Maternal effects of egg size on emu Dromaius novaehollandiae egg composition and hatchling phenotype
1 Department of Biological Sciences, University of North Texas, PO Box
305220, Denton, TX 76203, USA
2 Department of Biology, Kalamazoo College, Kalamazoo, MI 49007,
USA
* Author for correspondence (e-mail: edzial{at}unt.edu)
Accepted 17 November 2003
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Summary |
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Key words: emu, Dromaius novaehollandiae, egg, development, maternal effect, life history, allometry, scaling
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Introduction |
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The trajectory followed by an embryo from zygote to hatchling stages is
influenced by an interaction between genetic instructions in the nuclei of the
embryo's cells and conditions in the environment surrounding those cells.
Conceptually similar to evolutionary paths blazed by populations of organisms
through phenotypic space over several generations
(Raup, 1966), developmental
trajectories (Burggren, 1999
)
of oviparous amniotes can change as a result of biotic and abiotic factors
encountered outside the eggshell and factors, initially maternal in origin,
found within the eggs. Phenotypes of these embryos, developing toward hatching
and toward metamorphosis into a more independent (e.g. self-feeding,
thermoregulating and ambulatory) phase in their lives, are shaped both by
genetic and environmental effects
(Burggren, 1999
). Acquiring
in-depth knowledge of the sensitivity of developmental trajectories to
environmental perturbations, including maternal investment of nutrients and
energy in eggs, will improve our understanding of the genesis and importance
of maternal effects manifested in phenotypes of hatchlings.
Requiring only heat and oxygen from the environment and containing all
nutrients and water necessary to sustain developing embryos, avian eggs are
attractive models for investigating effects of maternal investment on
phenotypes of embryos and hatchlings. Variation in the composition of avian
eggs among species is correlated with functional maturity of hatchlings
(Carey et al., 1980;
Sotherland and Rahn, 1987
).
The quantity and composition of parental investment varies significantly
within species and is frequently correlated with hatchling mass
(Williams, 1994
).
Investigating how intraspecific variation in egg size and composition affects
hatchling attributes can provide useful insights into the importance of
maternal effects in oviparous amniotes.
In this study we examined consequences of natural variation in maternal
investment egg size and composition on emu hatchling
phenotypes. Emu eggs and hatchlings make good experimental subjects for a
study of parental investment because they are large (egg mass approx. 600 g;
hatchling mass approx. 400 g), facilitating measurements of hatchling
characteristics (e.g. blood volume) that are otherwise difficult to quantify.
In addition, intraspecific variation in egg mass from 400 g to>700 g
provides a reasonably, but not unusual, wide range of egg size. Female emus
lay between 5 and 20 eggs, typically incubated by the males during the
breeding season. After emerging from their eggs the precocial hatchlings
forage for food under guidance from the males
(Davies, 1975). Thus, like
other precocial birds (Williams,
1994
; Hill, 1995
),
emus should produce eggs having component masses that vary isometrically with
egg mass, as well as hatchlings, emerging from those eggs, that vary
isometrically with egg mass. Therefore we tested the following hypotheses: (1)
maternal investment, in the form of nutrients and water in eggs, is positively
correlated with egg size and varies in such a way that the proportional
composition of eggs remains constant; (2) morphological and physiological
phenotypes of hatchlings correlate positively with egg size such that
proportional composition of hatchlings remains constant regardless of
hatchling size; (3) maternal investment in eggs provides for greater energy
use in larger eggs during development while provisioning hatchlings with
similar amounts of residual yolk regardless of hatchling size.
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Materials and methods |
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Egg components
Fresh egg mass was determined by drilling two small holes through the shell
over the air cell, filling the air cell with water, and then weighing the eggs
on a Denver Instruments (Denver, CO, USA) digital balance. Short of weighing
eggs immediately after oviposition, this is the most reliable method of
obtaining fresh egg mass (Ar and Rahn,
1980). Fresh eggs were then separated into shell, yolk and albumen
following the methods described in Finkler et al.
(1998
). The intact yolk was
weighed with the balance to determine yolk mass. Yolk, albumen and shell were
then dried to a constant mass in a drying oven at 60°C. Shell mass was
measured by weighing the dry shell on the balance, and albumen wet mass was
determined by subtracting yolk wet mass and shell dry mass from the mass of
the egg. Water contents of yolk and albumen were determined by subtracting dry
mass of each from the respective wet mass; the sum of water mass in the yolk
and water mass in the albumen yielded total water content of each egg. Mass of
egg solids was computed by adding yolk and albumen dry masses.
