cross section of a shell fragment from an unhatched egg showing a pore traversing the
thickness of the shell. The outer shell membrane is attached to this shell fragment and is marked with an
arrow. Note that each shell unit is intact and rounded a t its base. X 216.
X
6
Near-median cross section through two shell units from a hatched egg. Note that the base of each shell
unit is flat. rather than rounded. x 352.
5 Near-median cross section through a shell unit from an unhatched egg. The outer shell membrane is
clearly visible a t the base of the shell unit. Note that the membrane extends into the central area. x 352.
4
3 Surface view of eggshell showing pore a t the intersection of four shell units.
E X P l A N A T I O N OF FIGURES
PLATE 1
-1
m
139
X
880.
10 Inner surface of shell from an unhatched egg. Outer shell membrane detached from shell after desiccation, hut this detachment led to little disruption in shell structure. X 352.
9 Outer surface of outer shell membrane from an egg decalcified in HC1 for 60 seconds. The surface of the
membrane is dotted with the cores that ordinarily would occupy the central area of intact shell units.
X 88.
8 Outer surface of outer shell membrane from a hatched egg showing two cores and the crystallites associated with them. X 432.
7 Higher magnification view of central area of a shell unit.
EXPLANATION OF FIGURES
PLATE 2
STRUCTURE OF SOFTSHELL TURTLE EGGSHELLS
Mary J. Packard and Gary C. Packard
PLATE 2
c
Q
Inner shell membrane showing two (or 3) fibrous layers. x 432.
14 Outer shell membrane from the air cell of a hatched egg. A layerk) of the membrane was sheared away
during preparation, revealing the tip of a shell unit. The outer shell membrane is less fibrous than the
inner shell membrane and resembles a reticulate mat. x 432.
13
12 Transition zone from inner surface of shell of a hatched egg. The area a t the bottom of the picture is
from beneath the air cell whereas that a t the top is from an area adjacent to, hut not included in, the air
cell. Shell fragment was treated with NaOH to remove the outer shell membrane from the air cell. x 56.
11 Inner surface of shell from hatched egg. Separation of outer shell membrane from shell during incubation led to a disruption in shell structure. These shell units are not intact (compare with fig. 10).The
tips separated from the rest of the shell units, and the central area has been exposed as a result. x 432.
EXPLANATION OF FIGURES
PLATE 3
STRUCTURE OF SOFTSHELL TURTLE EGGSHELLS
Mary J Packard and Gary C. Packard
PLATE 3