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First published online August 17, 2006
Journal of Experimental Biology 209, 3476-3483 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02343
Intraspecific variation in tracheal volume in the American locust, Schistocerca americana, measured by a new inert gas method
1 Department of Biology, University of New Mexico, Albuquerque, NM 87131,
USA
2 School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501,
USA
* Author for correspondence (e-mail: hlease{at}unm.edu)
Accepted 22 May 2006
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Key words: respiration, trachea, scaling, American locust, Schistocerca americana
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) the volume of the tracheal system (VT)
also determines the amount of oxygen available during breath-holding, and thus
may determine the duration of the closed phase of discontinuous gas exchange.
Insects and many arthropods have developed complex tracheolar respiratory
systems that deliver oxygen directly to tissues and cells
(Chapman, 1998). The design and
performance of these systems probably varies with animal size, body plan, and
lifestyle, but relatively little is known about such variation, and how it
affects VT.
The relationship between VT and other insect life
history characteristics is almost certainly not a simple one. For example,
insects with increased metabolic rate are likely to have more extensive gas
delivery systems. Insect VT may vary with organism size,
metabolic rate, species type, mode of respiration (e.g. cyclic, non-cyclic, or
discontinuous ventilation), presence of air sacs, and developmental stage.
Tracheal volume may also change with reproductive or digestive status of an
organism. For example, oogenesis or a full crop may cause temporary reduction
in VT in insects with air sacs. In addition, it has been
previously shown that hemimetabolous insects accumulate mass within an instar,
and that because of a relatively inelastic exoskeleton, this growth can reduce
VT and reduce oxygen delivery in some insects
(Greenlee and Harrison, 2004a).
However, in general, the magnitude of such effects on VT
and oxygen delivery capacity are unknown.
Tracheal volumes of insects have been estimated using water displacement
(Wigglesworth, 1950),
stereology (Schmitz and Perry,
1999) and inert gas-mass spectrometry
(Bridges et al., 1980). The
water displacement method is quick and inexpensive, but requires death of the
animal, and may mis-measure VT due to water adherence to
the cuticle and lack of fluid infiltration of the tracheoles. Bridges, Kestler
and Scheid developed an inert gas wash-out and mass spectrometry technique to
assess VT (Bridges et
al., 1980). Their method provided the conceptual foundation for
our approach, as it demonstrated the practical use of inert gases to measure
VT. However, the flow-through mass spectrometer system
used is expensive, notoriously difficult to maintain, and not widely
available. Also, their method relies on the insects exhibiting discontinuous
gas exchange (DGC); chamber flushing occurs during the closed DGC phase of the
respiratory cycle. Since many insects do not predictably exhibit DGC
(Lighton, 1998), comparative
studies using this method are limited. Schmitz and Perry
(Schmitz and Perry, 1999)
developed a stereological approach to assessing VT. Using
light microscopy and electron microscopy, they made direct morphometric
estimates of VT. However, this approach is labor intensive
and is challenging to use on large insects, hampering its utility for
comparative studies. To date, no methods are available that allow repeated
estimates of VT on living insects that do not exhibit
discontinuous gas exchange.
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We first establish helium equilibration times for large and small grasshoppers, and characterize the system conditions that allow chamber flushing without significant loss of helium from the trachea. We then use this method to investigate how VT varies with body size, developmental stage, gender, and reproductive status in S. americana.
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).
For between-instar comparisons of S. americana, early instar animals
and adult males were used. For within-instar comparisons of S.
americana, fifth instar animals ranging from early to late instar were
used. The instar stage of each animal was determined by taking the ratio of
femur length to abdomen length, which decreases with age within an instar (S.
D. Kirkton, and K. J. Greenlee, personal communication); all animals
designated as early stage had femur to abdomen ratios of greater than 1. Wings
were removed with scissors from sixth instars and adults prior to inert gas
volumetry, because preliminary tests showed that they may hinder chamber
flushing of heliox during the rapid nitrox flush period.