Incubation
Eggs were stored at 4°C for no more than 7 days before incubation. Eggs
were incubated in forced draft incubators with automatic rotation at Cross
Timbers Emu Ranch until approximately day 40 of incubation. They were then
transferred to the University of North Texas, where incubation continued until
hatching in forced draft emu incubators (GQF Manufacturers, Savannah, GA,
USA). Eggs were incubated at 36.5±1°C and a relative humidity of
approximately 30%, corresponding to the relative humidity experienced in the
nest. Prior to internal pipping, all eggs were transferred to a hatching
incubator maintained at 36.5°C and a relative humidity of
3540%.
Gas exchange of near-term embryos
Metabolic rates
(O2) of 15 eggs
were measured on day 46 of incubation (i.e. 92% of incubation) using a
flow-through system similar to the methods of Dzialowski et al.
(2002
). Eggs were placed in
individual PVC respirometers (approx. vol. 1 l) and then into a constant
temperature chamber regulated at 37.5°C. Air was pumped through the
individual chambers and flow was measured at the inflow side of the chambers
using a calibrated Brooks (Hatfield, PA, USA) flow meter. Outflow
O2 concentration from each respirometer was measured using a
Beckman OM11 O2 analyzer (Anaheim, CA, USA). Inflow O2
concentration to the respirometers was determined from the outflow of an empty
respirometer. Metabolic rate (i.e. rate of oxygen consumption) was calculated
using the equation of Hill
(1972
), corrected to
STPD and expressed in units of ml O2
h1.
Air cell PO2 was measured in eight emu eggs on day 46 of incubation. On day 40 of incubation a 5 mm diameter hole was drilled in the air-cell end of each egg using a drill press. A square patch of 0.4 mm thick Thera-bandTM latex was glued over the hole using Duro Quick GelTM and the egg was replaced into the incubator for 6 days. Using a 1 ml syringe and a 27-gauge needle inserted through the latex, a 1 ml sample of gas was withdrawn from the air cell and then promptly analyzed for PO2 using a Cameron Instruments (Port Aransas, TX, USA) BGM2000 blood gas meter.
Eggshell conductance
We measured water vapor conductance (GH2O) of fresh
eggs of mass 487778 g (N=16). Eggs were initially weighed and
then placed in individual desiccators (approx. vol. 6 l). Each desiccator
contained an ample amount of DrieriteTM desiccant in the bottom of the
desiccator to ensure that water vapor pressure around each egg was near 0 kPa.
The mass of each egg, desiccator temperature and atmospheric pressure were
measured daily for 5 days. Whole eggshell GH2O was
determined following the protocol of Ar et al.
(1974). Finally, initial egg
mass was measured as above by filling the air cell with water and then
weighing the egg.
Hatchling morphology and composition
All measurements of morphology and composition were made on hatchlings that
were less than 1 day old. Hatchlings were euthanized by exposure to either
halothane or isoflurane, and then weighed to the nearest 0.1 g to obtain
hatchling mass (yolk-free hatchling mass plus residual yolk and yolk sac). The
yolk sac was carefully dissected from each hatchling and weighed to measure
the quantity of residual yolk; yolk-free hatchling mass was determined by
subtracting residual yolk mass from hatchling mass. Culmen length and right
tibiotarsus length were measured to the nearest 0.1 mm on each hatchling using
digital calipers (Mitutoyo, Aurora, IL, USA) as a means of quantifying
hatchling structural size. Heart, gizzard and liver were dissected from the
body, weighed separately, and then dried to a constant mass in an oven at
60°C. The yolk sac and what remained of the hatchling were dried to a
constant mass in a similar way. Water contents of the various components were
determined by subtracting dry mass of each from the respective wet mass. Mass
of yolk-free hatchling solids was computed by adding heart, gizzard and liver
dry masses to the dry mass of the dissected carcass. We estimated the quantity
of yolk consumed by an embryo during incubation by subtracting the measured
dry yolk sac mass from the calculated mass of dry yolk that the egg from which
a neonate hatched would have contained at the outset of incubation, using
initial egg mass and the equation for dry yolk mass in
Fig. 1.
