Development of respiratory function in the American locust Schistocerca americana : II. Within-instar effects
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: kendra.greenlee{at}asu.edu)
Accepted 22 October 2003
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Summary |
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Key words: ontogeny, insect, locust, Schistocerca americana, ventilation, gas exchange, hypoxia
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Introduction |
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Oxygen delivery demands depend not only on body mass but also on
mass-specific oxygen consumption
(O2). Therefore,
the within-instar variation in gas exchange requirements depends on how
mass-specific metabolic rate changes during that period. In arthropods, this
pattern can be complex. For example, in the crustacean Nephrops
norvegicus, mass-specific metabolism does not vary within a molt cycle,
so changes in total oxygen consumption parallel the increase in body mass
(Alcaraz and Sarda, 1981
). In
grasshoppers (Locusta migratoria), shrimp (Xiphonpenaeus
kroyeri) and spider crabs (Hyas coarctatus and Libinia
ferreirae), variation in mass-specific metabolic rate within the instar
depends on the animal's developmental stage
(Clarke, 1957b
;
Jacobi and Anger, 1985
;
Anger et al., 1989
;
Carvalho and Phan, 1998
).
Insects with air sacs may be particularly susceptible to developing a deficit
in respiratory capacity relative to tissue oxygen needs because, as they grow
and increase in mass within an intermolt period, the chitinous integument may
constrain increases in animal volume, leading to compression of the larger,
air-filled tracheae and unsclerotized air sacs. In fact, tracheal system
volumes decrease by 90% during the fourth instar in Locusta
migratoria (Clarke,
1957a
).
In the present research, we tested the hypothesis that Schistocerca
americana grasshoppers at the end of an instar (late-stage animals) have
reduced capacity for oxygen delivery relative to oxygen demand. We predicted
that the late-stage animals would have decreased safety margins for
O2 delivery compared with early-stage individuals due to reduced
maximal tracheal conductances relative to tissue gas exchange needs.
Specifically, if grasshopper body mass doubles during an instar, then (1)
metabolic rate should increase by about 70%
(Schmidt-Nielsen, 1984). If
there is no ventilatory or morphological compensation, then (2) the safety
margin for O2 delivery should decrease by a similar percentage as
animals develop through an instar. We also predicted that late-stage animals
would have (3) reduced tidal volumes due to increased tissue mass and
decreased air-space and (4) increased ventilatory frequencies to compensate
for the reduced tidal volumes and greater gas exchange needs. Finally, we
predicted that (5) tracheal or air sac compression should result in decreased
maximal tracheal conductance during an instar and perhaps a greater decrease
in the safety margin for oxygen delivery than predicted by the rise in
metabolic rate.
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Materials and methods |
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Statistics
Statistical tests were performed with SYSTAT 10.2.01. We used one-tailed
significance tests (P<0.1) for all the tests, since we predicted
directions of change. To detect variation in normoxic ventilatory parameters
and maximal tracheal system conductance, we used analysis of variance with
instar and stage as independent variables. We also used t-tests to
identify the effect of stage within specific instars. To determine how stage
within an instar affected the response to hypoxia, we used repeated-measures
analyses of variance (ANOVAs), since each animal was exposed to multiple
levels of PO2. In these tests, instar and stage
were independent variables and PO was the repeated
variable. We also used repeated-measures ANOVAs to test for significant
differences within a particular instar. We used non-parametric tests for
statistical tests of Pc values, because these were
discrete variables. To test for the effect of stage within an instar on the
Pc for mass-specific CO2 emission rate
(CO2) and for
abdominal pumping, we used the Scheirer-Ray-Hare extension of the
Kruskal-Wallis test, a non-parametric two-way ANOVA
(Sokal and Rohlf, 1995
). We
also used Mann-Whitney U tests to detect differences within a
specific instar.
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Results |
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Response of CO2 emission to hypoxia
The effect of stage on mass-specific
CO2 during
progressive hypoxia depended on the animal's instar (repeated-measures ANOVA,
PO x instar x stage interaction,
F9,540=2.4, P<0.02;
Fig. 4). When we tested each
instar separately, there was a significant interaction between
PO and stage for each instar (repeated-measures ANOVA,
PO x stage interaction within each instar; instar 1
- F10,140=1.65, P<0.1; instar 3 - F11,154=5.32,
P<0.001; instar 5 - F11,154=3.71, P<0.001;
adult - F11,154=4.07, P<0.001), indicating that animals
at different stages within the instar responded differently to hypoxia.
