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First published online April 18, 2006
Journal of Experimental Biology 209, 1639-1650 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02180
Metabolic, respiratory and cardiovascular responses to acute and chronic hypoxic exposure in tadpole shrimp Triops longicaudatus
1 Department of Environmental and Molecular Toxicology, Oregon State
University, Environmental Health Sciences Center, 1011 ALS, Corvallis, OR
97331, USA
2 Department of Biology, University of Nevada, Las Vegas NV 89154,
USA
* Author for correspondence (e-mail: harpers{at}science.oregonstate.edu)
Accepted 20 February 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: invertebrate, development, physiology, hypoxia, oxygen partial pressure
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
McGaw et al., 1994;
Reiber, 1995;
Reiber and McMahon, 1998;
Wilkens, 1999). Oxygen
conformers are able to reduce metabolic demand so that O2
consumption (
O2)
is coupled to environmental O2 partial pressure
(PO2)
(Loudon, 1988;
Hochachka et al., 1999;
Boutilier, 2001). Oxygen
regulators, by contrast, maintain
O2 independent
of environmental PO2 down to a point where the
O2 required for aerobic processes becomes limiting
(PCRIT) (Herreid,
1980; Hochachka,
1988). Below PCRIT, either tissue oxygen
demand decreases or anaerobic processes become increasingly important in
meeting energy requirements (Hochachka,
1988). Above PCRIT, O2 uptake and
delivery may be enhanced through a variety of compensatory mechanisms
including adjusting ventilatory parameters (rates and volumes), increasing
internal convective processes such as heart rate and stroke volume and
changing perfusion patterns (McMahon,
1988; Hervant et al.,
1995; Willmer et al.,
2000; Boutilier,
2001; Hochachka and Lutz,
2001; Hopkins and Powell,
2001; Hoppeler and Vogt,
2001). Additionally, some animals can modulate the
O2-binding affinity of their respiratory proteins, which enhances
O2 uptake at the respiratory surface and increases total
O2 capacitance (Wells and
Wells, 1984; Kobayashi et al.,
1988; Hochachka et al.,
1999; Willmer et al.,
2000).
Hypoxic exposure during development (embryonic or larval periods) may
elicit a similar suite of compensatory responses or may result in long-term or
permanent changes to the morphology and/or physiology of the animal and thus
impact the adult hypoxic response (Bamber
and Depledge, 1997; Bradley et
al., 1999; Willmer et al.,
2000; Gozal and Gozal,
2001; Peyronnet et al.,
2002). The physiological consequences of hypoxia-induced
developmental plasticity can be a reduced metabolic rate
(Hochachka et al., 1999;
Boutilier, 2001), greater
respiratory gas exchange surface area
(Loudon, 1988;
Loudon, 1989), increased
respiratory capacities and/or enhanced convection and O2 delivery
mechanisms (Banchero, 1987;
McMahon, 1988;
Graham, 1990;
Szewczak and Jackson, 1992;
Childress and Seibel, 1998;
Pelster, 1999;
McMahon, 2001).
Transcriptional regulation accounts for many of these long-term physiological
and morphological changes associated with developmental exposure to hypoxia. A
well-documented example of this type of developmental hypoxic response in
crustaceans is the increase of existing O2-binding proteins as well
as the production of new higher-affinity proteins that result in long-term
increases in O2-uptake and capacitance
(Wells and Wells, 1984;
Snyder, 1987;
Kobayashi et al., 1988;
Hervant et al., 1995;
Astall et al., 1997;
Hou and Huang, 1999;
Wiggins and Frappell, 2000;
Barros et al., 2001).
The degree to which developmental PO2
influences adult metabolic, respiratory and cardiovascular hypoxic responses
was investigated using tadpole shrimp Triops longicaudatus. Several
features of tadpole shrimp make them well suited to study the effects of acute
and developmental hypoxic exposures. They are often faced with environmental
PO2s below their PCRIT, yet
the mechanisms by which they tolerate such conditions are poorly understood
(Horne and Beyenbach, 1971;
Hillyard and Vinegar, 1972;
Eriksen and Brown, 1980;
Scholnick, 1995;
Scholnick and Snyder, 1996).
Tadpole shrimp have high metabolic rates and respiratory structures
(epipodites) that are thought to be inadequate to maintain O2
uptake in their euryoxic habitats (Fryer,
1988; Horne and Beyenbach,
1971; Hillyard and Vinegar,
1972; Scott and Grigarick,
1978; Eriksen and Brown,
1980; Scholnick,
1995; Scholnick and Snyder,
1996). A tubal, myogenic heart devoid of vasculature produces the
only internal convective currents in tadpole shrimp
(Yamagishi et al., 1997;
Yamagishi et al., 2000).
Large, extracellular hemoglobin (29 subunits) is produced in response to
hypoxic exposure, but the mechanisms of this hypoxic induction are not known
(Horne and Beyenbach, 1971;
Scholnick and Snyder, 1996).
Finally, tadpole shrimp have a comparatively rapid generation time and
amenability to laboratory culture, which make them tractable organisms for
developmental investigations (Fryer,
1988; Horne and Beyenbach,
1971; Scott and Grigarick,
1978).
