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Department of Biological Sciences, University of North Texas, Denton, Texas 76203-5220
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ABSTRACT |
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 Top
 Abstract
 Introduction
Material and methods
Results
Discussion
References
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Body mass, length, oxygen consumption
(
O2)
and heart rate
(fH) were
measured in "embryos" (prior to hatching), "larvae" (days
10-20),
"juveniles" (days 30-70 in
10-day intervals), and "adults"
(day
100) of the zebrafish
Danio
rerio. Fish were chronically reared at
either 25, 28, or 31°C and then acutely exposed to hypoxia at
different developmental stages. We hypothesized that at any given rearing and measurement temperature,
D.
rerio would maintain O2
at lower ambient PO2 [i.e.,
have a lower critical partial pressure
(Pcrit)] as development
progressed and that at any given developmental stage individuals reared
and measured at higher temperatures would show a more pronounced
hypoxic bradycardia. O2
in normoxic fish at 28°C peaked at ~40
µmol · g
1 · h1
at day
10, thereafter falling to 4-5
µmol · g1 · h1
at day
100. The
Q10 for
O2
was 4-5 in embryos, falling to 2-3 from
day
10 to
day
60 and rising again to 4-5 at
day
100.
Pcrit at 28°C was ~80 mmHg
in embryos but decreased sharply to 20 mmHg at 100 days, supporting the
hypothesis that more mature fish would be better able to oxygen
regulate to lower ambient PO2 levels.
Pcrit increased sharply with
measurement temperature. Heart rate
(fH) at
28°C increased from about 125 beats/min in embryos to a peak of
~175 beats/min at days 10-30
and then fell to ~130 beats/min by
day
100. Unlike for
O2,
the Q10 for
fH was more
constant at 1.2-2.5 throughout development. Hypoxic exposure at
any temperature had no effect on
fH until
~day
30, after which time a hypoxic
bradycardia was evident. As evident for
O2,
the bradycardia in older larvae was more profound at higher
temperatures. On the assumption that bradycardia is indicative of
hypoxic stress, the increasing prevalence of a hypoxic bradycardia in
older, warmer individuals supports the hypothesis that increasing
hypoxic susceptibility with development would be exacerbated by
increasing temperature. Collectively, these data indicate that the
ability to regulate O2
and fH in
response to the compounding demands of increased temperature
and/or decreased oxygen availability first develops after ~20
days in D.
rerio and, thereafter, the ability to maintain O2
in the face of ambient hypoxia progressively builds through to
adulthood. Additionally, the temperature responses of metabolism and
heart rate differ substantially at different phases of development, suggesting a loose coupling between the respiratory and cardiovascular systems, at least early in development.
development; hypoxia; embryos
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INTRODUCTION |
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Top
Abstract
Introduction
Material and methods
Results
Discussion
References
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THE ZEBRAFISH, Danio rerio (Brachydanio), is a tropical Cyprinid teleost fish that recently has been the focus of increasing numbers of developmental studies. Physiological interest in this species has been spurred, in part, by the relative ease with which cardiovascular and other mutants can be induced by chemomutagenesis (see Ref. 8). However, our understanding of the basic physiology of D. rerio, in particular the normal processes that occur during development, lags far behind that of other vertebrate models for development (see Ref. 2). This paucity of information for Danio, combined with a fragmentary knowledge of developmental physiology in fishes generally (28, 29) argues for basic studies of developmental physiology in this species.
Temperature and hypoxia, known to fluctuate in the environments in which lower vertebrates develop, can profoundly affect the physiology and morphology of lower vertebrate embryos (for review, see Refs. 5, 23, 28). Moreover, the effects of chronic exposure to environmental challenge may be quite different from acute effects, given the considerable developmental plasticity of embryonic and larval organ systems (see numerous chapters in Ref. 2). D. rerio presents an excellent model for investigating environmental influences on physiological development in lower vertebrate embryos and larvae. Because zebrafish reach adulthood in under 100 days (37), the impact of environmental perturbation can be assessed over relatively short experimental periods.