Apparatus
Gas mixtures were supplied from pre-mixed tanks. These flowed via
Bevaline tubing and two computer-controlled solenoid valves (Clippard EVO-3;
Clippard Instrument Laboratory, Cincinnati, OH, USA) through a glass animal
chamber, and exited the chamber via a third computer controlled
solenoid valve (Fig. 1). Sable
Systems Data Acquisition hardware and software (Sable Systems International,
Las Vegas, NV, USA) were used to control the valves. In most cases, the volume
of the animal chambers used was approximately 7 ml, but for trials with larger
insects we used chambers with volumes up to 100 ml. Threaded aluminum end caps
with o-rings and vacuum grease (Dow Corning, Midland, MI, USA) were used to
seal both ends of animal chambers. Clippard barb fittings (Clippard 1/8 inch
to 10/32 inch) at the proximal ends of the chambers allowed gas entry and
exit.
First, the chamber containing the insect was ventilated with heliox gas mixture (21% oxygen, 79% helium) at a flow rate of 1 l min-1 (Brooks mass flow controller; Brooks Instruments, Hatfield, PA, USA) until the insect respiratory system had equilibrated with heliox (0-10 min; see Results). The chamber was then flushed with a nitrox gas mixture (21% oxygen, 79% nitrogen) at a flow rate of 5 l min-1 until essentially all helium had been eliminated from the chamber (0-3 s; see Results), after which time the chamber was sealed. After a period of time (0-10 min; see Results), sufficient to allow at least 99% of the helium to evolve from the insect respiratory system, a 2 ml sample of air was withdrawn from the chamber using a gas-tight Hamilton syringe, and then injected into a gas chromatograph (Varian Inc., Palo Alto, CA, USA; Model 3400) to measure helium concentration (CHe, mol l-1). The carrier gas used was ultra high purity N2 (30 ml min-1). Helium was separated from oxygen and carbon dioxide using a Gas Chrom MP-1 column (Varian), and the helium peak measured with a thermo-conductivity detector. Sable Systems Data Acquisition hardware and software (Sable Systems International, Las Vegas, NV, USA) were used to collect the gas chromatograph output and integrate the areas under the helium peaks. Calibration injections of helium (0-1000 µl), which overlapped the range of VT values we measured yielded linear calibration curves (R2=0.99) relating peak areas to moles of helium injected.
Validation of method parameters
It was necessary to optimize and standardize the time periods of the method
to ensure: (1) that helium was fully equilibrated in the insect respiratory
system during the pre-equilibration phase; (2) that nitrogen flushing was long
enough to remove helium traces from the chamber (and outside surface of the
animal), yet short enough that the insects had not begun to eliminate
significant amounts of helium from their respiratory systems; and (3) that all
of the helium had evolved from the insects during the wash-out phase. We did
this by varying heliox pre-equilibration time, nitrogen flushing time and
wash-out duration, while holding the other two variables constant. We also
tested whether these phase durations varied with size of organism by running
the optimization tests on both large (0.9-1.5 g) and small (0.04-0.20 g)
grasshoppers.
