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First published online February 27, 2009
Journal of Experimental Biology 212, 778-784 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.026575
Hemoglobin enhances oxygen uptake in larval zebrafish (Danio rerio) but only under conditions of extreme hypoxia
Department of Biology, Brandon University, Brandon, MB, Canada R7A 4X8
* Author for correspondence (e-mail: rombough{at}brandonu.ca)
Accepted 16 December 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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O2), critical
dissolved oxygen level (Pc) and residual oxygen level
(Pr) were determined and used, respectively, as indicators
of response in normoxia, moderate hypoxia and extreme hypoxia.
rO2 was defined
as the average rate of O2 uptake before O2 became
limiting (i.e. at high PO2s).
Pc is the PO2 at which
rO2 first
becomes O2-limited and Pr is the
PO2 below which larvae are no longer able to
extract O2 from the ambient medium. CO poisoning had no significant
impact on rO2 or
Pc at any age, indicating that the lack of functional Hb
does not impair routine O2 usage in normoxia or at moderate levels
of hypoxia [down to at least 25–50 torr (1 torr
0.133 kPa), depending
on age]. Pr, however, was significantly lower overall for
control larvae (6.7±1.1 torr; mean ± 95%CI) than for CO-poisoned
larvae (11.2±2.1 torr). It would appear that the presence of functional
Hb allows zebrafish larvae to extract O2 from water down to lower
PO2s under conditions of extreme hypoxia. This
is the first documented (as opposed to inferred) benefit of Hb in developing
zebrafish. However, given the relatively small magnitude of the effect it is
unclear if this benefit on its own is sufficient to balance the costs
associated with Hb production and maintenance.
Key words: zebrafish, Danio rerio, hemoglobin, larva, O2, aerobic metabolism, cost–benefit analysis
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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), nitric oxide
transport (Allen and Piantadosi,
2006), oxygen storage and buffering
(Baumann and Dragon, 2005) as
well as a number of indirect functions arising from the metabolic machinery
associated with the red blood cell in which the hemoglobin resides
(Hume et al., 1996). Of these
functions, blood O2 transport generally is consider the most
critical (Burggren, 2004). This
perception is based on observations that poisoning of hemoglobin in higher
vertebrates (birds and mammals) rapidly leads to death from asphyxiation. In
mammals Hb, depending on its concentration, can increase the O2
carrying capacity of the blood >100-fold compared with what can be
transported dissolved in the blood plasma
(Burns et al., 2007). Without
functional Hb, there is simply not enough O2 dissolved in the
plasma to meet metabolic demand. Adult fishes typically have lower Hb
concentrations so the increase in blood transport capacity is not as great as
in mammals, but is still usually in the range of 15- to 30-fold (Gallauger and
Farrell, 1998). To date, the only adult vertebrates found not to use Hb to
supplement plasma O2 transport are Antarctic fishes of the family
Channichthyidae. It is believed that they are able to get by without Hb
because of the special circumstances of their habitat and life history, namely
the fact that they inhabit very cold water (–1.8°C to +1.5°C)
and have a sedentary life style (Sidell
and O'Brien, 2006). Cold and a sedentary life styles reduce the
metabolic demand for O2, and cold water (and cold blood plasma) is
able to hold more dissolved O2 than warm water (the dissolved
oxygen content of water at 0°C is about 1.9x that at 28°C, the
temperature at which the zebrafish used in this study were reared).
Zebrafish, Danio rerio is a small, active tropical fish that is
widely used as a model of vertebrate development, including the ontogeny of
the blood and circulation (Zon,
1995). Zebrafish begin to produce hemoglobin at a very early age.
Erythrogenesis is initiated at about 15 h postfertilization (hpf) and red
blood cells containing hemoglobin are present in the circulation by 24 hpf,
24–48 h prior to hatching (Brownlie
et al., 2003). Given the large metabolic costs associated with
hemoglobin production (Paul et al.,
2004), one would suspect that the hemoglobin produced by zebrafish
embryos was performing some critical function.