The purpose of this study was to investigate oxygen consumption
(O2)
and heart rate
(fH) as a
function of acute hypoxic exposure throughout development in
D.
rerio chronically reared at 25, 28 (the preferred temperature), and 31°C. Our experiments were
designed to test the hypotheses that at any given rearing and
measurement temperature, older
D.
rerio would be able to maintain O2
at lower ambient PO2 [i.e.,
have a lower critical partial pressure
(Pcrit)] and that, at any
given stage, individuals reared and measured at higher temperatures
would show a more pronounced hypoxic bradycardia. These experiments
have additionally provided new data on the changes in the temperature
responses of body mass, length, metabolism, and
fH over a very
wide developmental span in D.
rerio.
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MATERIAL AND METHODS |
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Top
Abstract
Introduction
Material and methods
Results
Discussion
References
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Animals
Newly laid eggs (<24 h) of D. rerio obtained from commercial suppliers (Scientific Hatcheries, Huntington Beach, CA) were reared in the laboratory under 14:10-h light-dark conditions during the period January-July of 1996. Three separate groups were maintained: one reared at 28°C, the preferred temperature for this species (37); one group reared at 25°C; and a final group reared at 31°C. Larvae,1 juveniles, and adults from all groups were fed twice daily, except for the 24-h period preceding measurement, when fish were fasting.O2
and fH were
measured in unhatched embryos (<72 h) and at ages 10, 20, 30, 40, 50, 60, 70, and 100 days. Body mass (wet) and length (snout to tail) was
measured immediately after each series of metabolic measurements and
was additionally measured in each group at 150 and 200 days of
development. Accurately measuring the extremely small body mass of
embryos presented particular difficulties. Embryos were first removed
from the egg capsule, leaving the yolk sac intact. The collective
weight of 10 individual embryos was then determined using a
microbalance, and the average weight of that group was calculated. This
procedure was repeated 10 times, providing 10 separate body mass
measures from which was calculated an average and SE for embryos.
Body length measurements were made to the nearest 0.1 mm using a dissecting microscope fitted with a calibrated scale in one eyepiece. Body length was not measured in the embryos, which are normally in a highly curled natural posture within the egg.
O2
O2
determinations were made at the rearing temperature of each group (25, 28, and 31°C) over the range from unhatched eggs to
day
100. To determine
O2,
animals were placed in closed respirometers created from glass syringes
containing aerated water. Respirometers were covered with opaque
material to minimize visual disturbances to the fish and were placed in
a temperature-regulated water bath. Syringe water volume was matched to
fish biomass within the syringe to regulate the rate of oxygen
depletion during the experiments (see below). Because of the extremely
small size of the embryos and, to a lesser extent,
day
10 larvae, measurements of
O2
in individuals were not practical. Consequently, for these two early stages a known total mass comprising several individuals of the same
developmental stage was placed in each respirometer, with the
overall calculated
O2
within the syringe divided by the number of individuals to generate a
single point for mass-specific O2
for an individual fish at that developmental stage. Thus each
O2
measurement derived from one respirometer run was considered one data
point for that developmental stage, regardless of the number of animals
within the syringe that had contributed to the calculation of that
datum. For subsequent stages of days
20-100, only individual fish were placed in each
respirometer and, again, each
O2
measurement was considered one data point.
All fish were allowed a 2-h acclimation period in the respirometers
before measurements. Although this period is unlikely to have
alleviated all stress from handling in this very active species of
fish, preliminary experiments indicated that there were no significant
differences in
O2
measured after 1, 2, or 3 h of acclimation, so at least the fish were
in a relative steady state if not completely acclimated. At the end of
the acclimation period, one-half of the water in each respirometer was
very gently and slowly exchanged with air-saturated water (normoxia),
taking great care not to disturb the fish within. Before commencement of the first
O2
measurement period, the initial water
PO2 (in mmHg) of each respirometer
was measured in a 100-µl water sample injected directly from the
respirometer into a water-jacketed O2 electrode (Microelectrodes)
connected to a Radiometer pHM71 gas meter. At this time, the initial
water volume was also recorded. Water
PO2, which then began to decline over
time due to the
O2
of the fish, was measured in each respirometer over seven or eight
successive 20-min intervals. On the basis of
O2 data from preliminary experiments on each developmental stage, different-sized syringes and/or different initial volumes of
water were employed to ensure that the
O2
by the fish in each respirometer volume produced a
PO2 drop of no more than 15 mmHg
during the 20-min measurement period. This ensured that each animal
contributed one point to the 15-mmHg-wide
PO2 "bins" used in subsequent analysis (see below).