Calculation of tracheal volume
The gas chromatograph measures helium concentration,
CHe, in the sample of air taken from the chamber. This
concentration can be converted to a quantity of helium evolved from the
tracheal system (VTHe, µl), when corrected for
displacement of air within the chamber by the animal. The method requires
minimization of chamber volume to maximize chamber helium concentration and
reduce flush time, so a relatively small chamber volume was selected. To
calculate the effect of displacement of air within the chamber by solid
components of the animal, we placed dead grasshoppers of various sizes (with
sealed spiracles) into the chambers. 100 µl of helium was injected into the
chamber, and after equilibration, the concentration of helium in the chamber
was measured. As the size of animals in the chamber increased, the measured
helium concentration increased linearly, consistent with a simple displacement
mechanism. For example, with our most commonly used 7 ml chamber, we
calculated a displacement correction factor (DCF, unitless) that varied with
mass and corrects a measured helium concentration back to that which would be
calculated using an empty chamber as:
 | (1) |
Therefore, the volume of helium in the chamber (VCHe,
µl) was calculated as:
 | (2) |
In addition to the problem of animal displacement of space, the presence of
animals in the chamber can increase residual helium left after flushing due to
trapping of air in animal boundary layers, and the quantity of such trapped
helium is expected to increase with animal size. Therefore, we measured the
amount of residual helium after a 2 s nitrox flush for 14 animals, ranging in
body mass from 0.1 to 2.5 g, and found that residual helium volume (RHV,
µl) could be predicted by animal mass (g):
 | (3) |
RHV must be calculated for each combination of chamber, flush rate, and flush time, and DCF should be calculated for each chamber.
We calculated the volume of helium in the tracheal system
(VTHe, µl) as:
 | (4) |
We calculated tracheal volume (VT, µl) as:
 | (5) |
Experimental design
We measured VT using the IGV method on early-instar
second, third, fourth, and fifth instar S. americana, mid to
late-instar fifth instar S. americana, and adult male and female
S. americana. Based on preliminary data, a standardized IGV method
protocol of 3 min of helium:oxygen wash-in, 2 s of nitrogen:oxygen chamber
flushing, and 5 min helium wash-out in the sealed chamber was used for each
animal. For each individual, the wet mass (g), dry mass (g), femur length
(mm), abdomen length (mm), and, where relevant, sex and egg mass were
measured. For comparative purposes, VT was also estimated
for a subset of individuals using the water displacement method
(Bartholomew and Barnhart,
1984). Animals were placed in a 60 ml plastic syringe filled with
soapy water. The plunger was moved back and forth to draw air out of the
tracheal system and replace it with fluid. The outside of the insect was
blotted dry, and the increase in insect mass was used as a measure of
respiratory system volume, assuming water density=1 g ml-1.
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, Eqn 2);
this was probably because of helium trapped in boundary layers of the animal
cuticle. This was a minor correction (<5%) for adult grasshoppers, but was
a major correction (
50%) for the smallest, first instar grasshoppers.
Determination of time period required for helium wash-out from insects
To determine the length of time required for helium to wash-out of the
animal and equilibrate with the chamber air, we varied helium wash-out periods
(x axis; Fig. 2C)
while maintaining other parameters constant (3 min heliox exposure, 2 s nitrox
flush time). This was calculated using seven animals, ranging from second
instar to adult; six to ten different wash-out time periods were tested
independently for each animal. We found that a period of 2 min was sufficient
to allow for complete elimination of helium from the tracheal system of S.
americana, regardless of body size
(Fig. 2C).
One of the advantages of the IGV method is that it allows repeatable measurements of VT without damage to the animal. Six repeated measurements yielded coefficients of variation of 0.076 for a 0.5 g S. americana, and 0.12 for a 1.1 g animal.
Inert gas volumetric method parameters for Schistocerca americana of variable size
Differences in wash-out and wash-in times between grasshoppers of different
sizes were relatively small, although there was a trend towards shorter times
in smaller animals (e.g. 100 s to plateau in third instars vs
180 s to plateau in adults). Thus, throughout the remaining studies (i.e.
for all age classes of S. americana) we used method times established
as sufficient for the larger animals, to ensure helium equilibration.
Comparison of inert gas and water displacement methods for assessing insect VT
Tracheal volumes estimated using the water displacement method increased
each time the syringe plunger was pumped
(Fig. 3). This suggested that
repeated compression and expansion stretched the abdomen, allowing more water
to enter the body. To minimize this problem, for subsequent analyses, we
stopped applying a vacuum to the system when air bubbles stopped leaving the
spiracles. With both methods, VT increased with animal
mass (Fig. 4A). Within
individuals, there was a curvilinear relationship between
VT measured with IGV and water displacement (WD) methods
(Fig. 4B). In general,
VT measured for smaller animals were similar with both
methods, but the WD method provided VT estimates that were
15% to 250% higher than those obtained through the IGV method for larger
animals (Fig. 3C).