This supposition led Pelster and Buggren
(Pelster and Buggren, 1996) to
examine what effect blocking hemoglobin function would have on zebrafish
embryos. Somewhat surprisingly, functional ablation of hemoglobin using either
carbon monoxide (which competes with O2 for the O2
binding site on Hb) or phenylhydrazine (which blocks the production of
erythrocytes and hence Hb synthesis) had no significant effect on heart rate,
blood pressure or, most significantly, whole body metabolic rate at any age up
to 4.5 days postfertilization (dpf), the last stage tested. Jacob et al.
(Jacob et al., 2002) extended
these observations to older larvae, up to 15 dpf. They found that functional
ablation of hemoglobin had no effect on heart rate, stroke volume and cardiac
output or, presumably, on metabolic rate since larvae without functional
hemoglobin did not appear to have to resort to increased anaerobic metabolism
to make up for any deficiency in aerobic metabolism (i.e. there was no
increase in whole body lactic concentration). Both studies looked at the
response to functional ablation of the O2 carrying capacity of
hemoglobin under normoxic condition. Based on these experiments, it would
appear that under normoxic conditions direct diffusion of O2 from
the environment combined with any O2 transported in the blood
plasma is sufficient to meet the metabolic demands of zebrafish larvae up to
at least 15 dpf. This, however, does not necessarily mean that there is no
selective advantage to using hemoglobin to increase the O2
transport capacity of the blood during embryonic and larval development.
Zebrafish are a tropical flood plain species that lay their eggs on the
bottom of shallow ephemeral ponds and in slow moving streams where
O2 levels are likely to be low
(Spence et al., 2008). The
advantage of Hb in terms of increasing the O2 transport capacity of
the blood is greater at moderate levels of hypoxia than in normoxia. For
example, the blood of adult rainbow trout at 20°C transports about 15
times as much O2 in the form of oxyhemoglobin as is dissolved in
the plasma at a partial pressure of oxygen
(PO2) of 150 torr (Burggren et al., 1992). The
fractional increase in carrying capacity at 35 torr, however, is >30-fold.
It is not until PO2 drops below about 15 torr
that the fractional increase in O2 carrying capacity falls below
the 15-fold value seen at 150 torr. It could be that developing zebrafish are
able get by without Hb when PO2 levels are
relatively high but that when PO2s are lower,
the higher O2 transport capacity engendered by the presence of Hb
becomes a distinct advantage. The aim of this study was to test this
hypothesis.
Routine metabolic rate
(rO2), critical
oxygen level (Pc), total body conductance (G) and
residual O2 levels (Pr) were determined using
closed-system respirometry for zebrafish larvae aged between 5 dpf and 42 dpf
after exposure to 5% carbon monoxide (CO) for 2–4 h. Values for these
indicators were compared with those of control larvae. Acute exposures were
chosen to exclude the possibility of developmental adaptations that might mask
the direct effects of carbon monoxide on blood O2 transport. For
example, increases in heart size and blood volume have been shown to
compensate for a lack of Hb in Antarctic ice-fishes
(Sidell and O'Brien, 2006). A
minimum of 2 h exposure was deemed sufficient. The hemoglobin of the
red-blooded Antarctic fish Pagoathenia bernacchii was 100%
CO-saturated within 3 min of exposure to 7% CO
(DiPrisco et al., 1992). The
blood of adult rainbow trout Oncorhynchus mykiss exposed to 5% CO was
93–95% CO-saturated when tested after 3 h (Holeton, 1971a). Larvae of
rainbow trout displayed increases in both heart rate and ventilation rate
within 10–15 min of being exposed to 5% CO (Holeton, 1971b). Routine
metabolic rate
(rO2) was
defined as the average rate of O2 consumption in the respirometer
before O2 became limiting in closed-system, `rundown' tests. The
critical oxygen level (Pc) is the
PO2 at which metabolic rate first becomes
O2 limited on exposure to graded hypoxia. It was assumed that if Hb
played a role in O2 transport, Pc would be
lower for larvae with functional Hb than for those in which the O2
binding capacity of Hb was blocked (i.e. CO-exposed larvae would not be able
to satisfy routine O2 demand down to as low a
PO2 as control fish). Total conductance
(G) is a measure of the total resistance (R) to
O2 flux from the body surface to the mitochondria
(G=1/R). It can be calculated from the general transfer
equation
O2=G
PO2,
where PO2 is the
PO2 at the body surface
(Po) minus the PO2 at the
level of the mitochondria (Pi)
(Dejours, 1981). When
Po=Pc, Pi by
definition is zero (or very close to it). Total conductance can therefore be
estimated as
G=rO2(Pc)–1.