Mass-specific
O2
for individual fishes was calculated from the rate of decrease in water
PO2 in the respirometer, volume of
the respirometer before water sample, elapsed time between successive
measurements, mass of the fish in each respirometer, and
O2 capacitance of water at
measurement temperature.
O2
data, including the Pcrit for each
developmental group, were plotted and analyzed as described in
Ref. 11. Despite matching of respirometer volume to fish
biomass, subtle differences in metabolism between identically aged fish
might produce, say, a water PO2 of
130 mmHg in one respirometer at the end of the 20-min measurement period but a water PO2 of 124 mmHg in
another respirometer. Thus, to allow for simplification of calculation
as well as greatly enhanced clarity in graphical presentation, each
O2
value successively generated as hypoxia developed during a respirometer
run was assigned to one of eight 15-mmHg-wide bins described by a mean
PO2 of 130, 115, 100, 85, 70, 55, 40, or 25 mmHg. This process resulted in an
O2
value being generated for each animal for each
PO2 bin. Mean
O2
values ±SE for a given stage at a given temperature were then
calculated by averaging each of the 10 individual
O2 values for each fish in each bin. In unusual cases, a particularly low
rate of metabolism by an individual fish would result in two O2
values calculated from two successive 20-min measurements falling
within a single PO2 bin. In this
instance, the two
O2
values were averaged to produce a single estimator for that fish in
that bin.
The Pcrit for a specific
temperature group at a specific developmental stage was calculated from
the intersection of two lines (determined by least-squares linear
regression) passing through the mean
O2
values representing each PO2 bin
(11). Initially, one line was plotted through the obviously decreasing values of mean
O2
at the lower PO2 values, and the other line was plotted through the obviously unchanging values of mean
O2
at high PO2 values just slightly
below air saturation. Then the mean values at the intermediate
PO2 values were alternately included
in the data for the upper line and then lower line until the combined
values of the r2
for each line were minimized, indicating the best overall fit of the
two lines through all mean values for
O2.
The intersection of the two best-fitting lines was determined to be the
Pcrit. The margin of error for
this method of Pcrit calculation
is estimated to be ±4 mmHg, which is, for example, just 5% of the
Pcrit difference calculated
between 25- and 31°C-acclimated embryos.
fH
fH was measured in a range of developmental stages of D. rerio reared at 25, 28, or 31°C. fH measurements in embryos and day 10 larvae were made on individuals placed in water-filled holding chambers constructed from petri dishes. Water flow was maintained through the chambers, with both water temperature and PO2 constantly regulated and monitored. Because embryos and early larvae are translucent, fH was measured visually through the body wall. Video recordings of the in vivo beating heart were made using a Javelin JE3010 color camera and were subsequently analyzed (after Ref. 24). During the acclimation period, water PO2 was maintained at 150 mmHg and fH was measured at normoxic PO2. Water PO2 was then lowered over a 5-min period to 105 mmHg and held for 30 min before fH was again measured. This process was repeated in sequence for PO2 values of 60, 30, and 10 mmHg. Water PO2 flowing into the holding chamber was controlled by equilibration with gas produced by a GF-3 gas mixing flowmeter (Cameron Instruments).Individual older larvae (
20 days), juveniles, and adults were placed
in a 15-mm-diameter glass tube irrigated at a rate of 30 ml/min with
water of appropriate temperature and
PO2 (regulated as described above for
embryos and young larvae). Although fish from 20 days to adult were no
longer translucent, appropriate obliquely directed fiber-optic lighting
clearly revealed cardiac-induced pulsations of the ventral body wall,
which were recorded with a Sony AF charge-coupled device
video camera mounted vertically under the glass tube. Each individual
was allowed to acclimate overnight (~14 h) before measurements, and
the same protocol of hypoxic exposure and
fH measurement
described above for embryos and young larvae was employed. In all
experiments, a 1-min-long videotape segment was analyzed, which, for
the highest possible fH recorded
(~200 beats/min), would result in a margin of error of less than
±4 beats/min.