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Hematology and blood volume
To obtain blood for hematological measurements, hatchlings were
anesthetized using halothane and blood was taken from the heart by direct
cardiopuncture. Hemoglobin was measured with a Radiometer
(Brønshøj, Denmark) OSM2 Hemoximeter. Hematocrit was measured by
centrifuging blood in heparinized capillary tubes. Osmolality of the blood was
measured using a Wescor (Logan, UT, USA) 5500 vapor pressure osmometer. Two
measurements of each variable were made and averaged for each animal.
Blood volumes were measured in 11 hatchlings using the Evan's Blue dilution
technique (El-Sayed et al.,
1995). Hatchlings were anesthetized with iso-flurane and attached
to a ventilator that maintained an iso-flurane concentration of 1% in the
inspired air. Both the right and left jugular veins were exposed and
non-occlusively canulated with tips of 26-gauge needles attached to PE50
tubing. The right jugular vein was used as the injection site for the Evan's
Blue solution, and the left jugular vein was used to withdraw subsequent blood
samples. Initially, 500 µl of blood was withdrawn into a heparinized
syringe from the right jugular vein. This was followed by an injection of 400
µl of an Evan's Blue solution (5 mg ml1 dissolved in 0.9%
heparinized saline) into the right jugular vein. The Evan's Blue injection was
followed by a 200 µl injection of heparinized saline to wash the tubing.
Samples of blood were then taken from the left jugular vein at 10, 15 and 20
min after the initial injection of Evan's Blue.
After each blood sample was collected, a portion of blood from the sample was added to an equal amount of heprainized saline and centrifuged for 15 min. All volumes were gravimetrically determined using a Denver Instruments digital balance to increase measurement accuracy. A 200 µl sample of the supernatant was added to 800 µl of heprainized saline and the absorbance was measured at 610 nm using a Bausch and Lomb (Rochester, NY, USA) Spectronic 88 spectrophotometer. A subsample of plasma from the initial blood sample, taken before injection of Evan's Blue, was used to create a blank for zeroing the spectrophotometer for each hatchling's measurement.
A standard curve (r2=0.93) relating absorbance to
Evan's Blue concentration was generated using plasma from four additional
hatchlings. Blood volumes were calculated from the measured Evan's Blue
concentrations according to the methods in El-Sayed et al.
(1995).
Statistical analyses
Linear regressions of parameters on egg mass and yolk-free hatchling mass
were carried out using SPSS 11.0. Additionally, loglog regressions were
performed on data to determine if component masses varied in simple linear
proportion to body mass (slope of loglog regression, b=1.0) or
if component masses showed a positive (b>1.0) or negative
(b<1.0) allometry with egg mass or hatchling mass. The regressions
were considered to vary in simple proportion to body mass if the 95%
confidence interval of the slope of the loglog regression included 1.
In order for the loglog relationship to hold true the intercept of the
untransformed data must pass through the origin. Prior to loglog
transformation all relationships were examined using non-linear power fits
(y=y0+axb; Sigmaplot 8.02) to
the untransformed data. The 50% confidence interval for the intercept
(y0) was used to determine if it differed significantly
from zero. Loglog regressions were carried out on data when the
intercept was not significantly different from zero. A significance level of
P<0.05 was adopted for all regressions. Linear regression
equations are provided in the figure legends and when determined allometric
slopes are provided in the text. All values are presented as means ±
S.D. except for the slopes of the loglog regressions, which
are presented as the slope (b) ±95% confidence interval.
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Results |
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The relative contribution of albumen and yolk to the eggs did not vary with
initial egg mass. Slopes (b) of the loglog regressions of log
albumen wet mass (b=1.04±0.13; r2=0.87),
log dry albumen mass (b=1.22±0.22;
r2=0.75), log yolk wet mass (b=0.93±0.14;
r2=0.82), and log dry yolk mass
(b=1.05±0.18; r2=0.76) against log initial
egg mass were not significantly different from 1. As a result, fraction of
yolk in the contents (0.47±0.03), typically correlated with
developmental maturity of hatchlings
(Sotherland and Rahn 1987),
did not change (F1,47=0.49; P=0.49) with egg
mass.