Tadpole shrimp were reared under normoxic (19–21 kPa O2),
moderate (10–13 kPa O2) or severe (1–3 kPa
O2) hypoxic conditions to determine whether differences in
developmental PO2 were sufficient to change
adult metabolic, respiratory or cardiovascular system physiology and hypoxic
sensitivity. Compensatory respiratory and cardiovascular processes that
enhance O2 uptake, internal convection and perfusion should
increase in response to hypoxic exposure. We hypothesized that tadpole shrimp
reared under hypoxic conditions would have decreased metabolic sensitivity to
PO2 change, increased physiological responses
to hypoxic exposure and increased hemoglobin concentration and
O2-binding affinity relative to those reared under normoxic
conditions (Wells and Wells,
1984; Snyder,
1987; Kobayashi et al.,
1988; Hervant et al.,
1995; Astall et al.,
1997; Hochachka et al.,
1999; Hou and Huang,
1999; Barros et al.,
2001; Boutilier,
2001).
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Materials and methods |
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).
Three 150-liter insulated aquaria (normoxic=19–21 kPa O2,
moderate hypoxic=10–13 kPa O2 and severe hypoxic=1–3
kPa O2) were set up in the laboratory using deionized water and
sediments taken from the pool. A model GF-3/MP gas flowmeter (Cameron
Instruments Company, Ontario, Canada) was used to regulate a mixture of
N2 and room air. Oxygen partial pressure in each aquaria was
monitored for a 48-h period each week using data-logging dissolved
O2 meters (Model 810 Orion Dissolved Oxygen Meter; Orion Research,
Boston, MA, USA).
Each aquarium was equipped with ultraviolet (3% UVB and 7% UVA) enhanced
lights (Super UV Reptile Daylight Lamp; 20 W; Energy Savers Unlimited Inc.,
Chicago, IL, USA) that established a 13 h:11 h L:D cycle. A timed circulating
water bath (Model VT513; Radiometer, Copenhagen, Denmark) and heat exchange
coil were used to cycle temperature with the lights. The aquaria started to
warm two hours after the lights were turned on and continued for five hours
each day to establish a 23–28°C temperature cycle. Dry sediments
(100 g) containing tadpole shrimp cysts were added to each aquarium weekly.
Animals ate algae, detritus and small invertebrates that hatched from the
sediment. Tadpole shrimp were identified as Triops longicaudatus
(Sassaman, 1991). Aquaria were
drained and refilled monthly.
Metabolism
Standard metabolic rate was measured when animals were active because they
were infrequently quiescent. To assess the confounding effect of
PO2 on activity, individual animals
(N=10) were placed in a marked cylindrical 10-ml flow-through chamber
and videotaped during progressive hypoxic exposure. Animals were acclimated
(30 min) to the chamber under normoxic conditions. A gas flowmeter (Model
GF-3/MP) was used to regulate the mixture of N2 and air. Chamber
PO2 was decreased from 20 to 2 kPa
O2 at a rate of 5 kPa O2 h–1. The
number of times the animal crossed defined marks on the chamber was averaged
over one-minute intervals to produce an index of activity in response to
PO2.
Individual animals from each rearing group (N=7 per rearing group)
were placed in a 125-ml closed-system darkened respirometry chamber at
28°C. The chamber had a plastic grate on the bottom under which a magnetic
stirring rod was placed to ensure thorough mixing of the chamber water.
Animals were acclimated for 30 min and then the chamber was sealed. Oxygen
content of the chamber was monitored using a model 781 Strathkelvin
O2 meter (Strathkelvin Instruments, Glasgow, UK). Oxygen
consumption was calculated based on the following equation:
 | (1) |
O2 is
oxygen consumption, Vr is the volume of water in the
respirometer, 
PWO2 is the change in
oxygen concentration of the water, ßWO2 is the
capacitance of oxygen in water, t is duration in minutes, and
Md is the dry mass of the animal measured in grams
(Piiper et al., 1971).
Individual tadpole shrimp dry mass was determined by drying the animal at
60°C until three constant mass measurements were obtained. Oxygen
consumption rates were calculated for successive 5-min intervals and expressed
as mass-specific O2 uptake (µl O2
g–1 h–1). The experiment was performed
without animals in the chamber, and the
O2 rates
obtained from three replicates were used to calculate a mean correction factor
for microbial respiration. Animals reared under normoxic (N=10) and severe hypoxic (N=10) conditions were used to assess anaerobic lactate metabolism in tadpole shrimp. Lactate concentration was measured for five animals pre-treatment and five animals after exposure to severe hypoxic conditions (2 kPa O2) for 12 h at 23°C. Lactate concentrations in the experimental chamber water were also determined. Hypoxic conditions were maintained using a gas flowmeter (Model GF-3/MP) to control the mixture of N2 and room air. Hemolymph was collected in glass capillary tubes by dorsal heart puncture. Lactate concentrations were determined for 10 µl water samples and 10 µl hemolymph samples mixed with 1.0 ml lactate reagent solution (#735-10; Trinity Biotech, St Louis, MO, USA). Hemolymph lactate concentrations were measured enzymatically (Sigma Diagnostics, St Louis, MO, USA; Sigma Lactate Kit #735) at 540 nm.
Ventilation
Ventilatory rate and amplitude were measured in response to hypoxic
exposure in order to assess the effects of developmental
PO2 on adult ventilatory hypoxic response.