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).
Within an instar, however, VT decreases dramatically with
tissue growth due to the constraints of a semi-rigid exoskeleton. In adults,
we found that gender has a strong effect on VT; males and
gravid females had VT that were 40% and 7% of body volume,
respectively. Both ontogenetic and reproductive developmental changes thus
have the capacity to impact VT in insects, and may have
strong effects on respiratory function and life history variables.
Critique of inert gas volumetric method
The most significant potential error in the IGV method seems to be the
correction for residual helium in the chamber after the nitrox flush. For the
smallest insects we used (mass about 0.06 g, VT=13 µl),
helium remaining in the chamber after the nitrox flush was more than 50% of
VT. Although repeatability of VT
estimates was relatively good, even for small grasshoppers, it seems probable
that this could provide an important, difficult to resolve error. Thus we feel
that our system was pushed to near its limit of resolution for measuring
VT, which is unfortunate as most insects are under 1 mg in
size. However, this system could likely be adapted to function for very small
insects by substantially reducing chamber and tubing size, as we did not
challenge the resolution of the gas chromatograph.
A related source of error in the IGV method is increased helium in boundary layers of the insect due to structures such as wings and hair. We found that wings needed to be removed from adult grasshoppers in order to reduce residual helium to acceptable levels. Potentially this would be a major issue with insects with substantial cuticular hair such as bumblebees. We assessed the magnitude of helium adherence to the outside of the animal by measuring VCHe for dead animals whose spiracles had been sealed with wax, and we recommend this approach to gauge the magnitude of this potential error.
A final potential source of error in the IGV method is the correction for animal body displacement. When known quantities of helium are injected into chambers with animals (with plugged spiracles) and allowed to equilibrate with chamber air, a body displacement correction factor (DCF) can be calculated; this was done because animal body size will affect chamber helium concentration (via body displacement of total chamber volume). In our system, this turned out to be a minor correction factor for both large and small grasshoppers. The DCF ranged from 0.6 µl He (for a 0.07 g hopper; <5% VT) to 10.2 µl He (for a 1.2 g hopper; also <5% VT). However, reducing the chamber volume to body volume ratio might increase the importance of this correction factor.
Like all gases, helium is soluble in body fluids, and so helium dissolved
in body fluids has the potential to produce an overestimate of
VT. The solubility of helium in biological fluids is
approximately 67% of that of N2, and 33% of O2, so
tissue solubility is less of an issue than for other gases. Helium solubility
in blood plasma is approximately 0.007 atm-1
(Hlastala et al., 1980), and
this value provides a reasonable estimate of the solubility of helium in
grasshopper body water. Thus, grasshopper body water equilibrated with heliox
at STPD should contain 0.79x0.007=0.0055 ml of helium per ml of body
water. Total body water is 66% of the wet mass of Schistocerca
grasshoppers (Harrison, 1989).
Because proportional VT increases with mass (Figs
4,
5), the fraction of total
animal helium localized in body water will be greatest for small grasshoppers.
For the smallest grasshoppers (0.06 g, 13 µl VT, 0.3
µl He in body water) helium dissolved in the body water represents 2.3% of
the estimated VT, whereas for the largest grasshoppers
(1.6 g, 542 µl VT, 5.8 µl He in body water), helium
dissolved in body water represents only 1.1% of VT. In
either case, this is a small error compared to the variation among individual
grasshoppers, although for insects with a very small VT
[e.g. stick insects have VT only 1.3% of body volume
(Schmitz and Perry, 1999)]
this error could become relatively more important.