However, much of the difference appeared to be due to lower mass-specific
CO2s later in
the stage, regardless of PO2. There was no
overall effect of stage within an instar on Pc for
CO2. For first,
third and fifth instars, early- and late-stage animals did not differ
significantly in the Pc for
CO2; however,
for adults, animals later in the stage had a higher Pc
(Fig. 5; Mann-Whitney
U test, U=12.5, P<0.02).
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Tidal volume in hypoxia
We used percent change in abdominal height as an index of tidal volume and
were able to statistically compare the responses of first-instar and adult
grasshoppers at three common PO levels (21, 5 and 1 kPa;
Fig. 3). The response of
abdominal pumping height to PO varied with stage depending
on the instar (repeated-measures ANOVA, PO x stage
x instar interaction, F2,56=3.3,
P<0.05). When we analyzed each instar separately, we found a
significant interaction between PO and stage for adults
(repeated-measures ANOVA, F4,56=12.9, P<0.001;
Fig. 3B) but not first instars
(Fig. 3A). Late-stage adults
had lower tidal volumes in normoxia but had similar maximal tidal volumes as
early-stage adults in 5 kPa PO
(Fig. 3B).
Ventilation frequency
Abdominal pumping frequency in hypoxia varied with stage depending on the
animal's instar (repeated-measures ANOVA, PO x stage
x instar interaction, F10,600=4.6, P<0.001;
Fig. 6). When we examined each
instar separately, we found significant interactions between
PO and stage on ventilation frequency in every instar
(repeated-measures ANOVA, instar 1 - F10,140=2.5, P<0.01;
instar 3 - F10,140=9.7, P<0.001; instar 5 -
F10,140=8.5, P<0.001; adult - F10,140=20.3,
P<0.001), indicating that for all instars, stage within an instar
significantly affected the ventilatory response to hypoxia. First-instar
animals showed no variation in breathing frequency from the beginning to end
of an instar except at PO levels below 6 kPa
(Fig. 6A). Below an air
PO of 6 kPa, the abdominal pumping rates of late-stage
first instars dropped off more dramatically than those of early-stage first
instars. In all of the other instars, breathing frequencies were 30-50% higher
in normal air for late-stage animals compared with early-stage animals. Also,
abdominal pumping frequencies of late-stage grasshoppers dropped at a higher
ambient PO compared with those of early-stage grasshoppers
(Fig. 6B-D). All animals at the
end of each instar had a significantly higher Pc for
abdominal pumping frequency than animals at the beginning of an instar
(Fig. 7; Scheirer-Ray-Hare
extension of the Kruskal-Wallis test, H=14.58, P<0.05;
for individual instars - Mann-Whitney U test,
P<0.01).
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Maximal total tracheal system conductance
Maximal total tracheal system conductance µmol g-1
h-1 kPa-1; calculated at the Pc)
decreased by an average of 35% from the beginning to the end of an instar,
depending upon the animal's instar (ANOVA, stage x instar interaction,
F1,60=3.25, P<0.08;
Fig. 8). When we analyzed each
instar separately, we found significant effects of stage in every instar
(instar 1 - F1,14=4.98, P<0.05; instar 3 -
F1,14=5.80, P<0.04; instar 5 -
F1,14=3.80, P<0.08; adult -
F1,14=4.07, P<0.08).
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Discussion |
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Effects of developmental stage on ventilatory parameters CO2 emission rate
Total gas exchange rates increased within each instar by 13-67%. However,
the percent increases in total CO2 emission were not matched to
percent increases in body mass, as mass-specific
CO2 decreased
from beginning to end of an instar (Table
1; Fig. 2B). The
greatest decrease in mass-specific
CO2 of 30%
occurred during the third instar, indicating that a large part of the third
instar mass increase may have been due to increases in non-metabolic tissue,
such as cuticle or fat.
The relationship between metabolic rate and mass throughout development may
be dependent upon species, as relationships reported in the literature are
highly variable. For example, mass-specific oxygen consumption decreased
strongly within most instars of Potamophylax nigricornis (caddisfly
larvae) and Galleria mellonella (wax moth larvae; cited in
Sehnal, 1985). The results we
report here for Schistocerca grasshoppers differ from those of Clarke
(1957b
), who found that, in
Locusta migratoria grasshoppers, oxygen uptake rate
(
O2) increases
were matched exactly by increases in body mass for first- to third-instar
animals. The parallel relationship between
O2 and mass
disintegrated by the fifth instar, when mass-specific
O2 decreased by
a third (Clarke, 1957b
).