Adult tadpole shrimp from each rearing group (N=13 per rearing group)
were held in a 30-ml flow-through chamber (temperature, 25°C). Tadpole
shrimp were secured in the chamber with an applicator stick and cyanoacrylate
glue on the lateral carapace. They were inverted in the chamber to allow the
ventral surface to be viewed. Movements of the respiratory appendages were
videotaped (60 Hz sampling speed) under a dissecting microscope (Leica
Stereozoom 6 Photo) using a video camera (Oscar Color Camera Vidcam), super
VHS video recording system (Panasonic PV-54566) and Horita time code generator
(VG 50; Horita Co., Inc., Mission Viejo, CA, USA). Tadpole shrimp were
acclimated for 30 min and then exposed to four environmental
PO2s (20, 13.3, 10 and 1 kPa O2) in
random order. Thirty minutes was allowed for acclimation once a new
PO2 was achieved. Animals were not returned to
normoxia between exposures. Time-encoded video was analyzed frame-by-frame on
an editing tape player (Panasonic AG-DS550) to determine ventilation rate and
amplitude. Ventilation rate (frequency) was measured as number of appendage
beats per minute. The amplitude of appendage beats was determined as the mean
distance that the 4th and 5th appendages separate during five subsequent
ventilatory strokes (Harper,
2003).
Cardiac physiology
Heart rate, stroke volume and cardiac output were measured in response to
hypoxic exposure in order to assess the effects of developmental
PO2 on adult cardiac hypoxic responses. Adult
tadpole shrimp from each rearing group (N=13 per rearing group) were
secured in a 30-ml flow-through experimental chamber as previously described.
Animals were acclimated for 30 min and then exposed to five environmental
PO2s (26.7, 20, 13.3, 10 and 4 kPa
O2) in random order. Thirty minutes was allowed for acclimation at
each PO2. Cardiac contractions were videotaped
as previously described. Heart rate (fH; beats
min–1) was measured when the time-encoded video was advanced
frame-by-frame on an editing tape player (Panasonic AG-DS550, Cypress, CA).
The tadpole shrimp heart was modeled as a cylinder with a volume of
r2h, where r is half the width of
the heart and h is length. Images of the heart were collected during
maximal [end diastolic volume (EDV)] and minimal [end systolic volume (ESV)]
distention. Those images were dimensionally analyzed using Scion Imaging
software (National Institutes of Health, Bethesda, MD, USA). Stroke volume
(VS; µl beat–1) was calculated as the
difference in heart volume between EDV and ESV. Cardiac output
(
; µl min–1) was
calculated as the product of fH and
VS.
Hemoglobin
Hemoglobin is the major protein in tadpole shrimp hemolymph
(Horne and Beyenbach, 1971).
Protein concentrations of animals from each rearing group (N=10 per
rearing group) were determined to assess the influence of developmental
PO2 on hemoglobin production. Protein
concentrations were determined for tadpole shrimp reared under normoxic
conditions and exposed to severe hypoxic conditions for 5, 7 and 10 days
(N=7 per day). Likewise, protein concentrations were determined for
tadpole shrimp reared under severe hypoxic conditions and exposed to normoxic
conditions for 5, 7 and 10 days (N=7 per day). Protein concentrations
were determined using a Micro BCA Protein Assay Reagent Kit (#23235; Pierce,
Rockford, IL, USA). Protein standards (2.0 mg ml–1 BSA in a
solution of 0.9% saline and 0.05% sodium azide) were diluted with tadpole
shrimp saline (5.84 mg NaCl, 7.45 mg KCl, 11.09 mg CaCl2, 9.52 mg
MgCl2, 3.65 mg HCl and 1 ml H2O)
(Yamagishi et al., 2000) to
form solutions with final BSA concentrations of 200, 40, 20, 10, 5, 2.5, 1 and
0.5 µg ml–1. Working reagent was prepared by mixing 12.5
ml Micro BCA Reagent MA (sodium carbonate, sodium bicarbonate and sodium
tartrate in 0.2 mol l–1 NaOH) and 12 ml Micro BCA Reagent MB
[bicinchoninic acid (4.0%) in water] with 0.5 ml Micro BCA Reagent MC (4.0%
cupric sulfate, pentahydrate in water). One milliliter of each standard was
added to appropriately labeled test tubes. A water blank and tadpole shrimp
saline were used as controls. In each test tube, 1.0 ml working reagent was
added and mixed. The tubes were covered with ParafilmTM and placed in a
60°C water bath for 60 min and then cooled to room temperature (23°C).
Absorbance was measured at 562 nm, with corrections made for reference. A
standard curve was produced to obtain hemoglobin concentrations of hemolymph
samples.
Hemoglobin O2-binding affinities were measured for animals from
each rearing group to determine differences dependent on developmental
PO2. Hemolymph (60 µl) was collected in
glass capillary tubes from large tadpole shrimp (N=7 per rearing
group) by dorsal puncture of the heart. Hemolymph was added into a small
flow-through tonometer that opened into a narrow chamber (1 mm inner diameter)
inside a cuvette (1 cmx1 cmx5 cm). The tonometer was placed on its
side when hemolymph was added and during each equilibration step. This allowed
the hemolymph to flow into the bulbous region of the tonometer. A stirring
flea powered by a magnetic stirrer was placed into the tonometer to ensure
thorough mixing of the hemolymph with inflowing gas mixtures. During
spectrophotometric measurements, the tonometer was held upright so that the
hemolymph flowed into the narrow chamber. The
PO2 of humidified inflowing gas (1000 sccm) was
adjusted using a gas flowmeter (Model GF-3/MP; Cameron Instruments, Guelph,
ON, Canada). Hemolymph was equilibrated for 20 min with normoxic air (20 kPa)
and analyzed spectrophotometrically using a Turner spectrophotometer (Model
340; Mountain View, CA, USA) at a wavelength of 570 nm. This is the wavelength
of maximal absorbance for oxy- and deoxy-hemoglobin for Triops
(Horne and Beyenbach, 1974).