Finally, the various parameters of the IGV method should be checked when applying this method to different taxa, and animals of different sizes. In general, insects with higher metabolic and ventilation rates will require shorter, faster nitrox flushes to ensure that exhaled helium is not removed from the chamber during the nitrogen flush period. On the other hand, animals with particularly low metabolic and respiratory rates may require longer helium equilibration periods and wash-out periods.
Comparison of inert gas volumetric and water displacement methods for assessing insect VT
Wigglesworth developed (Wigglesworth,
1950), and others have since utilized
(Clarke, 1957;
Weis-Fogh, 1964;
Bartholomew and Barnhart, 1984;
Harrison, 1989), a water
displacement method to estimate VT. This method replaces
tracheal air with fluid, and the subsequent increase in insect mass is used as
a measure of respiratory system volume. The VT may be
underestimated by this method if tracheoles are not infiltrated with fluid
because of their small size. The VT may be overestimated
by this method because water adheres to cuticle. The magnitude of these errors
is difficult to estimate, but both are likely to be relatively greater issues
for animals without air sacs (and lower VT), whereas
water-adherence seems likely to be a greater problem for smaller insects
because of their higher surface/volume ratios. We also identified another
potentially major problem with the water displacement method here. It is
difficult to know whether one is actually expanding the animal when applying
syringe pressure; this causes over-inflation of air sacs and over-estimation
of VT when multiple syringe pumps are used with
grasshoppers (Fig. 3). Even
when we attempted to control for this problem by ceasing syringe pumps when no
further air was removed by suction from the animal, we found that this method
can substantially overestimate VT
(Fig. 4C).
Despite the mismatch in VT between measures made by the IGV and WD methods (Fig. 4C), across individuals, both the IGV and the WD methods predicted a similar increase in VT with animal mass (Fig. 4A). Thus the inexpensive and field-portable WD method is still of use for comparative studies, especially for animals that lack air sacs.
The IGV method may be particularly useful for measuring within-individual
(physiological) variation in VT. For example, the effect
of digestive status on VT can be investigated using the
IGV method. Reduction in VT after a meal may be important
since feeding increases metabolic rate
(Zanotto et al., 1993),
increasing the need for gas exchange. Feeding status effects on
VT may be particularly interesting for flying insects,
since a full crop simultaneously increases body mass (increasing the metabolic
cost of flying) and compresses air sacs (potentially impacting the safety
margin for oxygen delivery). Inter-individual developmental assessment of
VT might also be interesting using the IGV method for
holometabolous insects that have large and flexible trachea (but lack air
sacs) during their larval stage, and have air sacs and thin, rigid trachea in
their adult stages.
Finally, the IGV method may allow determination of VT
variation as a function of mode and intensity of gas exchange. Gas-exchange
patterns (e.g. discontinuous and continuous gas exchange) can change quickly
within individual insects and are linked to variation in metabolic rate
(Gibbs and Johnson, 2004).
Average VT measured over multiple cycles (e.g.
discontinuous gas exchange cycles, or abdominal pumps) may vary with the
magnitude of pressure differentials, and the IGV method provides a new and
exciting method to assess such variation.
Scaling of tracheal volume with body size in Schistocerca americana
Proportional VT increases as body size increases across instars,
but falls dramatically as mass increases within an instar
(Fig. 4). A similar pattern was
found using the water displacement method
(Clarke, 1957), and may
partially explain why oxygen delivery becomes more problematic as insects
progress through an instar stage (Greenlee
and Harrison 2004b). We speculate that the decrease in
VT within an instar is a common phenomenon as many insects gain
mass within each life stage and yet have relatively rigid cuticles.
Our data provide the strong quantitative data that relative investment in
the tracheal system increases across instar with age and size in S.
americana (Fig. 4). This
is a particularly interesting result given that lung and cardiac volumes
change isometrically during development in vertebrates
(Altman and Dittmer, 1974;
Weibel et al., 1981;
Gehr et al., 1981), whereas
mass-specific capillary densities decrease with age/size
(Weibel et al., 1981). We
found that VT scales with mass to the 1.3 for this species.