According to our measurements, within-instar scaling of
CO2 varied
slightly with each instar in S. americana but did not differ much
from across-instar patterns and the classic scaling of metabolic rate in
animals (Fig. 9).
|
Index of tidal volume
We predicted that late-stage animals would have decreased tidal volumes due
to increased tissue mass and smaller air sacs. In normal air, abdominal
pumping height was 50% lower in late-stage adults compared with early-stage
adults (Fig. 5B). However, we
found no difference in abdominal pumping height for the first instar
(Fig. 5A). First-instar
grasshoppers had such low tidal volumes that tissue growth may have little
effect.
Interestingly, late-stage adults were able to increase abdominal pumping
height to levels exhibited by early-stage adults. Perhaps the late-stage
adults achieved maximal inhalation volumes similar to those of early-stage
adults by using their inspiratory muscles. During most situations,
grasshoppers use only the expiratory muscles
(Hustert, 1975), and, if this
were the case, reduced air sac volumes may have translated to reduced tidal
volume. However, inspiratory muscles tend to be active during very heavy
breathing (as would occur during hypoxia exposure) in grasshoppers
(Hustert, 1975
), and the
consequent expansion of abdominal volume could allow the maintenance of
maximal tidal volume.
CO2 loss per breath and expired PCO
Does ontogenetic development within an instar affect the use of diffusive
versus convective gas exchange or result in varying internal gas
tensions? We lack direct data to answer this question but we can indirectly
address this issue by examining the effect of stage on CO2 loss per
breath µmol breath-1; calculated from
CO2/ventilatory
frequency). Across air PO levels above 5 kPa,
CO2 emission per breath did not vary between early- and late-stage
animals for third and fifth instars and adult grasshoppers
(Fig. 10). For adults,
convection can account for all trans-spiracular CO2 transport
(Greenlee and Harrison, 1998
),
therefore:
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Ventilation frequency
First-instar animals showed no difference in ventilation frequency between
early and late stages, a pattern similar to that seen with tidal volume
(Fig. 6A). The lack of a
developmental effect in first instars could reflect a different strategy for
responding to hypoxia. As noted above, late-stage first-instar grasshoppers
have a greater µmol CO2 breath-1, suggesting either
increased use of diffusion or higher internal PCO s
(Fig. 10). Measurement of
internal gases or quantification of the amount of diffusive gas exchange using
anaesthetized animals would allow discrimination of these possibilities.
Late-stage animals (except first instars) increased ventilation frequency
compared with early-stage animals (Fig.
6B-D), probably to compensate for decreased tidal volumes
(Fig. 3). Maximal ventilation
frequencies were significantly higher in late-stage fifth-instar animals and
late-stage adults when compared with early-stage animals (t=-2.77 and
-3.2, P=0.019 and 0.007, respectively;
Fig. 6C,D). This ability to
increase ventilation frequency partly ameliorates the effect of tracheal
compression/fixed spiracle size on the maximal tracheal system conductance. It
is unclear what mechanism would facilitate the increase in maximal breathing
frequency at the end of an instar. Perhaps animals at the end of the instar
have an increased chemical drive for ventilation (i.e. lower internal
PO or higher PCO) or there may be
changes in ventilatory muscle properties (such as increased mass or
recruitment). Another possibility is that the sensitivity to neural excitation
in response to hypoxia is increased at the end of an instar. This hypothesis
could be tested by measuring the effect of hypoxia on the frequency of action
potentials from ventilatory motor nerves
(Bustami et al., 2002).
Maximal tracheal system conductance
Total mass-specific tracheal system conductance was lower in late-stage
relative to early-stage grasshoppers at every instar
(Fig. 8). The decrease in
conductance could have been due to air sac or tracheal compression from tissue
growth (Figs 3,
8). However, the lack of an
effect of stage on tidal volume suggests air sac compression could not account
for a decrease in maximal conductance in first instars. Perhaps tissue growth
causes an increase in the length of secondary tracheae and possibly even a
decrease in diameter if tracheae were stretched as a consequence of tissue
growth. Another possibility is that tracheole density may decrease during the
intermolt period.
Pc within an instar
In general, the Pc for abdominal pumping was lower than
the Pc for CO2 emission. For the first and
third instars, the Pc values for abdominal pumping for
late-stage animals were significantly and substantially higher than those of
early-stage animals (fourfold; Fig.