Water and solid content of eggs increased with egg mass (Fig. 2), but the fraction of water and solids did not vary significantly over the range of egg masses examined. Water in eggs (343.02±46.21 g; N=45) increased significantly (F1,43=648; P<0.001) with egg mass, as did the solid content of eggs (163.97±25.84 g; N=45; F1,43=245; P<0.001). Approximately 71% of water in eggs was found in the albumen (244.56±36.70 g; N=47), which was composed of an invariant fraction of water (0.90±0.01; F1,45=1.8; P=0.19). Similarly, neither the fraction of water in the yolk (0.42±0.03; N=47) nor the overall fraction of water in the eggs (0.68±0.02; N=45) changed significantly with egg mass. However, the total amount of water in the yolk (99.93±13.84 g; N=47) increased significantly (F1,45=66; P<0.001) with egg mass as did the total amount of water in the albumen (F1,45=265; P<0.001).
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Gas exchange of near-term embryos
Metabolic rate (107.8±11.6 ml O2 h1;
N=15) of pre-pip embryos, measured on day 46, was positively
correlated with initial egg mass Me
(F1,13=7.7; P=0.016;
O2=0.11Me+33.9;
r2=0.37) and with the yolk-free mass of the hatchlings
Mh when they emerged from the same eggs
(F1,13=14; P=0.003;
O2=0.20Mh+48.8;
r2=0.51). Metabolic rate scaled with a negative allometry
with egg mass (b=0.56±0.41) and yolk-free hatchling mass
(b=0.50±0.25). In a separate set of eggs, eggshell water vapor
conductance (447.4±76.7 mg kPa1
day1; N=14;
GH2O=0.09Me+4.5;
r2=0.51) increased significantly
(F1,13=9.4; P=0.01) with initial egg mass. In
contrast, pre-pip air cell PO2 (15.0±0.8
kPa; N=8) did not vary significantly with egg mass
(F1,6=2.4; P=0.17) (not shown).
Hatchling morphology and composition
Hatchling mass (yolk-free hatchling plus residual yolk) increased with egg
mass (Fig. 3). Hatchling mass
(403.59±45.67 g; N=48) increased significantly
(F1,46=214; P<0.001) with egg mass, as did
yolk-free hatchling mass (303.47±37.06 g; N=48;
F1,46=83.6; P<0.001) and residual yolk mass
(100.13±23.96 g; N=48; F1,46=14.3;
P=0.001). Dry mass of yolk-free hatchling (80.76±11.73 g;
N=45) and dry mass of residual yolk (57.13±13.93 g;
N=46) also increased significantly (F1,43=32.8;
P<0.001 and F1,44=17; P<0.001
respectively) with egg mass. Hatchling mass increased in simple linear
proportion to egg mass. The slopes (b) of the loglog
regressions of log yolk-free hatchling wet mass (b=0.96±0.20;
r2=0.66) and log yolk-free hatchling dry mass
(b=0.94±0.29; r2=0.47) against log initial
egg mass were not significantly different from 1.
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Large hatchlings were composed of more water and solids than were small hatchlings (Fig. 2), but the fraction of water in hatchlings remained unchanged regardless of hatchling size. Mass of water in yolk-free hatchlings (225.20±27.2 g; N=44) increased significantly (F1,43=747.2; P<0.001) with mass of yolk-free hatchlings, but the fraction of water in those hatchlings (0.74±0.02; N=44) did not vary significantly (F1,43=0.05; P=0.82) with hatchling mass. Mass of solids in hatchlings (i.e. dry mass of yolk-free hatchling) also increased significantly (F1,43=120; P<0.001) with hatchling mass.
Yolk consumed by developing embryos, i.e. difference between mass of the dry yolk (estimated using measured initial egg mass and the equation for dry yolk mass provided in Fig. 1) and measured mass of residual yolk remaining in the yolk-sac (83.64±14.44; N=46) increased significantly (F1,44=48.4; P<0.001) with yolk-free hatchling mass (Fig. 4A). The combination of initial yolk mass increasing with egg mass and yolk consumed increasing with yolk-free hatchling mass yielded a constant residual yolk mass across all hatchling masses (mean dry residual yolk 57.8±14.6). However, the statistical residuals from regressions of yolk-free hatchling dry mass and residual yolk dry mass on egg mass revealed that, independent of initial egg mass, larger hatchlings had less residual yolk upon hatching than smaller hatchlings (Fig. 4B).