Water was used as a reference. The absorbance of hemoglobin was determined
after equilibration with air of 30, 4.0, 2.7, 1.3, 1.1, 0.8, 0.5 and 0 kPa
O2. Standard curves were constructed from the absorbance of
hemoglobin at 0% and 100% saturation. Percent saturation for hemoglobin at
each PO2 was calculated using standard curves.
Curve fitting of O2 binding was calculated using SigmaStat 2.03
(SPSS Inc., Chicago, IL, USA). The P50 for each rearing
group was determined as the PO2 at which 50%
saturation occurred (Bruno et al.,
2001). Cooperativity (nH) was calculated as
the maximal slope of log[saturation/(1–saturation)] against
log[PO2]
(Bruno et al., 2001).
Oxygen-dependent changes in hemolymph pH were used to determine the significance of a Bohr shift in altering hemoglobin O2-binding affinity. Hemolymph pH was measured using a PHR-146 Micro Combination pH Electrode (Lazar Research Laboratories, Inc., Los Angeles, CA, USA) inserted into the base of a flow-through chamber (20 µl). The PO2 of inflowing humidified gas (1000 sccm) was adjusted using a gas flowmeter (Model GF-3/MP) to control a mixture of N2 and room air. Hemolymph pH was determined after 10 min equilibration at 30, 4.0, 2.7, 1.3, 1.1, 0.8, 0.5 and 0 kPa O2.
Oxygen-carrying capacity and delivery potential
The O2-carrying capacity of 20 µl of O2-saturated
hemolymph was determined for animals from each rearing group (N=7 per
rearing group) using methods described previously
(Tucker, 1967). Hemolymph was
saturated by equilibration with 30 kPa O2. A potassium ferricyanide
solution {6 g potassium ferricyanide [K3Fe(CN)6], 3 g
saponin (Sigma) and 1 kg water} was added to a 10 ml glass syringe and
degassed by plugging the syringe needle with a rubber stopper, pulling back
gently on the plunger to create a vacuum and shaking. Extracted gas was
expelled and the process was repeated a minimum of five times to ensure
complete degassing. A microrespirometry chamber (400 µl) with O2
electrode (Model 781 Strathkelvin oxygen meter) was filled with degassed
potassium ferricyanide solution, plugged and stirred. After five minutes
equilibration, the PO2 in the chamber was
measured. The stopper was removed from the chamber and 20 µl of hemolymph
was injected into the chamber with the degassed potassium ferricyanide. After
five minutes equilibration, the PO2 in the
chamber was determined. Hemolymph and degassed potassium ferricyanide solution
were removed. Aerated potassium ferricyanide solution was added to the
chamber, removed and added again. The chamber was left unplugged and the
PO2 determined after 20 min equilibration. The
potassium ferricyanide solution was removed from the chamber and a solution of
sodium sulfite and sodium borate [1 mg sodium sulfite
(Na2SO3) and 5 ml 0.01 mol l–1 sodium
borate (Na2B4O7) (Sigma)] was added before
the chamber was plugged again. After five minutes equilibration, the
PO2 of the chamber was determined. Hemolymph
O2 content (ml O2 100 ml–1 hemolymph or
Vol%) was calculated using the equation:
 | (2) |
PO2 is the change in
PO2 after injecting the hemolymph into the
degassed potassium ferricyanide solution, 
is the solubility
coefficient of O2 in the potassium ferricyanide solution,
V is the chamber volume and Vs is the sample
volume (Tucker, 1967).
Oxygen-delivery potential was calculated as the product of O2
content and cardiac output and reported in µl O2
min–1 (Ronco et al.,
1991). Calculations for the determination of O2 content
and cardiac output were described above.
Oxygen consumption/oxygen transport coupling
The amount of coordination between O2 demand and delivery was
determined by comparing the ratio of
O2 to
cardiovascular transport. The degree of coupling was compared among rearing
groups to determine the effects of developmental
PO2 on respiratory and cardiovascular system
coordination. Indices of the relationship between
O2 and hemolymph
O2 transport
(O2) were
calculated using the equation:
 | (3) |
O2 is
oxygen consumption in µl O2 g–1
h–1, CO2 is oxygen content of
fully saturated hemolymph (µl O2 µl–1
hemolymph) and is cardiac output
(µl g–1 h–1)
(Territo and Altimiras, 1998;
Territo and Burggren, 1998).
The ratio is unitless because
O2 and
O2 are both
expressed in µl O2 g–1 h–1. A
value of one suggests a strong coupling between O2 demand and
convective transport. Values below one indicate that circulatory O2
transport capacity exceeds total
O2. Values above
one indicate that convective transport may limit O2 supply.
Statistical analyses
All statistical analyses were run with SigmaStat 2.03 unless otherwise
specified. Results are presented as means ± s.e.m. with statistical
significance accepted at the level of P<0.05. Multiple pairwise
comparisons were made using Bonferroni t-tests when rearing effects
were significant, unless otherwise specified.
