Plastic and evolved responses of larval tracheae and mass to varying atmospheric oxygen content in Drosophila melanogaster
Section of Organismal, Integrative and Systems Biology, School of Life Sciences, Arizona State University, PO Box 874601, Tempe, AZ 85287-4601, USA
* Author for correspondence (e-mail: joanna.henry{at}asu.edu)
Accepted 9 July 2004
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Summary |
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Key words: Drosophila melanogaster, tracheae, hypoxia, hyperoxia, selection, hypertrophy
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Introduction |
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In addition to affecting the tracheal system, rearing oxygen level may also
affect insect body size. There is now considerable interest in the possibility
that increased atmospheric oxygen level might correlate positively with
maximal body size, across historical periods and present-day environments
(Graham et al., 1995;
Dudley, 1998
;
Chapelle and Peck, 1999
). In
support of this hypothesis, adult fruit fly body size is increased by
hyperoxia and decreased by rearing in hypoxia
(Frazier et al., 2001
). Such
correlation could be driven by developmental plasticity of body size (as in
Frazier et al., 2001
) or by
evolutionary changes in insect size in response to changes in atmospheric
oxygen. Interestingly, because changes in tracheal diffusing capacity could at
least partially compensate for change in atmospheric
PO2, plastic or evolved changes in tracheal
dimensions seem likely to reduce effects of atmospheric
PO2 on body size.
In the present study, we test for both a developmentally plastic response and a heritable response to atmospheric oxygen (10, 21 and 40% oxygen) for dimensions of the main dorsal tracheae (DT) and larval masses of the fruit fly, D. melanogaster. The simplicity of the system, ease of visualization (the tracheae of larvae are visible through the cuticle with a dissecting microscope) and short generation time make larval fruit flies an ideal model for the study of oxygen effects on tracheal morphology. We hypothesize that, like mealworms, dimensions of the DT are regulated during development in order to compensate for oxygen availability, and such compensation will be heritable and maintained after sustained, multiple-generation exposure to hypoxic or hyperoxic atmospheres. We further propose that oxygen can influence the evolution of body size in fruit flies and can act as a limiting factor if flies are artificially selected for large body size. In one experiment, flies were reared for multiple generations in different atmospheric oxygen levels, with females being randomly mated. In an artificial selection experiment designed to test whether oxygen is limiting, only the largest females were allowed to mate and found the next generation. In these experiments, flies were reared for 56 generations in the test oxygen levels and then subsequently reared for two generations in common normoxic conditions. This two-generation common-garden design at least partially controls for plasticity and parental effects and allows us to test for heritable responses of larval mass and DT diameter to atmospheric oxygen level. In this paper, we report the effect of two trials of these experiments on the masses and diameters of the DT of third-instar larvae.
During the 1st, 2nd and early 3rd instars, D. melanogaster possess
only two functional spiracles, located at the posterior end of the animal,
that open into two DT (Manning and
Krasnow, 1993). While late 3rd instars use two anterior spiracles
in addition to the aforementioned posterior spiracles
(Manning and Krasnow, 1993
),
diameters along the DT may be more affected by
PO2 depending on proximity to the posterior
spiracles if oxygen affects tracheal dimensions during the earlier stages of
development. Unlike many other insects, the spiracles of fruit fly larvae lack
valves and do not close except by fine hydrophobic hairs that fold over the
spiracular openings when the animal becomes submerged in liquid medium
(Manning and Krasnow, 1993
).
Since the posterior spiracles are continuously open, and the diameters of the
spiracles are similar to the diameters of the DT, major resistance to gas
exchange must occur in the tracheae and tracheoles. DT diameters tend to
decrease posterior-to-anterior by segment in larval flies
(Beitel and Krasnow, 2000
).
Because the posterior sections of the DT must supply oxygen to an entire
larva, while the anterior sections of the DT need only supply the cranial
regions of a larva, it seems possible that the posterior regions of the DT may
be more sensitive to atmospheric oxygen level.
Gas exchange is likely to be primarily diffusive in larval D.
melanogaster (Krogh,
1920) since these insects are small (<2 mg) and lack air sacs
(Manning and Krasnow, 1993
).