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Linear dimensions of heavier hatchlings were greater than those of lighter hatchlings (Fig. 5). Length of both the right tibiotarsus (70.44±3.79 mm; N=48; F1,46=107.12; P<0.001) and culmen (36.78±1.90 mm; N=48; F1,46=9.6; P=0.003) increased significantly with yolk-free hatchling mass.
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Wet and dry masses of heart, liver, and gizzard all increased significantly with yolk-free hatchling mass (Table 1).
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Hematology of hatchlings
Blood volume (27.8±7.0 ml; N=11), which constituted an
essentially constant proportion (approximately 9.2%) of the yolk-free
hatchling mass, increased significantly (F1,9=12;
P=0.007) with yolk-free hatchling mass
(Fig. 6). The increase in blood
volume was proportional to the increase in yolk-free hatchling mass
(b=1.05±0.46; r2=0.69). However, none of
the other blood parameters measured [osmolality (304.6±15.6 mOsm
kg1; N=44), hematocrit (38.4±4.1%;
N=45), or hemoglobin concentration (12.9±1.8 g%;
N=44)] varied significantly with yolk-free hatchling mass
(Fig. 6).
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Discussion |
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Yolk and albumen in eggs
One measure of parental investment in bird eggs typically is expressed as
the fraction of yolk in the contents (FYC) of eggs. Emus in this study laid
eggs containing nearly 50% yolk (FYC=0.47), which is within the range of yolk
content for precocial birds but larger than that predicted for precocial eggs
of the same mass. Sotherland and Rahn
(1987) examined the
relationship between egg mass and energy content for a wide variety of birds
and found that FYC for precocial species ranges from 0.32 to 0.69. Based on
the equation for yolk content in precocial species
(Sotherland and Rahn, 1987
),
we predicted the FYC for an average sized emu egg (512 g wet contents) to be
0.39, which is less than the FYC of emu eggs measured in this study. Thus,
female emus provision their eggs with relatively more yolk and less albumen
than would be predicted for a typical large precocial egg. If we compare emu
eggs with those of closely related species, emu eggs tend to have a larger FYC
than either the ostrich Struthio camelus (1.2 kg egg, FYC=0.38;
Romanoff and Romanoff, 1949
)
or cassowary Casuarius casuarius (546 g egg, FYC=0.42;
Carey et al., 1980
). This
finding is not surprising, however, because the incubation period of the emu
is longer than that of the ostrich, suggesting that emu embryos require more
energy than ostrich embryos to complete incubation. In contrast, emu eggs have
a lower FYC than the smaller kiwi eggs (Apteryx australis; 440 g egg,
FYC=0.61; Reid, 1971
;
Calder et al., 1978
), which
has an incubation period about 25 days longer than the emu.
Another related comparison among species entails examining the contribution
of albumen and, therefore, water (albumen in all bird eggs is about 90% water;
Sotherland and Rahn, 1987) to
avian egg contents. We suggest here that always focusing on yolk and FYC
diverts attention from albumen and its important contributions to embryo
development and hatchling phenotype. The fraction of albumen in the contents
(FAC=1FYC) of eggs is very high (about 80%) in altricial species and
drops to less than 50% in more precocial species. Emu egg contents are about
half albumen (FAC=0.53), and at the middle of the range observed in ratites,
where FAC varies between 0.4 (kiwi) to 0.6 (ostrich).
Scaling of egg composition
Maternal investment in avian eggs varies both interspecifically and
intraspecifically in two ways. First, the absolute size of eggs and egg
contents can vary among and within species. Second, the relative contribution
of yolk, albumen and shell to the mass of an egg can vary with egg size and
with maturity of neonate at hatching.
Intraspecifically, emu eggs exhibit isometric scaling between egg size and
all egg components. Large eggs contained more yolk and albumen
(Fig. 1) as well as water and
solids (Fig. 2) than small
eggs, but yolk and albumen mass increased isometrically with egg size; the
slope of loglog regressions of these components on egg mass did not
differ significantly from 1. Thus, emu eggs in this study followed the
precocial pattern (Williams,
1994) where eggs of all sizes had the same relative amount of yolk
and albumen.