Metabolism
The strength of the relationship between activity level and
PO2 was measured using Pearson Product Moment
Correlation. Comparisons of metabolic response to graded hypoxia were made
among rearing groups using one-way repeated-measures analysis of variance
(ANOVA). Comparison of lactate concentrations was made between normoxic and
severe hypoxic rearing groups using a Student's t-test.
Ventilation
Comparisons of ventilatory amplitude and frequency among rearing groups
were made using Kruskal–Wallis one-way ANOVA on ranks because data had
unequal variance. Multiple pairwise comparisons were made using a Tukey
test.
Cardiac physiology
Comparisons of heart rate, stroke volume and cardiac output to graded
hypoxia were made among rearing groups using one-way repeated-measures
ANOVA.
Hemoglobin
Comparisons of hemoglobin concentration were made among rearing groups
using one-way ANOVA. The hemoglobin concentration of animals that had been
switched from normoxic to severe hypoxic, and from severe hypoxic to normoxic
conditions (after 5, 7 and 10 days), was analyzed using one-way
repeated-measures ANOVA. Comparison of the O2-binding affinity of
hemoglobin from each rearing group was made using Friedman repeated-measures
ANOVA on ranks because of unequal variance. Hemoglobin P50
was compared among rearing groups using Kruskal–Wallis one-way ANOVA on
ranks because data were not normally distributed. Multiple pairwise
comparisons were made using a Tukey test. Hemoglobin nH
was compared among rearing groups using analysis of covariance (StatView,
5.0.1; StatView Software, Cary, NC, USA). Multiple pairwise comparisons were
performed using a Scheffe test and StatView. Comparisons of hemolymph pH with
PO2 were made among rearing groups using
one-way repeated-measures ANOVA.
Oxygen-carrying capacity and delivery potential
Comparisons of the O2-carrying capacity of hemolymph were made
among rearing groups using Kruskal–Wallis one-way ANOVA on ranks because
data did not have equal variance. Multiple pairwise comparisons were made
using a Tukey test. Comparisons of the O2-delivery potential of
hemolymph were made among rearing groups using Kruskal–Wallis one-way
ANOVA on ranks because data had unequal variance. Multiple pairwise
comparisons were made using a Tukey test.
Oxygen consumption/oxygen transport coupling
Comparisons of the O2 consumption/oxygen transport coupling were
made among rearing groups using one-way repeated-measures ANOVA.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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O2 measurements.
Developmental PO2 had no effect on metabolic
rate or metabolic response to hypoxic exposure
(Fig. 1). All groups gradually
decreased O2
down to a PCRIT of 2 kPa O2 (691±20
µl O2 g–1 h–1 at normoxia to
274±9 µl O2 g–1 h–1 at
2 kPa O2 in normoxic animals; 714±14 µl O2
g–1 h–1 at normoxia to 277±14 µl
O2 g–1 h–1 at 2 kPa O2
in moderate hypoxic animals; and 728±3 6 µl O2
g–1 h–1 at normoxia to 253±12 µl
O2 g–1 h–1 at 2 kPa O2
in severe hypoxic animals). Baseline lactate levels for animals reared under
normoxic (2.971±0.29 mmol l–1) and severe hypoxic
(3.142±0.33 mmol l–1) conditions were not
significantly different after 12 h of severe hypoxic exposure
(3.810±0.45 mmol l–1 and 3.304±0.59 mmol
l–1, respectively). No lactate was observed in the any of the
water samples.
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Cardiac physiology
Heart rate response to hypoxic exposure was significantly different among
the rearing groups. At low PO2,
fH decreased in tadpole shrimp reared under normoxic
conditions from 281±6 beats min–1 at 20 kPa
O2 to 226±5 beats min–1 at 2 kPa
O2 (Fig. 3A).
Animals reared under moderate hypoxia also showed a significant decrease in
fH, from 262±11 beats min–1 at 20
kPa O2 to 223±8 beats min–1 at 2 kPa
O2. However, animals reared under severe hypoxic conditions did not
exhibit a change in fH with decreased
PO2 (275±9 beats min–1
at 20 kPa O2 to 238±10 beats min–1 at 2 kPa
O2). Tadpole shrimp reared under normoxic (0.23±0.01 µl
beat–1) and moderate hypoxic conditions (0.28±0.01
µl beat–1) had significant differences in VS at
10 kPa O2 (Fig. 3B).
Those reared under normoxic conditions decreased VS, whereas those
reared under moderate hypoxic conditions increased VS in response
to severe hypoxic exposure. Yet neither hypoxic response was statistically
significant due to the high variability of VS; mean standard
deviation was greater than the hypoxic response. Cardiac output was maintained
in animals reared under severe hypoxic conditions down to 2 kPa O2
(Fig. 3C). However, animals
reared under moderate hypoxic conditions decreased
from 64±6 µl
min–1 at 20 kPa O2 to 53±2 µl
min–1 at 2 kPa O2, and animals reared under
normoxic conditions decreased from
67±4 µl min–1 at 20 kPa O2 to
59±3 µl min–1 at 2 kPa O2.
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Hemoglobin
Hemoglobin concentrations measured as protein concentration were dependent
on rearing PO2 and were significantly altered
by chronic hypoxic exposure. Animals reared under normoxic (9.6±0.7
µg µl–1) and moderate hypoxic (2.8±1.4 µg
µl–1) conditions had significantly less protein
(hemoglobin) than those reared under severe hypoxic (19.8±0.9 µg
µl–1) conditions (Fig.