However, when we separate out the instars with the smallest
VTs (log VT<0.6), we find that the
slope of the line describing the relationship between VT
and body mass is no longer significantly different from 1
[VT=2.536+1.011xlog(mass), where
VT is in µl and mass in g; P<0.0001,
R2=0.79]. This `log VT<0.6' cut-off
may be arbitrary; when we exclude individuals with log mass<0.9, we get a
slope of 1.1 instead of 1.01 for the larger individuals. Regardless, the
exclusion of small instars indicates a smaller slope for the relationship
between VT and mass than when all individuals are
included. Our data thus suggest that very early instars have considerably
reduced tracheal volumes relative to older animals, but that tracheal volume
may change isometrically with body size for larger instars and adults. Despite
this, we think that the higher slope of 1.3 more accurately reflects the
overall trend of VT scaling for this species. At this time
we cannot determine to what extent the observed increase in
VT is due to increases in relative air sac and/or
tracheole content. Hartung et al. (Hartung
et al., 2004) showed a proportional increase with age in the
tracheal volumes of S. americana legs, which indicate increased
investment in tracheae, especially tracheoles. Greenlee and Harrison
(Greenlee and Harrison, 2004a)
showed that the percent compression of the abdomen during ventilation of
S. americana increased with age (first instars and adults), strongly
suggesting an increase in relative air sac content with body size.
Additionally, recent X-ray synchrotron imaging suggests that first instars
lack air sacs, and that second instars have very few air sacs (J.F.H.,
unpublished data from Argonne National Laboratory).
Increased relative investment in tracheal system structure in larger/older
S. americana explains many developmental patterns in respiratory
capacities. Tracheal system respiratory volumes do not appear to scale
isometrically with resting metabolism, which scales with mass0.8
(Greenlee and Harrison, 2004a).
However, mass-specific metabolic rates during jumping increase strongly with
mass (Kirkton et al., 2005),
and flying adults have metabolic rates much higher than juveniles
(Rascón and Harrison,
2005). Increased relative investment in the tracheal system
provides a partial mechanistic explanation for this increased metabolic
capacity. Also, this increased relative investment in the tracheal system
explains the greater respiratory capacity of older grasshoppers, observed as
larger safety margins for hypoxia [lower critical oxygen partial pressures;
tested across first, third and fifth instars
(Greenlee and Harrison,
2004a)]. This trend of increased tracheal investment with
increased age might allow insects in general to overcome diffusion limitations
as age and size increases. If so, we should see such increases in proportional
VT in interspecific comparisons as well.
Effect of sex on tracheal volume
Female S. americana clearly show reduced VT
compared to males (Fig. 6),
probably due to displacement of air sac space by eggs
(Fig. 7). Currently it is
unknown whether this reduced volume translates into a performance deficit. Egg
mass displacement of respiratory volume is probably widespread in other
species. It is also interesting to note that egg accumulation may temporarily
increase the oxygen consumption needs of insects
(Taylor and Leelapiyanart,
2001) and at the same time, as our data suggests, possibly
compromise oxygen delivery capacity. Clearly an important next step is to
examine how this phenomenon affects performance.
Conclusions
Variation in metabolic capacity in insects can be reflected by respiratory
structure and volume in diverse ways. We have documented here a new,
repeatable method to measure insect VT. We have also shown
here that proportional investment in the tracheal system increases with
age/size in this species. The scaling exponent of 1.3 for
VT exceeds scaling relationships of 0.8 for metabolic
rate, and partially explains the enhanced respiratory capacity found in larger
grasshoppers (Greenlee and Harrison,
2004a). Both of these findings contradict the argument that gas
exchange is more difficult for larger insects. The trend we found, of
increased proportional investment to VT in larger insects,
probably interacts with the increased use of convection that has been observed
in larger insects (Greenlee and Harrison,
2004a) to allow insect respiratory systems to compensate for
increasing size.
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Acknowledgments |
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