In the present study, we calculate tracheal diffusing capacities of the DT to
gain insight into the degree to which changes in diameters of the DT
compensate for changes in atmospheric oxygen. Using larval metabolic rates
(Berrigan and Pepin, 1995
), we
also estimate the drop in PO2 from the
spiracles to the anterior termini of the DT.
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Materials and methods |
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To test for possible evolved responses, D. melanogaster were
reared in three different oxygen levels (10, 21 and 40% oxygen) over five to
six generations in two trials each of two types of experiments
(Fig. 1). In the oxygen
selection experiment (OS), females were randomly mated with males; this study
is a `laboratory natural selection experiment'
(Gibbs, 1999) in which oxygen
may serve as a selective agent affecting larval body or tracheal sizes. In the
artificial selection experiment (AS), the females with the largest body masses
were chosen to mate and found the next generation. After the flies were reared
under the OS or AS conditions for 56 generations, flies were reared for
an additional two generations (generations 78) in normoxic (21%
O2) conditions. We tested for significant evolutionary effects of
rearing oxygen level on larval masses or tracheal diameters by comparing these
values within generations 78. Investigation of the effect of OS and AS
on adult body mass is continuing and these results will be reported
elsewhere.
|
Study organism
To ensure that stock flies were variable and to increase the likelihood
that experimental responses observed are general to D. melanogaster,
rather than specific to a particular strain, our experiments were conducted
with fly stocks recently collected from a diversity of locations.
Drosophila melanogaster used in the first multi-generation trial were
captured from the wild by Melanie Frazier in the state of Washington. Fruit
flies used in the second multi-generation trial and first plasticity trial
were purchased from Carolina Biological Supply Company (Burlington, NC, USA).
Drosophila melanogaster used in the second plasticity trial were
captured from a compost heap in Tempe, Arizona. All populations were
maintained in the lab as outbred stocks for no more than five generations
prior to starting the experiment.
Rearing conditions
Flies were reared in 237 ml plastic vials containing 50 ml of a diet
composed of dextrose, agar, yeast, cornmeal, Tegosept antifungal agent
(Genesee Scientific, San Diego, CA, USA) and water. Ascorbic acid (0.1 g per
50 ml media) was also added to the media while boiling to possibly reduce
oxygen toxicity effects that the hyperoxic flies may experience
(Fridovich, 1998;
Sohal and Weindruch, 1996
).
The plastic vials were housed in 5 liter glass bottles that were all kept in a
single temperature-controlled incubator set at 25°C (range of
temperatures: 24.225.5°C) and lit using a fluorescent lamp set to a
14 h:10 h L:D cycle. At this air temperature, rearing oxygen level has strong
effects on adult body size (Frazier et
al., 2001
).
Drosophila melanogaster were reared from egg to adult in hypoxic
(10%; 9.7 kPa O2), normoxic (21%; 20.4 kPa O2) and
hyperoxic (40%; 38.8 kPa O2) conditions; the balance of the test
gasses was N2. For the first trial of the multi-generation
experiments, airflow from each gas cylinder was kept near 100% relative
humidity by bubbling air through a water reservoir. For the second
multi-generation trial and both of the plasticity trials, relative humidity
was kept near 65% by directing saturated 25°C air through a 19°C water
bath to reduce condensation of water along the sides of the rearing bottles.
Airflow rates through each glass chamber were set at 15 ml
min1, as measured by a Cole-Parmer flowmeter (model N032-41;
Vernon Hills, IL, USA). Excurrent air was monitored using an Ametek S-3A/I
oxygen analyzer (Paoli, PA, USA) to confirm that oxygen levels within each
chamber were very similar to air coming from the gas cylinders. Measurements
using similar vials and flow rates indicate that air within 1 mm of the fly
media had oxygen levels within 0.1% of the incurrent air
(Frazier et al., 2001).
Virgin collection and mating
Rearing vials were monitored each morning for the presence of newly emerged
flies. Because their age was unknown, these newly emerged adult flies were
anesthetized using CO2 and frozen. We collected flies no more than
810 h later, to ensure the virginity of collected females
(Ashburner, 1989a). We
anesthetized flies on a CO2 flowbed and sorted them by sex. After a
24 h isolation period, we placed 30 females individually in 12x75 mm
culture tubes containing two males and 1 ml of fly media. After 24 h, 15
pregnant females were placed in each plastic bottle and allowed to lay for two
days; this method ensured that population density remained nearly constant for
each generation.