Total conductance includes that component associated with convective
O2 transport. If Hb facilitates convective O2 transport,
the value of G should be higher for larvae with functional Hb than
for those without. When fish are placed in a closed-system respirometer, they
gradually deplete the O2 that was originally in the respirometer.
Consumption of O2, however, does not continue until all the
O2 is consumed. At some point PO2
becomes so low that there is no longer sufficient driving force to move
O2 into the body and O2 consumption stops even though
some O2 is still present. The PO2 at
which this occurs is termed the residual oxygen level, Pr
(Grigg, 1969). If Hb
facilitates O2 uptake at very low
PO2 values, one would expect residual
O2 levels to be higher for CO-poisoned larvae.
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MATERIALS AND METHODS |
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Respirometry
Routine metabolic rates, critical dissolved oxygen levels and residual
oxygen tensions were determined at 28±0.1°C for zebrafish larvae at
5, 7, 14, 21, 30 and 40–42 dpf using two closed-cell Stathkelvin RC300
glass respirometers and SI 130 microcathode oxygen electrodes (Stathkelvin
Instruments, North Lanarkshire, UK). Groups of larvae were placed in a
respirometer cell and the oxygen level allowed to decline naturally as a
result of consumption by the fish. Group size ranged from 15 to two
individuals depending on age (fewer older individuals) and was chosen so that
it took about the same length of time (40–60 min) for the oxygen in the
respirometer to become depleted. Test larvae were lightly sedated using 30 mg
l–1 neutral buffered MS-222 (Sigma-Aldrich, St Louis, MO,
USA) to reduce stress and limit activity. Larvae of all ages remained
responsive to external stimuli at this level of sedation and were able to swim
normally. Preliminary tests with 7 dpf larvae indicated exposure to 30 mg
l–1 MS-222 resulted in about a 90% reduction in the frequency
of spontaneous swimming activity (P.R., unpublished data). Oxygen levels in
the respirometer were recorded as a function of time both digitally and using
a chart recorder. Respirometers were reopened 3–5 min after
O2 levels in the respirometer ceased falling as a result of larval
O2 uptake. This typically occurred at a
PO2 of 5 to 10 torr. Larvae tolerated of this
relatively brief period of extreme hypoxia and recovered if placed back in
air-saturated water. Normally, however, larvae were removed from the
respirometer and placed directly in an air-saturated solution of 100 mg
l–1 MS-222. Once they were fully anaesthetized, larvae were
transferred to 5% Bouin's fixative and kept there for at least 7 days to allow
their preserved mass to stabilize. Larvae were individually weighed to the
nearest 10 µg using a semi-micro balance. Preserved samples of the oldest
group of larvae tested (42 dpf) were examined under a microscope at 10x
magnification and their total body length, maximum body depth and maximum body
width determined.
Blank chambers (i.e. no fish) were assayed at the beginning and end of each
daily set of experiments to account for bacterial and background oxygen
uptake. The chart recordings of dissolved oxygen level as a function time were
used to estimate routine metabolic rates, critical oxygen levels and residual
O2 tensions using the graphical technique outlined in Rombough
(Rombough, 2007). With this
technique the initial approximately linear portion of the curve relating
dissolved oxygen and time, excluding the first 5–10 min, is taken as
representative of routine metabolic rate. The point at which this linear
portion of the curve begins to deviate from a straight line is use to estimate
Pc. Pr was taken as the point at which
the curve flat-lined near the end of the test. Two regression methods for
estimating Pc were tested [asymptotic curve fitting using
the SPSS TableCurve 2.0 program and complex linear regression analysis using
the program provided by Yeager and Ultsch
(Yeager and Ultsch, 1989)] but
the graphical method was found to be generally superior.