A number of studies have examined intraspecific variation of egg
composition and have revealed patterns of how yolk and albumen content vary
with egg size along the altricialprecocial continuum
(Sotherland et al., 1990;
Williams, 1994
;
Hill, 1995
;
Carey, 1996
). For most species
of birds, the vast majority of which are altricial, variation in albumen mass
accounts for most of the variation in egg mass, but yolk contributes more to
variation in egg mass as FYC increases toward the precocial end of the
altricialprecocial continuum
(Sotherland et al., 1990
).
Williams (1994
) reviewed 22
studies that had examined intraspecific variation in egg components and found
that only half of these studies revealed an isometric relationship between egg
size and either yolk or albumen content. Hill
(1995
) found that wet albumen
mass and wet yolk mass tended to scale isometrically with egg mass in
precocial species, whereas in altricial species albumen showed positive
allometry (b>1.0) and yolk showed negative allometry
(b<1.0). Thus, it seems that altricial species change egg size by
increasing the amount of albumen while keeping yolk content relatively
constant, whereas precocial species tend to alter egg size by increasing both
yolk and albumen content with an increase in egg mass. Further support for
this pattern has been observed in precocial wood ducks Aix sponsa
(Kennamer et al., 1997
) and
ruddy ducks Oxyura jamaicensis
(Pelayo and Clark, 2002
),
which lay eggs having yolk and albumen varying isometrically with egg mass,
and in altricial great tit Parus major eggs, in which much of the
variation in egg mass is attributable to variation in albumen mass
(Lessells et al., 2002
).
Egg size and hatchling size
The developing emu embryo may partition the maternal investment of energy
and nutrients into growth and maintenance of the developing body or into
residual yolk. The energy and nutrients invested in an egg by a female that
the embryo uses for growth and maintenance are parental investment in
embryogenesis, whereas energy and nutrients left as residual yolk or hatchling
fat deposits comprise parental investment in care of the hatchling
(Congdon, 1989). Increased
parental investment in larger emu eggs
(Fig. 1) yielded larger
hatchlings (Fig. 3) that tended
to contain similar amounts of residual yolk as smaller hatchlings due to the
fact that the larger hatchlings consumed more of their yolk during incubation
(Fig. 4). Increased hatchling
size is attributable to increased total water content
(Fig. 2), increased dry mass
(Fig. 2), and increased
structural size as measured by the tibiotarsus and culmen lengths
(Fig. 5). Heart, liver and
gizzard masses were also larger in hatchlings from large eggs
(Table 1). Thus, the increased
maternal investment was used by the developing embryo for embryogenesis to
yield a larger hatchling that had the same level of post-hatching care in the
form of residual yolk, suggesting that egg size can be equated with egg
quality in emus.
In contrast with our findings here, a review of the literature by Williams
(1994) found that larger bird
eggs produce heavier hatchlings, but not necessarily structurally larger
hatchlings. However, many of the studies of the relationship between mass and
structural size in hatchlings examined only hatchling mass including residual
yolk and concluded that hatchlings from larger eggs were heavier because they
contained more residual yolk and not because they were structurally larger
(Williams, 1994
). Ankney
(1980
) found a significant
positive relationship between egg size and length of the tarsus and culmen of
lesser snow goose Anser caerulescens hatchlings, but the relationship
between hatchling size and linear dimensions was not reported. Larger eggs
laid by the thick-billed murre produced heavier hatchlings, but this was due
mainly to increased water content or residual yolk and not due to increased
linear dimensions (Birkhead and Nettleship,
1982
). In a number of alcid species, most of the variance in
hatchling mass in relation to pipped egg mass was attributed to differences in
residual yolk rather than increased water content or dry hatchling mass
(Birkhead and Nettleship,
1984
). In the king eider Somateria spectabilis, large
eggs produced larger hatchlings with larger wet and dry breast and leg muscle
mass than the hatchlings produced from small eggs
(Anderson and Alisauskas,
2002
). However, the relative structural size of larger king eider
hatchlings was less than that of small hatchlings. In the altricial blackbird
Turdus merula, large eggs produced both heavier and larger hatchlings
(Magrath, 1992
).