Multi-generation experiment protocols
The protocols for the OS and AS experiments are shown in
Fig. 1. At the beginning of
each experiment, 180 founding females were removed from the outbred stock and
placed in 12 new media bottles, initiating generation 0 (15 females per
bottle; two bottles per treatment). These bottles were kept in humidified 21%
O2 chambers until the generation 0 adults began to eclose. One
hundred and eighty randomly chosen virgin females were mated and allowed to
lay their eggs in 12 new bottles kept in the three different oxygen levels.
For each subsequent mating in the OS experiments, 90 of the females (30 per
oxygen level) were randomly chosen to found the next generation. For the AS
experiments, 120 newly emerged females were removed from their respective
oxygen levels, weighed and sorted. The 30 heaviest were mated and allowed to
found the next generation. After 56 generations, flies were once again
randomly chosen from each O2 level and selection group and were
reared for two generations in normoxia.
Collection of larvae
Larvae begin to search for a pupation site during the late third instar
when they are approximately 120 h old
(Ashburner, 1989b). This
behavior facilitated the identification of larvae that were of the same
developmental stage and also allowed them to be easily collected from the
sides of the plastic bottles. In both the OS and AS experiments, 30
wandering-stage third-instar larvae were removed from each O2 level
and were washed in 100 mOsm sucrose/200 mOsm NaCl to remove food particles and
debris. If measurements of the larvae were to be taken at a later date, the
animals were kept in the solution and frozen. If the larval masses and
tracheal dimensions were immediately analysed after collection, the larvae
were gently rolled on a paper towel to dry. Larvae were weighed using a
Mettler AE 240 microbalance (±0.01 mg; Mettler-Toledo, Inc., Columbus,
OH, USA). Preliminary experiments indicated that freezing did not affect
tracheal morphology so, in the second plasticity and multi-generation trials,
larvae were frozen after collection and images taken later.
Dimensions of the DT
Individual larvae were placed in separate wells on a microwell plate. Each
well contained two drops of isosmotic solution to improve visibility of the DT
through the cuticle and to prevent desiccation and oxidation of the frozen
animals. Images (26x and 70x) were taken with a Hitachi HV-C20M
3CCD camera (Hitachi Denshi, Ltd, Tokyo, Japan) on a Cambridge Instruments
dissection microscope (Leica Microsystems Inc., Bannockburn, IL, USA),
digitized with a Scion Image Grabber Card (model CG-7; Scion Corp., Frederick,
MD, USA) and analyzed using Scion Image software (Scion Corp.). A small piece
of 0.18 mm diameter Stren® fishing line placed at the bottom of a
microwell was used to convert tracheal dimensions in pixels to µm.
Diameters were measured at six different positions along one of the DT
during the first plasticity and multi-generation trials. Measurements were
taken at the anterior anastomosis, at the second, fourth, sixth and eighth
transverse connectives and at the posterior anastomosis (as defined by
Manning and Krasnow, 1993;
Fig. 2). Because results in our
first trials indicated that most morphological changes occurred in the
posterior regions (see Results), only the tracheal diameter near the posterior
anastomosis was measured during the second plasticity and multi-generation
trials (Fig. 2, between e and
f). The lengths of the DT were measured from the posterior to anterior ends.
While only the external tracheal diameters could be measured using this
procedure, the thickness of the cuticle is assumed to be negligible as
measurements of luminal diameters reported by Beitel and Krasnow
(2000
) are similar to those of
this study.
|
Calculation of diffusing capacity, and PO2 gradients
The mean diffusing capacity (GDT; µmol
kPa1 s1) for oxygen moving longitudinally
down a single DT was calculated as:
![]() | (1) |
We calculated the mean partial pressure gradient
(PO2; kPa) along the entire length of
both DT if gas exchange occurred totally by diffusion as:
![]() | (2) |
The molar rate of oxygen consumed
(O2; µmol
s1) for an active larval fly was estimated as:
![]() | (3) |
Statistical analyses
Statistical analyses were carried out using SYSTAT 10.2 (SPSS, Chicago, IL,
USA) software with our experimental type I error less than or equal to 5%. We
used analysis of variance (ANOVA) to compare larval responses to different
oxygen treatments. Post-hoc multiple comparisons of responses within
an oxygen treatment over multiple generations were done using a Bonferroni
correction. Analysis of covariance (ANCOVA) and linear regressions were also
used in the analysis of mass effects on tracheal diameter. Values are shown as
means ± S.E.M. unless otherwise noted.