Differences in
O2,
fH, and
Pcrit as a function of development
and differences in
O2
and fH at the
same developmental stage as a function of
PO2, were individually assessed using one-way ANOVA performed by Sigmastat statistical software (Jandel; San
Rafael, CA). A significance level of 0.05 was used for all tests.
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RESULTS |
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Top
Abstract
Introduction
Material and methods
Results
Discussion
References
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Body Mass and Length
Changes in wet body mass and body length as a function of chronological age (0-200 days) and rearing temperature (25, 28, or 31°C) are presented in Table 1. Not surprisingly, there are significant disparities in both mass and length induced by rearing temperature. At any given chronological age up to ~60 days, both body mass and length were lowest at 25°C and highest at 31°C. The exception was embryos, in which rearing temperature had presumably not had time to create an effect. Interestingly, from about day 70 on, the 31°C-acclimated fish actually showed leaner body mass than 28°C-acclimated fish, although the warmer fish were longer in length. This may reflect the greater cost of maintenance at the highest rearing temperature.
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Interpretation of these mass and length data is highly complex (see DISCUSSION), and we made no attempt in this study to correct the measured chronological age in days to the developmental age, which is properly assessed using a variety of morphological, physiological, and biochemical markers. Consequently, the metabolic and heart rate data reported below compare fish of identical chronological age at the three different rearing temperatures.
Influences of Development and Rearing Temperature
O2.
O2
in unhatched embryos at 28°C, ~3.6 µl · g1 · h1,
increased >10-fold with hatching and initial growth, reaching a peak
of 39.4 µl · g1 · h1
at day
10 (Fig. 1).
O2
then decreased progressively to ~5.8 µl · g1 · h1
at day
60 and showed little additional change
during the next 40 days of development. Zebrafish groups reared at 25 and 31°C showed this same general pattern of change in
O2
with development (Fig. 1). The effect of development on
O2
was highly significant (P < 0.0001)
in all three groups, with the single exception of a nonsignificant
change between days
20 and
30 at 25°C.
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O2
at a rearing and measurement temperature of 25°C was significantly lower than at 28°C, which in turn was significantly lower than at
31°C (P < 0.05).
Q10 for
O2
was calculated over the intervals 25-28°C and 28-31°C
(Fig. 2,
top).
Q10 for
O2
over the range 25-28°C in all developmental stages was equal
to or greater than values measured over the range 28-31°C.
Q10 for
O2
showed a highly distinctive, U-shaped pattern of change with
development over both temperature ranges.
Q10 in embryos was relatively high at ~4-5, but the temperature sensitivity of metabolism fell
sharply with continued development to a
Q10 of 2-3 in larvae from
~10-60 days. However, Q10
increased once again to values of 4-5 in
day 100 adults.
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fH.
fH in embryos at
28°C was ~125 beats/min but climbed to ~175 beats/min by
day
10 (Fig.
3). From a peak at
day
20,
fH at 28°C declined to ~130 beats/min at day
50 and older.
fH at 25°C
was significantly lower than, and at 31°C significantly higher
than, fH values
at 28°C at all development stages. The effect of development on
fH was
significant in all three groups (P < 0.03 at 25°C, P < 0.05 at
28°C, and P < 0.0001 at
31°C). However, no significant (P > 0.05) change in
fH occurred after
60 days with the exception of day
70 at 25°C, which was
significantly, but only slightly, higher than at
day
60.
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Influence of Acute Hypoxia
O2
in embryos and young larvae (days
10-30) at all three rearing temperatures
declined significantly (P < 0.001)
from normoxic (control) values as acute hypoxic exposure became
progressively more severe (Fig. 4). Only
O2
at days 20 and
30 at 25°C showed no significant
(P > 0.10) effect of hypoxia. Figure
5 illustrates Pcrit as a function of development
and measurement temperature. Older larvae, juveniles, and adults were
capable of regulating O2
at normoxic levels, even in the face of severe hypoxia. These findings
supported our hypothesis that the ability to regulate oxygen
consumption increases with developmental stage.
Although the patterns of decline in
Pcrit showed similar patterns at
all three temperatures, the absolute value of
Pcrit at any given developmental stage was highly temperature dependent, especially early in
development. Day
10 larvae at 25°C, for example,
could maintain
O2
to a Pcrit of ~50 mmHg, but this
value increased to ~65 mmHg at 28°C and to >90 mmHg at
31°C.