Emu hatchlings had a large residual yolk, which was positively correlated with initial egg mass (Fig. 3) but not yolk-free hatching mass. Female emus provisioned eggs with enough yolk to support development and maintenance of embryos and to provide sufficient residual yolk to support activity and survival after hatching. By factoring out the effect of egg mass on both residual yolk dry mass and yolk-free hatchling dry mass (Fig. 4B) we found that larger-than-average hatchlings, at any egg mass, have less residual yolk than smaller-than-average hatchlings. Therefore, there appears to be a trade-off for the developing embryo: produce more tissue and hatch with less residual yolk or hatch smaller with more residual yolk.
In general, emu hatchlings have more residual yolk, as a fraction of the
whole hatchling, than many of the other avian species studied
(Vleck and Vleck, 1996).
Whereas precocial hatchlings retain residual yolk of 0.150.18 of their
wet mass and 0.28 of their dry mass
(Carey, 1996
;
Vleck and Vleck, 1996
), emu
hatchlings in our study had residual yolk amounting to 0.25 of their wet mass
and 0.42 of their dry mass. There is also sizeable residual yolk in the
ostrich, accounting for 0.29 of wet mass and 0.56 of dry mass
(Gefen and Ar, 2001
). The
ostrich and emu are both ratites, suggesting that members of this clade have
noticeably high parental investment in hatchling care and residual yolk.
Though we did not examine survivorship consequences of the levels of
residual yolk measured here, it is plausible that the large amount of residual
yolk we measured would influence early growth and survival of these hatchlings
because adult emus do not feed the young
(Davies, 1975). Parental
investment in hatchlings via yolk can provide hatchlings with energy
used to grow and sufficient residual yolk (i.e. parental investment in care),
which serves as a post-hatching source of nutrients and energy that can affect
survivorship, especially during times of nutritional stress. A number of
studies of precocial species (Kear,
1965
; Ankney, 1980
;
Peach and Thomas, 1986
;
Thomas et al., 1988
;
Slattery and Alisauskas, 1995
;
Visser and Ricklefs, 1995
;
Dawson and Clark, 1996
;
Nager et al., 2000
;
Anderson and Alisauskas, 2001
)
have shown that an increase in residual yolk, correlated with increased egg
size, results in increased hatchling survival under limited food
conditions.
Water relations and hatchling mass
Water loss from avian eggs during incubation and metabolic water production
by the embryos occur at rates that cause the hydration of egg contents at the
end of incubation to be similar to that at the beginning of incubation
(Ar and Rahn, 1980); these
coincident rates also cause hatchlings and the eggs from which they emerge to
have similar water contents (Sotherland
and Rahn, 1987
). Emu eggs contained on average 68% water, which
comprised 74% of the hatchlings they produced
(Fig. 2). Both of these values
are in close agreement with the water content of precocial eggs and hatchlings
(Sotherland and Rahn, 1987
).
There was an isometric increase in the water content of both the egg and the
yolk-free hatchling with an increase in initial egg mass and yolk-free
hatchling mass (Fig. 2); a
similar relationship was observed in the dry mass of eggs and yolk-free
hatchling solids (Fig. 2).
Japanese quail Coturnix coturnix hatchling water content scales
isometrically with egg size, but the proportion of water in laughing gull
Larus atricilla chicks increases with a positive allometry such that
larger hatchlings are composed of more water
(Ricklefs et al., 1978
).
The quantity of water in avian eggs, found mainly in the albumen, has a
significant influence on the mass of developing embryos and hatchlings.
Variation in emu hatchling mass is attributable in part to variation in mass
of water in yolk-free hatchlings (Fig.
2). Studies examining the effects of water loss from eggs during
incubation have shown that differences in wet embryo mass tend to be
correlated with water content of the embryo and that eggs losing the most
water tend to produce embryos with the lowest mass
(Davis et al., 1988;
Tullett and Burton, 1982
).
Removing albumen from chicken Gallus gallus eggs caused a reduction
in hatchling size (Hill, 1993
;
Finkler et al., 1998
) and
resulted in hatchlings with a reduced yolk-free wet body mass
(Finkler et al., 1998
). Though
hatchlings emerging from eggs from which albumen had been removed were smaller
(i.e. length of the tibiotarsus was shorter), much of the decrease in wet body
mass was attributed to the presence of less water in the smaller hatchlings.