P-values are not reported when not significant.
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Results |
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Oxygen effects on larval mass
In general, higher rearing oxygen levels produced larger larval mass in the
plasticity trials but did not produce consistent heritable effects on larval
mass. In the plasticity trials, there was no effect of trial on larval masses
(ANOVA, F1,538=0.445, P>0.05); therefore, the
data were pooled across trials. After one generation, atmospheric oxygen had
significant effects on larval mass (Fig.
3; ANOVA, F2,528=10.85, P<0.001).
Pair-wise comparisons of the larval masses showed that those raised in 21% and
40% O2 were different from those raised in 10% O2
(post-hoc Bonferroni corrected, ANOVA, P<0.001) but not
different from each other (Fig.
3).
|
Because the larval body masses from the multi-generation trials were significantly different (ANOVA, F2,1137=2.38, P<0.001), these data were not pooled. In the first multi-generation trial including data for all generations, there was no significant effect of oxygen on mass (Fig. 4A). For the second multi-generation trial, there was a significant effect of oxygen on larval mass (Fig. 4B; ANOVA, F2,701=9.56, P<0.001), but the directional effect of O2 differed each generation (ANOVA, oxygen x generation, F8,689=6.16, P<0.001). Linear regression analyses showed that tracheal diameters did not significantly scale with mass in any generation or trial.
|
Position effects on tracheal diameters
The DT were narrower in the anterior portion of the larva (further from the
functional spiracles; Fig. 5).
Oxygen level affected tracheal diameters in the posterior but not anterior
regions of larvae (ANOVA, oxygen x position,
F10,466=3.03, P=0.001;
Fig. 5). Therefore, tracheal
diameters were measured only near the posterior anastomosis in the second
plasticity and multi-generation trials.
|
Scaling of tracheal diameters with larval mass
Because the larval masses and tracheal diameters differed significantly by
trial, the data could not be pooled. Linear regressions showed that tracheal
diameters did not significantly scale with mass in any generation or trial.
Within any given trial or generation, there was no significant effect on
tracheal diameters in different oxygen levels with mass as a covariate.
Developmental plasticity: effect of single-generation exposures to different atmospheric oxygen levels on tracheal diameters
Tracheal diameters were negatively correlated with rearing oxygen levels in
the plasticity trials. Because the second trial used only measures from the
posterior region while the first trial averaged all regions, the data were not
pooled across trials. In the first plasticity trial, mean tracheal diameters
from hypoxic larvae were 5% larger than those raised in normoxia, and those
from hyperoxic larvae were 4% smaller. In the second trial, hypoxic larval
tracheal diameters from the posterior region of the animal were 7% larger, and
hyperoxic larval tracheal diameters were 3% smaller than their normoxic
counterparts (Fig. 6). These
changes in the tracheal diameters with oxygen were significant in both trial 1
(ANOVA, F2,83=9.92, P<0.001) and trial 2
(ANOVA, F2,164=17.76, P<0.001).
|
Effect of multi-generation exposures to different atmospheric oxygen levels
Rearing oxygen level produced heritable, compensatory changes in tracheal
diameters. In all but one generation across both trials, flies raised in 10%
O2 had larger tracheal diameters than those raised in 21%
O2, while those raised in 40% O2 had the thinnest
tracheae (Fig. 7). The negative
relationship between rearing oxygen level and tracheal diameter persisted
after two generations of rearing in 21% O2, indicating that rearing
in 10% or 40% O2 for 56 generations produced heritable
changes in tracheal diameters. In both the first and second trials, there was
a significant negative linear relationship between oxygen content and diameter
that persisted after all flies were reared in the common-garden conditions
(trial 1 diameter=42.970.20O2,
r2=0.26, P<0.0001; trial 2
diameter=47.900.24O2, r2=0.20,
P<0.0001).
|
Effect of rearing oxygen level on tracheal diffusing capacities and required PO2 gradients
The diffusing capacities of the DT in the first plasticity and
multi-generation trials decreased linearly with similar slopes as atmospheric
PO2 increased
(Fig. 8; slopes not
significantly different). The changes in diffusing capacity partially
compensate for the change in air PO2
(Table 1). In hypoxia,
diffusing capacity increased 8% in the multi-generation trial and 12% in the
plasticity trial, while in hyperoxia it decreased 11% in the multi-generation
trial and 15% in the plasticity trial. In both trials, there was a significant
effect of oxygen on calculated PO2
across the length of the DT, with the % change in
PO2 being similar to the % change in
diffusing capacity (plasticity trial ANOVA,
F2,82=27.6, P<0.0001; multi-generation trial
ANOVA, F2,57=4.51, P<0.02).