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The influence of acute, graded hypoxia on
fH as a function
of development is shown in Figs. 6-8. Hypoxic exposure had no
significant effect on
fH in embryos and
days 10 and
20 larvae at any rearing temperature
(Fig. 6). In
day
30 juveniles there was no hypoxic bradycardia at 25°C, but at the two higher rearing temperatures a
bradycardia began to develop below a
PO2 of 30 (28°C) or 100 mmHg
(31°C) (Fig. 7). In both cases, the
bradycardia at the lowest tested PO2
was severe, with a drop in
fH of as much as
100 beats/min from normoxic values. This pattern, in which a
hypoxia-induced bradycardia became more pronounced as temperature
increased, similarly occurred in all groups from
day 40 to
day
100 (Figs. 7 and
8). Interestingly, in the most mature day
100 fish, a more profound level of
hypoxia was required to elicit bradycardia than at any of the juvenile
stages.
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On the assumption that the development of severe bradycardia indicates hypoxic stress, these data collectively support our hypothesis that at any given stage heart rate is more susceptible to hypoxic-induced change at warmer rearing temperatures.
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DISCUSSION |
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Top
Abstract
Introduction
Material and methods
Results
Discussion
References
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Body Mass and Length
Attempts to interpret development change as influenced by body temperature in poikilotherms will be fraught with pitfalls that stem from the deviation in chronological from actual developmental age. Clearly, for a given chronological age, zebrafish reared at 25°C are not as developmentally mature as zebrafish reared at 31°C, at least early in development. That is, there is a Q10 for development in larval fishes that impacts everything from body mass to energy assimilation, a fact that is widely recognized (see Ref. 27) but difficult to measure and even more difficult to extrapolate to experimental findings in growing animals reared at different temperatures. Rombough and others (22, 27) and Wells and Pinder (35) have attempted to circumvent this problem by reporting development as a function of "accumulated thermal units," or ACUs, which are calculated from the mean temperature multiplied by the number of days posthatch. Although this creative approach recognizes the problem inherent in measurements at different temperatures, it by itself cannot overcome the fact that the Q10 for development may be different over different temperature intervals (just as we found Q10 forO2
to differ between 25 and 28°C and between 28 and 31°C), making
less useful the simple arithmetic approach of multiplying time by
temperature, regardless of actual temperature. Moreover, calculating
ACUs cannot by itself correct for the fact that certain morphological,
physiological, and biochemical variables may each have different
Q10s, resulting in a larval or
juvenile animal that may have various organ systems at different stages
of development depending on rearing temperature! Our data for zebrafish
(Table 1) indicate that there is not a simple relationship between
temperature, body mass, and length, with, for example, older and warmer
fish being longer but also leaner. Similarly, Rombough's data on
energy assimilation, metabolic rate, and body mass in steelhead (27)
suggest that a fish larva at 6°C differs from one at 15°C by
more than a simple "van't Hoff equation" form of correction that
would require that all measured variables increase equivalently for the
9°C temperature difference.
We have elected in the present study not to introduce any corrections to our chronological ages for the zebrafish, on the assumption that no correction (i.e., presentation of the raw data) is at this point more appropriate than creating a correction for zebrafish that is grounded in the currently poorly understood thermal biology of this species. There will definitely be a difference in developmental age when comparing our data from different rearing temperatures at the same chronological age, in which, for example, fH at day 60 in a 25°C-reared fish may be more strictly comparable to fH at day 50 or even day 40 in a 31°C-reared fish. Yet the absolute differences between groups across time are so large that we feel our arguments and conclusions are not qualitatively affected. Obviously, understanding the full implications of temperature on development is an enormously complex problem that is beyond the scope of this study but which deserves additional comprehensive investigation in studies designed expressly for that purpose.