4). Concentrations in tadpole shrimp reared under normoxic
conditions and transferred to severe hypoxic conditions increased
significantly after 7 days (Fig.
5). After 10 days of severe hypoxic exposure, concentrations were
not significantly different from animals that had been reared under severe
hypoxic conditions. Adult tadpole shrimp reared under severe hypoxic
conditions and switched to normoxic conditions did not decrease the amount of
hemoglobin in their hemolymph for the 10 days investigated.
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The O2-binding affinity of hemoglobin was dependent on developmental PO2. Hemoglobin O2-binding affinity in animals reared under normoxic conditions was less than those reared under severe hypoxic conditions but not different from animals reared under moderate hypoxic conditions (Fig. 6). Hemoglobin cooperativity was significantly different among rearing groups (Fig. 7). Animals reared under normoxic conditions exhibited more cooperativity among hemoglobin subunits than those reared under moderate and severe hypoxic conditions. Oxygen-binding affinities and cooperativity values for hemoglobin from each rearing group are given in Table 1.
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Oxygen-carrying capacity and delivery potential
Tadpole shrimp O2-carrying capacity was dependent on
developmental PO2
(Table 1). Animals reared under
severe hypoxic conditions had 5.2x the O2-carrying capacity
of those reared under normoxic conditions. Maximal O2-delivery
potential for tadpole shrimp was dependent on developmental
PO2. Oxygen-delivery potential was
significantly lower in animals reared under normoxic conditions (274 µl
O2 min–1) relative to animals reared under severe
hypoxia (1336 µl O2 min–1)
(Table 1).
Oxygen consumption/oxygen transport coupling
The ratio of
O2/O2
was dependent on developmental PO2. Oxygen
consumption/transport ratio was significantly higher (consistently above 1) in
animals reared under normoxic conditions relative to those reared under
moderate or severe hypoxic conditions (Fig.
8). Ambient PO2 significantly
affected
O2/O2
in animals reared under normoxic and moderate hypoxic conditions. Animals
reared under moderate hypoxic conditions had a near coupling (1.18) of
O2 demand to cardiovascular supply at 20 kPa O2, which
decreased significantly with severe hypoxic exposure (2 kPa O2).
Animals reared under severe hypoxic conditions maintained
O2/O2
below 1 at each PO2 investigated.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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O2 may be
related to the frequency and duration of hypoxic exposure that an animal
typically encounters in its habitat (Chen
et al., 2001). Tadpole shrimp inhabit euryoxic environments that
can fluctuate daily by more than 26.7 kPa O2 or remain almost
anoxic (<1.3 kPa O2) for months
(Horne and Beyenbach, 1971;
Scholnick, 1995). In addition,
they vertically migrate from severely hypoxic (2 kPa O2) sediments
to hyperoxic (32 kPa O2) surface water in a relatively short period
of time (Scholnick and Snyder,
1996). Oxygen conformation to environmental
PO2 should be energetically favorable for
organisms frequently exposed to hypoxic conditions on many temporal and
spatial scales (Chen et al.,
2001). Tadpole shrimp would be considered oxygen conformers as
they did not tightly regulate
O2 when
PO2 was decreased
(Fig. 1). Instead, tadpole
shrimp reduced O2 demand to match O2 supply.
Sensitivity to hypoxic exposure may be altered in animals exposed to
hypoxic conditions throughout development due to ontogenetic changes in
physiological capabilities and metabolic demand
(Schulte, 2001). Adult animals
that develop under hypoxic conditions often have decreased metabolic rates and
decreased hypoxic sensitivity relative to animals that develop under normoxic
conditions (Pichavant et al.,
2001; Sokolova and Portner,
2001; Cech and Crocker,
2002; Chapman et al.,
2000; Hammond et al.,
2002). Yet developmental PO2 did
not affect adult tadpole shrimp metabolic rate or metabolic response to acute
hypoxic exposure. Tadpole shrimp that develop under hypoxic conditions must
balance immediate O2 demand with long-term requirements for
completing their life cycle within 30–40 days
(Horne and Beyenbach, 1971).
Therefore, transient decreases in
O2 in response
to ambient changes in PO2 may be more
beneficial than permanent decreases in
O2 that could
ultimately limit growth and development.
Anaerobic metabolism can be used to supplement aerobic metabolism and
decrease sensitivity to hypoxic exposure in many aquatic organisms
(Truchot, 1980;
Childress and Seibel, 1998).
Tadpole shrimp do not appear to utilize anaerobic metabolic pathways that end
in lactic acid production; hemolymph lactate levels did not increase after 12
h of severe hypoxic exposure (1.3 kPa O2). However, tadpole shrimp
may employ other anaerobic pathways that were not investigated by the current
study (i.e. pathways ending in pyruvate, valeric acid or alanine).
Ventilation
Organisms from a variety of phyla, including crustaceans
(Hervant et al., 1995), birds
(Faraci, 1991;
Maina, 2000), bats
(Maina, 2000), humans
(Gozal and Gozal, 2001;
Hoppeler and Vogt, 2001),
mussels (Chen et al., 2001),
fish (Galis and Barel, 1980;
Cech and Crocker, 2002) and
toads (Hou and Huang, 1999)
increase ventilation rate and/or volume in response to acute hypoxic exposure.
Tadpole shrimp apparently lack this typical ventilatory response or perhaps
the response was not observed due to the length of acclimation prior to data
collection (Fig. 2).