Experimental procedure
Experimental animals were exposed to 5% CO (balance air) for a minimum of 2
h and a maximum of about 4 h prior to being tested. The requisite number of
larvae for the age being tested along with water saturated with 5% CO and
containing 30 mg l–1 neutral buffered MS-222 were transferred
to one of the two respirometers. A simultaneous trial was conducted in the
other respirometer using control animals of the same age. Controls were
treated identically to experimental animals in terms of the acclimation
procedure except for exposure to CO After loading, the respirometers were
closed and oxygen levels allowed to run down as described previously. A
typical test series consisted of three or four parallel tests of control and
CO-exposed fish. Respirometers used for control and experimental animals were
alternated to avoid potential confounding affects arising from any slight
differences in the two respirometers. Two different batches (eggs from the
same stock but fertilized on different days) of larvae were used for all ages
except 30 dpf, for which only a single batch was used.
Statistics
Two-way ANOVA (SigmaStat, SPSS) followed by Holm–Sidak pairwise
comparisons was used to test for age and treatment effects for
rO2,
Pc and G. Allometric relationships between
metabolic rate and tissue mass were calculated using a log–log
regression model. Slope comparisons were conducted using ANCOVA. A sign rank
test was used to test for overall differences in Pr.
Paired t-tests were used to test for differences in
Pr at the various ages. Differences were considered
significant if P<0.05.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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O2 increased
about 14-fold over the period from 7 dpf to 40–42 dpf, from 9 nmol
h–1 at 7 dpf to 125 nmol h–1 at
40–42 dpf (Fig. 2A).
There were no significant differences between the
rO2 of control
larvae and larvae exposed to CO overall or at any of the ages tested
(Fig. 2A). There, similarly,
were no significant differences between control and CO-treated fish in terms
of Pc (Fig.
2B) or total conductance (Fig.
2C).
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O2 and fish
mass (M; mg) was not significantly different for control and
CO-treated fish (Fig. 3). The
equation of best fit for the two groups combined
(R2=0.909; N=109) was:
 | (1) |
Residual O2 levels were significantly higher overall (Sign rank test, P<0.001, N=52) for CO-treated larvae than for control larvae (Fig. 4) Overall mean (±95% CI) values were 11.2±2.0 and 6.7±1.1 torr, respectively. Paired t-tests indicated that differences at specific ages were significant only for 7 dpf and 14 dpf larvae (P<0.001 for both).
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The estimates obtained for Pc in this study (range
25–50 torr) are generally lower than those reported for similar age
larvae by Barrionuevo and Burggren (Barrionuevo and Burggren, 1999) (range
40–75 torr) and Bagatto et al. (Bagatto et al., 2001) (range 75–50
torr). However, this does not necessarily mean they are unreliable as each of
the three studies to date that have looked at how Pc
varies with age (this study) (Barrionuevo and Burggren, 1999; Bagatto et al.,
2001) have reported very different ontogenetic patterns. Barrionuevo and
Burggren (Barrionuevo and Burggren, 1999) indicated that
Pc declined at a fairly constant rate with age from hatch
to 100 dpf. Bagatto et al. (Bagatto et al., 2001), however, reported a
more-or-less continuous increase with age up to 35 dpf, the last age tested.