Metabolic and Heart Rate Patterns During Development
Mass-specificO2
in developing zebrafish showed an early peak followed by a sharp,
steady decline as development progressed toward adulthood. Direct
comparison of this pattern with published metabolic data for other fish
species during development is problematic, because prior studies have
typically focused extensively on the period of development equivalent
to just the first development interval (embryo to
day
10) of the eight intervals that we
measured. Consequently, very few data from a single study exist for the later larval, juvenile, and adult periods equivalent to our 10-day intervals through day
100. Nonetheless, sharply rising
mass-specific metabolism in the days after hatching similar to what we
observed in zebrafish appears typical of salmonids, which have probably been the most intensively studied (28, 31). In the Atlantic salmon,
Salmo salar, for example,
O2
at 10°C increases >10-fold from hatching (body wt ~30 mg) to
age 3 mo (body wt ~0.5 g). A similar early peak in metabolism occurs
in the steelhead, Salmo gairdneri
(27). This pattern of metabolic apex early in development persists over
a temperature range of 6-15°C but occurs about five times
later in chronological age in steelhead at the lower temperature (27).
An early peaking of mass-specific metabolism early in overall
development followed by a sharp decline has also been demonstrated for
anuran amphibians (see Refs. 5, 11) and reptiles (34, 38) and can be
calculated for the chick embryo using data from Refs. 15 and 26,
suggesting that this pattern of metabolic change may be a general
vertebrate pattern.
The initial rise in metabolic rate that occurs in zebrafish during the
first 10 days is likely associated with organogensis and the conversion
of egg yolk into new metabolizing biomass. Soon, however, the onset of
free swimming in zebrafish at about day 4 and of feeding a few days later could begin to influence metabolic
rate. The subsequent fall in
O2
in zebrafish after day
10 until
day
100 may be attributed in part to
allometry, as body mass rises nearly 400-fold during this period.
Developmental changes in
fH in
D. rerio approximately paralleled
those for
O2,
with fH peaking
at days
10-30 at all measurement temperatures and then falling with additional development. This particular developmental pattern in
fH differs
somewhat from some of the other teleost species that have been observed
(e.g., walleye, rainbow trout, Arctic char), where maximum
fH typically
peaks much earlier before or around hatching and then decreases with further development (12, 16-20, 29). Although different vertebrate species show considerable variation in
fH patterns
during development, there is a general tendency in lower vertebrates
for fH to rise sharply during early development to an apex and then subsequently decline (7). Qualitatively, then,
fH change during
development in the zebrafish does reflect that observed in other lower
vertebrates, although the timing of the apex is somewhat delayed. The
cause of these general changes in resting
fH during
development remains unknown, but, as for
O2,
may involve a complex interplay of early heart maturation followed by
the increasing influences of body mass that is increasing rapidly with
additional growth and development. Clearly, more detailed studies of
the relationship between body mass, metabolic rate, and
fH during
development in vertebrates are required to delineate the influence of
development per se from those resulting from the ubiquitous effects of
scaling on biological processes.
Influence of Temperature on Metabolism and Heart Rate
Typically, physiological processes show Q10 values of ~2, with temperature insensitivity revealed by values approaching 1, and enhanced temperature sensitivity revealed by Q10 values substantially >2 (25). As anticipated, rearing temperature had a profound effect on bothO2
and fH in
D. rerio. However, the analysis of the
Q10 relationship describing
temperature effects on these physiological processes revealed
intriguing patterns of change during development (Fig. 2). The
temperature sensitivity of
O2 declined sharply in early development, but increased again as the
animals approached maturity. Q10
values for metabolic rate in larval fish vary enormously, but of 21 species surveyed by Rombough (28), only 6 had values >4 while 9 had
values <2. Temperature sensitivity reflected in a high
Q10 is common in stenothermic animals at measurement temperatures outside of their narrow range of
preferred temperature. The high values of
Q10 of the present study suggest
that adult zebrafish are relatively stenothermal animals that live and
breed at rather constant temperatures in their natural tropical
habitat. Indeed, captive zebrafish rarely breed >31 or <25°C in
laboratory settings. Mortality at these temperatures is high and
surviving embryos show increased incidence of abnormal development (32,
37). The biochemical basis for these complex changes in
O2
responses to temperature during development in D. rerio remain unknown. However, almost all
temperature-related characteristics of metabolism ultimately derive
from the properties of key metabolic enzymes. Thus the changing
temperature sensitivity of metabolism during development in
D. rerio suggests complex and
interesting development-associated changes in isozyme populations and
warrants additional biochemical study.