The dry yolk-free body mass of hatchlings from control eggs was not different
from that of hatchlings emerging from eggs from which albumen had been removed
(Finkler et al., 1998
). Thus,
water availability in eggs may be one of the main determinates of yolk-free
hatchling mass in precocial species. A similar relationship between water
content and hatchling mass has been observed in turtle eggs, where increased
levels of water in eggs result in increased hatchling and organ sizes
(Packard, 1999
; Packard et
al., 1987
,
2000
;
Packard and Packard,
2001
).
Finkler et al. (1998)
postulated that some of the observed variation in body mass, correlated with
variation in water mass, might be accounted for by variation in extracellular
liquid volume, including blood volume. Blood volume of emu hatchlings
increased isometrically with hatchling size
(Fig. 6), but this increase in
blood volume was not accompanied by variation in other hematological
parameters (Fig. 6). Thus, a
portion of the water found in larger emu hatchlings appears in a larger volume
of blood
Metabolic rate, eggshell conductance, and air cell PO2
Maximum metabolic rates of bird embryos and their eggshell conductance are
typically matched in such a way that levels of respiratory gases in the air
cell vary over an amazingly narrow range at the end of incubation, regardless
of egg size, length of incubation, or degree of hatchling maturity
(Rahn and Paganelli, 1990).
Metabolic rates of developing emu embryos reported here, which reach a plateau
about 8 days prior to hatching (Vleck et
al., 1980
), agree with those reported previously by Beutel et al.
(1983
) and Vleck et al.
(1980
), and were significantly
correlated with initial egg mass and yolk-free hatchling mass. Larger eggs
produced larger hatchlings, and, not surprisingly, near-term embryos from
larger eggs had greater overall metabolic rates than those from smaller eggs.
Because metabolic rate of emu embryos and water vapor conductance of the eggs
in which they developed covaried with egg mass,
PO2 in air cells of emu eggs did not vary with
egg mass and were in close agreement with values calculated by Vleck et al.
(1980
). Using Fick's law of
diffusion and our measurements of shell gas conductance and metabolic rate, we
calculated that air cell PO2 should have
averaged about 14.3 kPa, which is less than 5% different from the values
measured.
Consequences of egg size variation
Emu egg size influenced the morphological and physiological phenotypes of
the resulting hatchlings. To summarize the consequences of emu egg size
variation we used the regressions from the results and Figs
1,
2,
3,
4 to predict a number of
parameters for a small emu egg (450 g) and a 44% larger emu egg (650 g;
Table 2). In support of our
hypotheses, hatchling phenotypic characters measured here were 3851%
larger in hatchlings from the larger egg and scaled proportionally with egg
size. Using the energy content of dry solids in eggs (29 kJ
g1; Sotherland and Rahn,
1987), we predict that a female emu would invest 3596 kJ in a450 g
egg, whereas a 650 g egg would contain 48% more energy (5336 kJ). If
mass-specific costs of producing eggs were the same for eggs of all sizes
within a species, then a female ovipositing a 650 g egg would allocate nearly
50% more energy per egg than a female ovipositing a 450 g egg. We hypothesized
that increased maternal investment in the form of increased yolk would result
in larger hatchlings. Embryos in larger eggs received more parental investment
in embryogenesis, which allowed them to consume more yolk solids and grow
larger during incubation but have sufficient yolk reserves to support them as
hatchlings (Table 2).
|
Parameters that did not scale isometrically with egg mass were near term-embryo metabolic rate and air cell PO2 (Table 2). Larger eggs had higher metabolic rates, but metabolism did not increase to the same extent with increases in egg mass as with hatchling body mass, suggesting that embryos in larger eggs may have responded more to limitations imposed by a relatively low eggshell conductance.
Our investigation revealed that female emus vary parental investment in their offspring through changes in the absolute amount of yolk and albumen in eggs, while keeping the proportion of the two constant. Embryos in larger eggs developed into hatchlings that were heavier and structurally larger than embryos in smaller eggs (i.e. greater parental investment in embryogenesis yielded larger hatchlings), but hatchlings from eggs of all sizes contained the same amount of residual yolk (i.e. emus invest similar parental care, via eggs, in their hatchlings). We do not know, however, if embryos that `find' themselves in larger eggs, containing more resources and a larger gas exchange surface, respond by growing more or if embryos that would normally grow more are put into larger eggs. Further research is needed to elucidate more clearly how maternal phenotypes affect developmental trajectories and ultimately fitness.
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