This study reveals a more complex pattern
(Fig. 2B). There was a modest
decline in Pc between 5 dpf and 7 dpf. This coincides with
a similar modest decrease in
rO2 over the
same period and is probably a reflection of reduced energy availability during
the transition from endogenous to exogenous feeding. Once the transition to
exogenous feeding was complete, Pc levels began to rise
and reached a maximum at around 21 dpf. Beyond 21 dpf, there was a gradual
decrease in Pc value with age. It is tempting to link the
rise followed by a decline in Pc centered on 21 dpf to
changes in the efficiency of branchial gas exchange. Gill lamellae begin to
form at about 12–14 dpf in zebrafish
(Rombough, 2002) and
proliferate rapidly until about 21 dpf to 28 dpf at which time the gill
assumes its definitive adult morphology and the rate of lamellar expansion
levels off (Tara Klassen, personal communication). Which of the three
ontogenetic patterns reported for Pc is `correct' is
difficult to predict at this time. Pc values in adult fish
are dependent on metabolic rate (Hoar,
1983). This probably holds true for larvae. Therefore, the
different patterns that have been reported could simply be a reflection of
variations in activity levels. In this regard, the current study is the only
one to have tested sedated larvae which probably explains the generally lower
Pc values. One of the major reasons larvae were sedated
was to try to standardize activity levels for different aged fish. Hopefully
this aim was achieved and the current study has captured the underlying
response to ontogenetic changes in the structure and function of the
respiratory system missed by the other two studies.
On reflection, it is not particularly surprising that CO had no significant
effect on rO2.
Larvae, even at 42 dpf, are still small enough that tissue demand should be
able to be met by direct diffusion across body surfaces. Harvey's formula
(Dejours, 1981) can be used to
calculate the maximum radius of a theoretical spherical animal without
circulation that could be supported solely by simple diffusion if it had the
same metabolic intensity of a 42 dpf zebrafish (35 nmol O2
h–1 mg–1). Using the value reported by
Kranenbarg et al. (Kranenbarg et al.,
2003) for Krogh's constant of diffusion for zebrafish embryonic
tissue (3.4x10–5 nmol O2
s–1 cm–1 torr–1) and a
partial pressure gradient of 150 torr, the maximum distance works out to be
about 0.6 mm. The maximum distance O2 would actually have to
diffuse in a 42 dpf larvae is only 0.5 mm (i.e. half the maximum body width of
1.0 mm), so theoretically a spherical larva should be able to fully satisfy
its metabolic demand for oxygen in air-saturated water using only simple
diffusion. The situation in terms of supplying tissues with oxygen in carbon
monoxide poisoned zebrafish larvae is actually considerably more favorable
than this simple model suggests. Larvae are not spheres so mean diffusion
distances in reality are considerably less than maximum diffusion distances.
The model also assumes no internal circulation, which is obviously not the
case. Circulation of blood, even without functional Hb, tends to transport
O2 from the periphery of the body towards deeper tissues where it
can be used in lieu of O2 that would otherwise have to reach these
tissues by simple diffusion. It was estimated, based on the solubility of
O2 in plasma and the value reported by Jacob et al.
(Jacob et al., 2002) for
cardiac output, that plasma O2 could supply between 13 and 21% of
metabolic oxygen demand at 15 dpf. This might seem like a relatively small
fraction of total O2 consumption, but it should be remembered that
O2 transported in the plasma would be largely reserved for central
tissues since peripheral tissues would continue to rely on direct diffusion
(Territo and Burggren, 1998).
The net result is that even a relatively simple circulation significantly
increases the maximum size a larva can attain before it requires some kind of
specific O2 transporter. Although a number of models have been
developed to predict maximum size in the absence of internal circulation (e.g.
Territo and Altimiras, 2001;
Kranenbarg et al., 2000), no
one, to our knowledge, has yet published a predictive model for the maximum
size of vertebrate embryos or larvae with an internal circulation but no
specific O2 transporter.
It is more difficult to explain why the lack of functional Hb did not
affect Pc. The concentration of O2 in blood
plasma at 28°C would be expected to decline at a rate of about 0.005 vol%
torr–1 based on Territo and Burggren's
(Territo and Burggren, 1998)
value for the O2 content of the plasma of Xenopus larvae.