Ventilation rates did not increase in response to hypoxic exposure down to 1
kPa O2. Further, developmental PO2
did not affect ventilation rates under normoxic conditions or in response to
hypoxic exposure. Tadpole shrimp appendages are used for locomotion and the
generation of feeding currents, in addition to respiratory gas exchange
(Fryer, 1988). If ventilatory
rates were increased in response to hypoxic exposure, it could cause the
tadpole shrimp to swim faster and/or may alter the currents used for food
collection. Typical ventilatory hypoxic responses may be lacking because those
alterations may have adverse effects on locomotion and feeding.
Ventilatory amplitude was used in this study as an index of ventilation
volume. Ventilatory amplitude did not increase in response to hypoxic exposure
in any of the rearing groups. Below PCRIT, amplitude was
lowest in tadpole shrimp reared under severe hypoxic conditions but was not
significantly different from amplitude under normoxic conditions. Lower
ventilatory amplitude in animals reared under severe hypoxic conditions may
result from direct effects of O2 limitation on the respiratory
appendage movement or may be a strategy to minimize metabolic demand under
severe hypoxic conditions (Maina,
2000). Additionally, alterations in the angle of the appendages
could account for ventilatory volume changes without observed changes in
amplitude.
Cardiac physiology
Tadpole shrimp cardiovascular responses to hypoxic exposure follow a unique
pattern unlike that documented for other crustaceans
(McMahon, 2001). In most
crustaceans, acute hypoxic exposure results in a decrease in
fH (bradycardia) and concomitant increase in
VS to maintain or increase
(Reiber, 1995;
McMahon, 2001). Smaller
crustaceans, such as water fleas (Daphnia magna)
(Paul et al., 1998) and grass
shrimp (Palaemonetes pugio)
(Harper and Reiber, 1999),
increase fH (tachycardia) to maintain
in response to hypoxic exposure. In
tadpole shrimp, however, cardiac function appears to be highly insensitive to
hypoxic exposure (Fig. 3).
Again, it should be acknowledged that this may be an artifact of the
experimental protocol in that an acute hypoxic response may have been present
but not observed because it took place during the acclimation period. Those
animals reared under normoxic and moderate hypoxic conditions did not change
fH or VS when exposed to 5 kPa
O2. Below this, a bradycardia was observed and resulted in
decreased . All cardiac parameters
were maintained down to 1 kPa O2 in tadpole shrimp reared under
severe hypoxic conditions.
The difference between the cardiac response of tadpole shrimp and those of
other crustaceans may result from differences in mechanisms of cardiac
regulation. The heartbeat of many crustaceans is regulated through periodic
bursting activity of cardiac ganglion, excitatory neurons that innervate the
myocardium (Yamagishi et al.,
2000). Hypoxia-induced bradycardia in those crustaceans may be
mediated by a direct effect of lack of O2 to the cardiac ganglion
(Wilkens, 1999). Tadpole
shrimp hearts have a myogenic mechanism of regulation whereby the cardiac
muscle has endogenous rhythmic properties and does not rely on neural impulses
to contract (Yamagishi et al.,
1997; Yamagishi et al.,
2000). The heart of tadpole shrimp does not respond to hypoxic
exposure in the typical compensatory manner observed in other crustaceans.
Myogenic regulation apparently supports cardiac function over a wide range of
PO2s, including exposure to severe hypoxic
conditions.
Hemoglobin
Hemoglobin concentrations obtained in this study were comparable to
previously reported concentrations for Triops
(Horne and Beyenbach, 1974;
Scholnick and Snyder, 1996;
Guadagnoli et al., 2005).
Hemoglobin concentration was higher in animals that developed under severe
hypoxic conditions, yet was not proportional to developmental
PO2 (Fig.
4). Animals reared under moderate hypoxic conditions produced less
hemoglobin than animals reared under either normoxic or severe hypoxic
conditions. Moderate hypoxic conditions may represent the level of
PO2 in which O2 uptake is sufficient
to meet metabolic demand. Alternatively, it may represent a trigger for
genetic up- or down-regulation of hypoxia-inducible genes prior to the
translation of hemoglobin protein subunits.
Hypoxia-induced hemoglobin synthesis appears to be a compensatory response
that allows T. longicaudatus and several other branchiopods to
regulate O2 uptake and transport in their euryoxic habitats.
Hypoxic exposure (4–5 kPa O2) induced more than a 10-fold
increase in hemoglobin concentration within 10 days in adult D. magna
(Goldmann et al., 1999) and a
threefold increase within three weeks in adult brine shrimp (Artemia
salina) (Gilchrist,
1954). Hypoxic exposure (1–3 kPa O2) induced a
significant increase in protein concentrations within seven days in adult
T. longicaudatus (Fig.
5). Since hemoglobin is the only major protein in tadpole shrimp
hemolymph, protein concentration was accepted to be representative of
hemoglobin concentration (Horne and
Beyenbach, 1971). Within 10 days, hemoglobin concentrations
increased to the levels observed in tadpole shrimp reared under severe hypoxic
conditions. The induction of hemoglobin synthesis observed in T.
longicaudatus was less than the induction observed in A. salina
or D. magna. These results indicate that tadpole shrimp exposed to
hypoxic conditions acclimate by increasing hemoglobin concentration. Once the
Hb is produced, though, it remains even when animals are returned to normoxic
water (Fig. 5)
(Guadagnoli et al., 2005).