The relationship describing Q10
for fH with
development in zebrafish does not resemble the U-shaped curve relating
Q10 for metabolism with
development. Indeed, Q10 for
fH ranged from
~1.2 to 2.5 over the temperature range of 25-31°C, a
temperature sensitivity for
fH that compares
favorably with very recent data on trout larvae (10-60 mg body
mass), which showed a Q10 of
~2.4 over the range 5-15°C (22). Why does this discrepancy
exist between Q10s for
O2
and fH in
zebrafish? Cardiac function in adult vertebrates is typically viewed as
tightly coupled to metabolism, which it supports through the transport
of nutrients, respiratory gases, and wastes. Accordingly, it might be
anticipated that temperature sensitivity of
fH would
generally reflect that of
O2
throughout development, yet such was clearly not the case for
zebrafish. Early in development teleost fishes rely heavily on
cutaneous gas exchange in support of
O2
(22, 24, 27-31, 35, 36). Because cutaneous gas exchange in very
small organisms does not rely on convective oxygen transport by blood,
but rather on direct diffusive supply of oxygen to metabolically active
tissues, there is no reason to assume that cardiovascular system
performance would be tightly linked to metabolism early in development.
Indeed, the tight "supply-and-demand" relationship between
cardiac output and metabolic demand for
O2 typical of mature vertebrates
is largely lacking in early trout larvae on the basis of the lack of
coupling of growth-induced increases in cardiac output and
O2
as these larvae increase in body mass (22). The apparent dissociation of cardiac and metabolic patterns of change in early development in
both zebrafish and trout is additionally supported by recent studies in
embryonic vertebrates showing that blood circulation and/or
convective blood oxygen transport is not required to maintain normal
metabolic rates in zebrafish embryos younger than
hour 108 (24), as well as in embryonic and
larval amphibians (6, 21, 33) and 3-5 day chicken embryos (W. Burggren, S. Warburton, and M. Slivkoff, unpublished). Thus it would
appear that metabolic and cardiovascular performance become more
tightly linked as development progresses, a sequence of events that
begs further description in any lower vertebrate.
Although a dissociation of cardiac and metabolic functions due to
cutaneous gas exchange occurs early in development, the lack of
correlation of Q10 in
O2
and fH in older,
maturing zebrafish remains intriguing, especially because there is a
tight link between cardiovascular performance and metabolism in adult
teleosts (3, 9). In the absence of data on the interplay between heart
rate and stroke volume, an explanation for the muted temperature
sensitivity of heart rate relative to
O2
in maturing zebrafish awaits further exploration of the comprehensive
cardiovascular response to temperature change.
Influence of Hypoxia on
O2
O2
in D. rerio was highly dependent on
both developmental stage and rearing temperature, as evident from
calculations of Pcrit. As would be
expected due to the significant O2
diffusion barrier presented by the chorion and the unstirred or poorly
stirred perivitelline fluid (27), unhatched embryos showed the highest
Pcrit and embryos at 31°C become oxygen conformers at just slightly below normoxic
O2 levels. In support of our
initial hypothesis, Pcrit declined
(i.e., the animals were able to regulate oxygen consumption at lower
PO2 values) as development
progressed. Rombough (27) has presented one of the very few other
studies to examine Pcrit in larval
fish. He reported that in larval steelhead (which develop much more slowly at much lower temperatures than zebrafish)
Pcrit at 15°C similarly
declines from a peak of ~140 mmHg (just under air saturation) at
hatching at 20 days postfertilization to a value of ~75 mmHg (1:2 air
saturation) after ~30 days postfertilization. A fall in
Pcrit with development has also
been documented in larvae of the anuran amphibian
Xenopus laevis, where
Pcrit at 20°C falls from near
air saturation in water breathing larvae to ~75 mmHg once air
breathing begins, terminating at a value of just 30 mmHg in adult frogs
(11).
As was evident for steelhead larvae (27), at any given developmental stage in zebrafish Pcrit was temperature sensitive, being highest at 31°C and lowest at 25°C. This reflects the higher tissue O2 demand and lower water O2 content at higher measurement temperatures. Nonetheless, the ability of zebrafish to obtain O2 from a hypoxic environment, particularly as older larvae, juveniles, and adults, is impressive, and the Pcrit of ~20 mmHg for day 100 zebrafish at 28°C indicates the potential for acute survival in very hypoxic waters.