At PO2s corresponding to the range of
Pcs observed in this study (25–45 torr), the blood
plasma of zebrafish larvae would only hold between 17% and 30% as much
O2 as it would at normoxia (150 torr). The amount of
O2 bound to Hb depends on the Hb concentration ([Hb]) which,
unfortunately is not known for zebrafish larvae. However, even a relatively
low [Hb] would increase the [O2] in the blood to several times that
dissolved in the plasma in normoxia and, because of the shape of the
Hb–O2 dissociation curve, by an even greater relative amount
at PO2s near Pc (see
Introduction). One, therefore, would have expected larvae with intact Hb to be
able to supply tissues with enough O2 to meet routine demand down
to lower PO2s than could CO-poisoned larvae
forced to rely solely on plasma O2. There are several possibilities
why this did not occur. The most obvious is that the methods used in the
current experiment were somehow deficient. For example, the concentration and
duration of the CO exposure could have been insufficient to fully block
O2 transport by Hb. However, as pointed out in the Introduction,
this appears unlikely based on the literature. Rainbow trout larvae exposed to
the same [CO] used in this study responded by increasing heart and ventilation
rates within 10–15 min of being exposed (Holeton, 1971b). Even partial
blockage of O2 transport capacity should have resulted in some
degree of impairment if Hb played a major role in O2 delivery. The
possibility of methodological error, however, was of enough concern that a
follow-up study was initiated to test whether the methods used here could
detect an effect of CO-poisoning on Pc in adult zebrafish.
That study found that Pc values are indeed significantly
higher for CO-poisoned adults than for controls just as one would expect (S.
Dorn and P.R., in preparation). This finding strongly suggests that the lack
of effect on Pc in the current study is a reflection of
differences in larval and adult physiology rather than an artefact of the
methodology.
One possible reason why CO-poisoning had no significant effect on
Pc could be the high affinity of larval Hb. The
O2 binding affinity of the Hb of zebrafish larvae has not been
determined but larval Hbs in general have higher O2 affinities than
adult Hbs (reviewed in Rombough,
1997; Baumann and Dragon,
2005). In some cases the differences can be considerable. For
example, the P50 of bullfrog (Rana catesbeiana)
larvae is only 9–10 torr whereas that of adults is about 35 torr
(Pinder and Burggren, 1983).
P50s for larval and adult Hb are 0.9 torr and 10.3 torr,
respectively, for the southern hemisphere lamprey Geotria australis
(Macey and Potter, 1982). The
P50 of larval Hb was only 53% of that of adult Hb in
rainbow trout (Iuchi, 1973).
Larval Hbs also tend to display little or no Bohr effect
(Rombough, 1997). The result
of a reduced Bohr effect and high affinity means that larval Hb is not able to
unload significant amounts of O2 until
PO2 falls to relatively low levels
corresponding to the steep part of the O2–Hb dissociation
curve. If the affinity of the Hb of zebrafish larvae is similar to that of
larvae of other lower vertebrates, the PO2 at
which unloading begins is probably well below the Pc for
zebrafish larvae (25–40 torr). This would mean that even though Hb may
transport a lot of O2 at higher ambient
PO2s, that O2 is effectively
unavailable to the tissues, at least at normal levels of physical activity
(O2 might become available during intense exercise but that is a
subject for future studies).
The high affinity of larval Hb could also explain why residual oxygen
levels (Pr) are lower for control larvae than for
CO-exposed larvae (Fig. 4).
Grigg (Grigg, 1969) noted that
in the adult bullhead, Ictalurus nebulosus, arterial
PO2 was essentially zero at
Pr in all fish but that Pr was lower
for fish with higher affinity Hb. Why higher affinity Hb should reduce the
partial pressure gradient needed for O2 uptake across the gill is
not clear but a similar phenomenon could be at work in larval zebrafish. If
this were the case, CO-poisoned larvae without functional Hb would require a
larger partial pressure gradient for net O2 uptake than would
larvae with Hb – the higher the affinity of the Hb, the greater the
disadvantage of the CO-poisoned larvae.