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Discussion |
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What mechanisms might be responsible for the capacity of tracheal diameters
to respond during development to rearing oxygen level? Tracheal diameters
generally enlarge during larval growth in insects
(Manning and Krasnow, 1993),
so oxygen mediation of tracheal diameters may represent modulatory control of
a normal morphogenic process. In Drosophila, no cell division is
observed when tracheal diameters grow during normal development, suggesting
that changes in tracheal diameter may be due to changes in tracheal cell size
(Madhavan and Schneiderman,
1977
) or cell shape. It has been shown that, during the embryonic
dilation of the DT, the luminal diameter increases while there is little
change in the basal surface of tracheal cells
(Beitel and Krasnow, 2000
). How
might oxygen affect tracheal cell growth? Fruit flies exhibit a heterodimeric
basic helix-loop-helix/Per-Arnt-Sim hypoxia-inducible factor (HIF) system
analogous to that seen in vertebrates
(Lavista-Llanos et al., 2002
).
Hypoxia induction of levels of the Similar protein (analogous to HIF1-
)
is greatest in tracheal cells, demonstrating that the tracheae have cellular
mechanisms for sensing and responding to oxygen
(Lavista-Llanos et al., 2002
).
Increases in tracheal length are induced by a nitric oxide/cyclic GMP pathway
(Wingrove and O'Farrell,
1999
), suggesting that activation of Similar may produce changes
in tracheal dimensions via this pathway. Increases in tracheal
diameter only occur during molts (Beitel
and Krasnow, 2000
), suggesting that exposure to hypoxia in prior
developmental stages and the subsequent activation of Similar may be
responsible for the observed hypertrophy of the DT in 3rd instars.
Heritable effects on tracheal diameters and diffusing capacities
Rearing fruit flies for 56 generations in atmospheres of 10, 21 or
40% O2 produced heritable changes in tracheal diameters observable
after two generations of rearing in 21% O2
(Fig. 7). These heritable
changes allowed 815% compensation for atmospheric
PO2 (Fig.
8; Table 1); again,
it should be noted that changes in other parts of the tracheal system are
likely to have increased the magnitude of compensation by the entire tracheal
system. Perhaps due to small differences in experimental protocol such as the
use of different humidities and initial fly stocks, diameters seem to increase
in one trial and decrease in the other regardless of rearing oxygen. Despite
these differences, hyperoxia produced the smallest DT diameters and hypoxia
produced the largest diameters in both trials.
Heritable effects of rearing oxygen levels on tracheal diameters suggest that atmospheric oxygen level caused differential mortality or growth rates of flies. Our data suggest that flies that had small-diameter tracheae were more likely to reach adulthood and successfully reproduce in 40% O2 atmospheres, while flies with larger-diameter tracheae were more likely to reproduce successfully in 10% O2 atmospheres.
Rearing D. melanogaster in 10 kPa
PO2 at 25°C reduces survival by 15% and
growth rates by 30% and increases time to eclosion by 5%
(Frazier et al., 2001
).
Moderate hypoxia may exert these effects by reducing aerobic ATP production
and/or by limiting feeding. Hyperoxia also has deleterious effects on flies.