Hemoglobin O2-binding affinity was enhanced (decreased
P50) in tadpole shrimp reared under severe hypoxic
conditions relative to those reared under normoxic conditions
(Fig. 6). Differences in
O2-binding affinity are thought to have resulted from changes in
subunit assembly of the functional hemoglobin molecule
(Guadagnoli et al., 2005).
This has been observed in other branchiopods such as D. magna and
A. salina (Bowen et al.,
1969; Waring et al.,
1970; Sugano and Hoshi,
1971; Kobayashi et al.,
1988; Goldmann et al.,
1999). Specific assembly of hemoglobin subunits could be dependent
on environmental PO2, internal chemistry or
protein concentration (Sugano and Hoshi,
1971; Kobayashi et al.,
1988; Fago and Weber,
1995; Goldmann et al.,
1999). Since each subunit differs in O2-binding
affinity and cooperativity, changes in assembly directly affect
O2-binding affinity of the functional hemoglobin molecule
(Kobayashi et al., 1988). The
observed differences in hemoglobin O2-binding affinity and
cooperativity support the hypothesis that different
PO2 levels induce different hemoglobin isoforms
or differential subunit assembly in T. longicaudatus (Figs
6,
7).
Oxygen-carrying capacity and delivery potential
Tadpole shrimp reared under severe hypoxic conditions appear to have an
enhanced ability to transport and possibly store O2 obtained from
the environment due to their increased O2-carrying capacity
(Table 1). Tadpole shrimp
reared under severe hypoxic conditions had a fivefold increase in hemolymph
O2-carrying capacity compared with those reared under normoxic
conditions. Increased hemolymph O2-carrying capacity resulted from
increased hemoglobin concentration and O2-binding affinity observed
in those animals. Hemoglobin with high O2-carrying capacity can
often serve a storage function (Fago and
Weber, 1995). Tadpole shrimp frequently surface and expose their
respiratory appendages to the air–water interface. Tadpole shrimp may
obtain and store O2 from the higher
PO2 surface water for use during feeding and
hunting in severely hypoxic regions of the pool. Surfacing behavior has been
shown to increase with decreased PO2, further
supporting a storage function of tadpole shrimp hemoglobin
(Scholnick and Snyder,
1996).
Oxygen-delivery potential, which takes into account hemolymph O2
content and , appeared to be dependent
on developmental PO2. Maximal
O2-delivery potential for tadpole shrimp was lowest in animals
reared under normoxic conditions and highest in those reared under severe
hypoxic conditions (Table 1).
However, this was not a direct effect of developmental environment since
hemoglobin synthesis could be increased throughout the life of the animal.
Adult tadpole shrimp reared under normoxic conditions significantly increased
hemoglobin concentrations in response to severe hypoxic exposure.
Oxygen consumption/oxygen transport coupling
The coupling of
O2 with
cardiovascular transport reveals the level of coordination in tissue
O2 demand relative to O2 delivery
(Territo and Burggren, 1998).
In tadpole shrimp, differences in cardiovascular contribution observed among
the rearing groups were due to increased hemoglobin concentration and
O2-binding affinity since there was no observed cardiovascular
hypoxic response. Oxygen consumption of tadpole shrimp reared under normoxic
conditions was not as dependent on cardiovascular transport as it was in
tadpole shrimp reared under hypoxic conditions
(Fig. 8). The coupling of
O2 with
transport was consistently above 1 in tadpole shrimp reared under normoxic
conditions; however,
O2/O2
decreased in response to hypoxic exposure. This reduction suggests that
O2 was reduced
relative to cardiac output since (1) cardiac output did not increase in
response to hypoxic exposure and (2) hemoglobin levels remain constant during
acute hypoxic exposure. Animals reared under normoxic conditions increased
convective processes to supply O2 demands or relied on diffusional
processes when exposed to severe hypoxic conditions.
Cardiovascular contribution to O2 delivery was greatest in
animals reared under severe hypoxic conditions. Oxygen delivery was enhanced
in those animals via increased hemoglobin concentration and
O2-binding affinity. The increased O2 delivery to the
aerobic, metabolically active heart muscle may have supported cardiac
contraction at normoxic rates under hypoxic conditions. Tadpole shrimp reared
under severe hypoxic conditions maintained fH and
when exposed to severe hypoxic
conditions while the other rearing groups did not. It was concluded that as
tadpole shrimp produce more hemoglobin, they increase O2 delivery
relative to O2 supply and enhance cardiac function during hypoxic
exposure.
Conclusions
Developmental PO2 may not be sufficient to
induce permanent changes in adult tadpole shrimp physiological capabilities.
Metabolic rate, ventilatory rate and metabolic response to acute hypoxic
exposure were independent of developmental PO2.
Differences in cardiac response to hypoxic exposure among the rearing groups
were probably due to compensatory increases in hemoglobin concentration and
O2-binding affinity rather than to developmental differences. Based
on the results of this study, hypoxia-induced hemoglobin synthesis represents
an effective compensatory mechanism that allows tadpole shrimp to flexibly
regulate O2 uptake and transport under euryoxic conditions.
Differences that result from increased hemoglobin concentration should not be
considered developmental effects until it is shown that (1) hemoglobin type or
subunit arrangement is dependent on developmental
PO2 and not directed by ambient
PO2 during hemoglobin synthesis or (2)
hypoxia-inducible genes were switched on during development and are
continually expressed in the adult even if the animal is exposed to normoxic
conditions.
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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7). In some cases, the error
bars were smaller than the symbols.