One would expect that larvae that produce hemoglobin would have a selective advantage because of the ability to continue to extract O2 from the environment down to lower ambient PO2 values. Residual O2 levels averaged about 4.5 torr lower for control than for CO-poisoned zebrafish larvae at 28°C (the temperature of the current study). The difference would probably be somewhat greater at higher temperatures (the upper lethal temperature for zebrafish embryonic development is about 34°C) because of higher metabolic rates and lower O2 solubility. However, even at high temperatures a reduction in residual O2 levels is unlikely to be the whole story behind why zebrafish larvae produce hemoglobin.
Larvae that produce hemoglobin incur costs as well as benefits. Hemoglobin
is an energetically expensive molecule to produce. In addition to the direct
cost of synthesis, there are costs associated with the metabolic machinery
necessary to produce and maintain it (e.g. methemoglobin reductase). There are
also costs associated with producing the red cell in which it is packaged.
Paul et al. (Paul et al.,
2004) have shown that in the small, pelagic crustacean
Daphnia there is a complex tradeoff between the costs and benefits of
producing hemoglobin. Daphnia balance costs and benefits by
upregulating or downregulating hemoglobin synthesis depending on O2
availability and predator presence. There is some suggestion that zebrafish
larvae might be able to upregulate Hb concentration in response to chronic
hypoxia (Schwerte et al.,
2005) although this capacity, if it in fact exists, appears to be
much less than that exhibited by Daphnia (<35% vs
<1600%). The situation in developing zebrafish is complicated in terms of
benefits by the fact that their hemoglobin is probably involved in more
physiological functions than just simple O2 transport. The high
O2 affinity of larval hemoglobin means that, like myoglobin, it
probably provides the larvae with some reserve O2 capacity at very
low PO2 values. In addition, hemoglobin has
been implicated in nitric oxide transport
(Allen and Piantodosi, 2006).
The vasculature of zebrafish larvae has been shown to be responsive to nitric
oxide (Fritsche et al., 2000)
suggesting a possible indirect role for Hb in vascular function. Hb may also
play an important role in larval acid–base balance. These other
functions need to be taken into account in any overall cost-benefit analysis.
It could turn out that the benefits associated with any one function on its
own is not sufficient to cover the cost of Hb production. It may be that it is
only when several benefits are combined that the balance is tipped in favor of
Hb production.
Trade-offs between cost and benefits probably explain why the larvae of
some marine fish species do not produce Hb until very late in development. For
example, larvae of halibut Hippoglossus hippoglossus
(Pittman et al., 1990),
dolphinfish Coryphaena hippurus
(Benetti and Martinez, 1993)
and spot Leiostomus xanthurus
(Govoni et al., 2005) do not
begin to produce hemoglobin until near metamorphosis. In spot, measurable
amounts of Hb are not observed until about 48 days post hatch
(Govoni et al., 2005). The
larvae of these species are all pelagic and, thus, are unlikely to experience
acute episodes of extreme hypoxia (or if they do the magnitude and duration of
such events are likely to be so large that Hb would be of little practical
use). Pelagic larvae also need to take into account that hemoglobin makes them
more visible to predators. Limited benefits and high costs provide a plausible
explanation for why larvae of these species delay Hb production. The fact that
larvae of some species can thrive without Hb indicates that none of the
services provided by Hb during development are essential in an absolute sense.
It also reinforces the importance of environmental context when it comes to
trying to understand the ontogeny of vertebrate physiological processes.
LIST OF ABBREVIATIONS
O2
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Footnotes |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Allen, B. W. and Piantadosi, C. A. (2006). How do red blood cells cause hypoxic vasodilation? The SNO-hemoglobin paradigm. Am. J. Physiol. Heart Circ. Physiol. 291H,507 -512.[CrossRef]
Baggatto, B., Pelster, B. and Burggren, W. W. (2001). Growth and metabolism of larval zebrafish: effects of swim training. J. Exp. Biol. 204,4335 -4343.[Medline]
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