Oxygen levels above 45 kPa inhibit development in D. melanogaster,
with stage sensitivity to O2 being first instar > later instars
> pupae > eggs (Smith and Gottlieb,
1975
; Kloek et al.,
1976
). Reproduction can be inhibited at a
PO2 of 38 kPa
(Kloek, 1979
), though
obviously these flies reproduced. Hyperoxia is believed to cause toxicity by
stimulating the production of reactive O2 species that induce
teratogenesis and generalized cellular damage
(Lane, 2002
). For example, in
D. melanogaster, exposure to pure O2 at 33 kPa, or
O2 above 45 kPa in nitrogen, induces striking brain degeneration
and accumulation of dense bodies in neurons
(Philott et al., 1974
;
Kloek et al., 1978
) due
primarily to degradation of mitochondria
(Miquel, 1998
). While the role
of reactive O2 species in aging remains controversial, there is
considerable evidence that O2 consumption and hyperoxia induce
reactive O2 species such as superoxide, hydrogen peroxide and
hydroxyl radicals in D. melanogaster
(Sohal and Weindruch, 1996
;
Miquel et al., 1975
). Prior
studies have shown that fruit flies can adapt to hyperoxia
(Kloek and Winkle, 1979
); our
studies indicate that compensatory changes in tracheal diameters are a
component of this adaptation.
Oxygen effects on larval mass
In a previous study (Frazier et al.,
2001), adult flies were significantly larger (heavier and with
longer thorax lengths) in 40% O2 than in 21% O2, and
similar results have been found for adults in our multi-generation experiments
(J. F. Harrison, unpublished data). We found no effect of hyperoxia on larval
body masses. The lack of a significant increase in fruit fly larvae masses
when reared in hyperoxic conditions seems unlikely to be due to low
statistical power since there was not even a trend in this direction. These
results suggest that hyperoxic effects on adult body size are due to effects
occurring during the pupal stage.
Rearing in hypoxic conditions did cause smaller larval masses in the
developmental plasticity study, a result similar to that found for adults by
Frazier et al. (2001). In
hypoxia, adult flies were roughly 20% lighter than their normoxic counterparts
at 24°C (Frazier et al.,
2001
); third-instar fruit fly larvae had 10% less body mass in
hypoxia, again suggesting that effects on the pupal stage may be important in
determining the magnitude of developmental plastic response of adult mass.
Interestingly, multi-generation exposure to hypoxia produced no evolutionary
effects on larval body masses despite strong effects on adult mass (J. F.
Harrison, unpublished data), again suggesting that O2 effects on
pupae may be very important.
Diffusion in the DT
The calculated partial pressure gradient for oxygen along the DT if gas
exchange occurs by diffusion ranged from 4 to 8 kPa (57 kPa for
normoxic-reared flies), with the gradient decreasing at lower rearing oxygen
levels (Table 1). Thus, in
normoxic conditions, a 1415 kPa oxygen gradient is available to drive
oxygen transfer from the DT to the mitochondria. While a much smaller
gradient, 45 kPa, is available to drive oxygen transfer in hypoxia, it
is still sufficient to support metabolism. Moreover, since our calculations do
not account for increased branching in the tracheoles, as found by Jarecki et
al. (1999), the degree of
compensation and, consequently, the available O2 gradient may be
larger.
Conclusions
The flexibility in tracheal diameters in response to atmospheric oxygen in
fruit flies suggests that maintenance of tissue
PO2 within a relatively narrow range is an
important physiological function for fruit flies and perhaps animals
generally. Scientific literature suggests that insect tracheal systems are
overbuilt relative to need, with gas-phase oxygen delivery allowing high
oxygen consumption rates and very large safety margins for oxygen delivery.
However, the fact that hyperoxia can increase insect size
(Greenberg and Ar, 1996;
Frazier et al., 2001
) and
moderate hypoxia strongly affects growth and survival
(Loudon, 1988
;
Frazier et al., 2001
) suggests
that tracheal morphology and physiology must be tightly regulated to ensure
proper tissue PO2. Modulation of tracheal
diameters provides a mechanism for insects to strike a balance between
adequate oxygen delivery and excessive, toxic tissue
PO2 levels.
The evolutionary effects of atmospheric oxygen on tracheal diameters that
we observed in the lab suggests that variation in atmospheric
PO2 has influenced insect tracheal dimensions
in nature. These variations in atmospheric PO2
might be historical (Dudley,
1998) or associated with environmental exposure to hypoxic
conditions such as high altitude, soils, water or burrows with limited oxygen
availability (Hoback and Stanley,
2001
). Fruit flies develop in rotting fruits, which may experience
hypoxia due to microbial respiration. This capacity for evolutionarily
flexible tracheal structures has probably aided the evolution of insect
diversity in form and habitat.
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List of symbols |
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Acknowledgments |
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