7). One potential explanation for the larger stage effect on the
Pc for abdominal pumping in younger animals is that the
younger animals were generally less tolerant of hypoxia. In other words, the
greater hypoxia sensitivity of metabolism in first instars relative to adults
(Fig. 4) can be thought of as
arising from the summed effects of a number of more hypoxia-sensitive tissues
in the first instars. Also, animals may have preferentially shut down less
critical tissues to respond to hypoxia. If first- and third-instar animals
exchange gases primarily by diffusion, then the ventilatory muscles may be
relatively non-critical and would be shut down at a higher
PO2. By contrast, adults, which are highly
dependent upon convection for gas exchange (Greenlee and Harrison,
1998,
2004
), had relatively low
Pc values for abdominal pumping and much less of a stage
effect, perhaps because the ventilatory muscles are critical for surviving
hypoxia.
Contrary to our prediction, the Pc for CO2
emission did not differ between the early- and late-stage juvenile instars,
although the predicted stage effect did occur in adults
(Fig. 5). Perhaps our
statistical power was insufficient to discriminate such an effect, since the
median Pc was higher for all late-stage animals
(Fig. 5). However, for the
juvenile instars, the percentage decrease in maximal conductance in late-stage
animals (Fig. 8) was similar to
the decrease in mass-specific
CO2
(Fig. 2B), suggesting that
respiratory capacity did match gas exchange needs throughout those instars.
For the first instars, the elevated CO2 emission per breath in
late-stage animals suggests that the matching may have occurred through
increased diffusive gas exchange (e.g. by increased spiracular opening in
late-stage animals). For the third and fifth instars, CO2 emission
per breath only increased in late-stage animals relative to early-stage
animals below the Pc, suggesting that, for these instars,
the increased ventilation frequencies were sufficient to allow matching of
respiratory capacity and gas exchange.
Possible implications of decreasing safety margins for O2 delivery within an instar
Our data suggest that young, late-stage insects may be particularly
sensitive to high-altitude hypoxia. The Pc for
CO2 emission found for the late-stage first-instar grasshoppers was
17.5 kPa (Fig. 5), the
PO of air at an altitude of 1500 m. This calculation
suggests that oxygen availability could limit gas exchange of late-stage
first-instar grasshoppers of many species over large portions of their range
(Branson and Redlin, 2001).
However, if diffusion is important for oxygen delivery in first-instar
grasshoppers, the increased oxygen diffusion coefficient at decreased
barometric pressure may potentially offset the decrease in
PO (Joos et al.,
1997
). The reduced maximal tracheal system conductance of
late-stage animals suggests that their maximal metabolic rates and locomotory
performance may also be compromised late in the instar. Thus, late-stage
insects may be more vulnerable to predators or less able to disperse or
migrate.
An intriguing possibility is that decreases in tissue oxygen levels late in
the intermolt period may be a signal that could trigger molting. Molting is
initiated by a complex pathway, currently thought to begin with a decline in
ecdysteroids. A declining level of ecdysteroids stimulates release of
pre-ecdysis-triggering hormone (PETH;
Zitnan and Adams, 2000) and
ecdysis-triggering hormone (ETH) from epitracheal glands
(Zitnan et al., 1996
). Release
of ETH initiates release of eclosion hormone (EH), and a positive feedback
system occurs between EH and ETH (Ewer et
al., 1997
). However, the initial trigger of the cascade remains
elusive. In the tobacco hornworm (Manduca sexta), molting can be
initiated by achievement of a critical body mass (Nijhout,
1975
,
1979
). In the milkweed bug
(Oncopeltus fasciatus), abdominal stretch receptors stimulated by
injecting the animals with fluid triggered molting (Nijhout,
1975
,
1979
). This finding supported
the hypothesis that not only body mass but also increased body size could
initiate molting.
An additional hypothesis is that decreased tissue oxygen levels late in the
instar may be part of the mechanism for the initiation of molting. The fall in
maximal tracheal conductance, the rise in the Pc for
CO2 in adults
and the indirect evidence for a rise in internal PCO all
suggest that internal PO levels may fall late in the
instar. This decreased tissue PO could serve as a logical
cue for triggering molting. The idea that decreased O2 availability
may trigger molting is supported by the findings of Greenberg and Ar
(1996
), whose studies of
mealworms (Tenebrio molitor) reared in hypoxia, normoxia and
hyperoxia showed that the duration of intermolt periods was directly
proportional to PO2. In addition, since the
epitracheal glands are located near each spiracle
(Chapman, 1998
), they are
conveniently located for sensing changes in O2 delivery. Direct
measurements of the effect of stage on tracheal PO and the
effect of manipulation of tracheal PO on EH and ETH will
be necessary to test whether decreased PO actually can
serve as an initiator of molting in insects.
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Acknowledgments |
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