It is tempting to declare one developmental stage of zebrafish as a
"better" or "more competent"
O2 regulator than another stage in
accordance with the contemporary trend of assuming that the lower the
Pcrit, the more capable is the
animal at the overall maintenance of its oxygen consumption in hypoxia.
However, a comparison of Pcrit
values across development is compounded by the profound differences in
mass-specific
O2
between younger and older fishes. For example, the
O2
in day
10 larvae is ~15-30 times
higher (depending on temperature) than in adults at
100 days, this despite the fact that the
early larvae are relatively lethargic compared with the normally
actively swimming adults. Thus the demonstrated ability for adult fish
to oxygen regulate to far lower levels of ambient
PO2 may as much reflect the
"advantage" of their much lower unit metabolic rate than any
inherent superiority of the oxygen- delivery mechanisms of adults
compared with larvae.
Influence of Hypoxia on fH
Hypoxic exposure caused complex changes in fH in D. rerio, the exact nature of which depended on developmental stage and, to a lesser extent, rearing temperature. Interestingly, embryos and early larvae showed virtually no hypoxic response in fH over the entire range of ambient PO2. The lack of a hypoxic bradycardia in early larval stages has also been noted for rainbow trout (12) and Arctic char (18). Similarly, very early larvae of anuran amphibians show little or no hypoxic bradycardia (1, 5, 10), although this capability appears with additional development. A hypoxic bradycardia developed only after ~20-30 days of development, suggesting that both cardiac control mechanisms and chemoreceptors for detecting oxygen have developed and are functioning.The severity of the bradycardia in zebrafish and the ambient PO2 at which it developed were related to measurement temperature, first appearing at higher PO2 values and being most severe at 31°C. It is unknown whether stroke volume changes in the same or opposite direction as fH during severe hypoxic exposure in zebrafish. In adult teleost fishes, bradycardia does not necessarily signify reduced cardiac output, because many fishes control cardiac output primarily through stroke volume (3, 9). However, larval trout regulate cardiac output primarily through changes in heart rate (22). Whether heart rate is the major modifier of cardiac output in most larval teleosts will require further investigation.
In conclusion, we initially hypothesized that
D. rerio would be more capable of
maintaining oxygen consumption as development progressed and that at
any given developmental stage, individuals reared and measured at
higher temperatures would be more susceptible (as evidenced by hypoxic
bradycardia) to a given level of acute hypoxia. These hypotheses have
been borne out by the present experiments. Our findings additionally
suggest that the direction and magnitude of
fH changes only
generally reflect those of
O2.
The lack of coupling of cardiac and metabolic patterns early in
development most likely reflects the heavy dependence on cutaneous gas
exchange by the larvae. However, the similar disparity in
temperature-induced heart rate and metabolic changes in older, maturing
fishes serves to highlight the need for more comprehensive
cardiovascular and metabolic data over a wide range of development if
we are to determine the changing interrelationships between cardiac
output, blood oxygen, and metabolism during ontogeny.
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ACKNOWLEDGEMENTS |
|---|
We are grateful to Brian Bagatto and Dane Crossley for critiquing the manuscript.
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FOOTNOTES |
|---|
This study was supported in part by dissertation grants from CAPES and Conselho Nacional de Desenvocuimente Cientifico e Tecnologico (Brazil) to W. Barrionuevo and by National Science Foundation Operating Grant IBN96-16138 to W. Burggren.
1 In this paper we use the terms "embryo" to describe an individual within an unhatched egg (generally <72 h) and "larva" for an individual after hatching up to day 20. "Juvenile" is reserved for a fish with a markedly adult appearance at 30-70 days of age, while "adult" is reserved for a sexually mature fish of 100 days.
Address for reprint requests: W. W. Burggren, Dept. of Biological Sciences, Univ. of North Texas, PO Box 305220, Denton, TX 76203-5220.
Received 8 December 1997; accepted in final form 12 October 1998.
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REFERENCES |
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Top
Abstract
Introduction
Material and methods
Results
Discussion
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