Respiratory changes throughout ontogeny in the tobacco hornworm caterpillar, Manduca sexta
1 Baylor College of Medicine, Department of Medicine, Section of Pulmonary
and Critical Care Medicine, One Baylor Plaza, Houston, TX 77030,
USA
2 Section of Organismal, Integrative, and Systems Biology, School of Life
Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501,
USA
* Author for correspondence (e-mail: greenlee{at}bcm.tmc.edu)
Accepted 31 January 2005
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Summary |
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Key words: insect respiration, metabolic rate, gas exchange, development
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Introduction |
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How can the insect tracheal system compensate for growth and increased gas
exchange needs across and within instars? The tracheal system may potentially
compensate for increased lengths by either morphological or physiological
mechanisms. Across instars, morphological mechanisms of compensation may
include increasing tracheal diameters at each molt
(Beitel and Krasnow, 2000). In
addition, tracheal branching and sprouting are sensitive to
PO2
(Wigglesworth, 1954
;
Locke, 1958
;
Jarecki et al., 1999
), and
tracheole density increases with age in grasshoppers
(Hartung et al., 2005
). Thus
if tissue PO2 were to decrease due to
inadequate O2 delivery, tracheole proliferation could be
stimulated, providing an opportunity for tracheal morphology to match
O2 delivery needs. Physiological changes in the mechanisms of gas
exchange are also possible. Grasshoppers increase their mass-specific tracheal
capacities as they grow across instars by increasing convective gas exchange
through increases in the rate of abdominal pumping and tidal volume
(Greenlee and Harrison,
2004a
). Caterpillars have traditionally been thought to breath by
diffusion (Krogh, 1920
), but
they may use hemolymph pulsations generated by micro-contractions of the
intersegmental muscles of the abdomen to drive convection through the
compressible tracheae (Slama,
1999
; Smits et al.,
2000
).
In addition to dramatic body size increases across instars, within each
instar, caterpillars increase in body mass at least 100% and up to 1000% in
the fifth instar (Goodman et al.,
1985). Do these size increases correlate with increasing
challenges for oxygen delivery? In Drosophila, tracheal diameters do
not increase during an intermolt period
(Beitel and Krasnow, 2000
).
Similarly, spiracles are sclerotized and can only increase in size at the
molt. Thus, the only likely morphological mechanism to allow increased
O2 delivery within an intermolt period is tracheole sprouting
(Locke, 1958
;
Jarecki et al., 1999
).
Physiological mechanisms, such as increasing convective gas exchange, could
compensate for increased O2 demands; however compression of
flexible tracheae by growing tissues may impede the animal's ability to
increase convection. For example, grasshoppers late in the intermolt period
have decreased ability to respond to hypoxia (i.e. critical
PO2 increases throughout the instar,
Greenlee and Harrison, 2004b
).
The decreased ability to respond to hypoxia may be caused by a decrease in the
volume of the air sacs that become compressed as tissue grows within the
sclerotized exoskeleton (Clarke,
1957
; Greenlee and Harrison,
2004a
). However, since the caterpillar exoskeleton is only
sclerotized at the head, leaving the rest of the body free to expand
throughout the instar (Eaton,
1988
), tissue growth seems less likely to compress the
tracheae.
If an insect exchanges gases by diffusion, then larger body sizes may lead
to increasing problems with gas exchange due to the well-documented
exponential decreases in diffusion rates with distance. This idea has
contributed to the suggestion that atmospheric oxygen levels may be linked to
insect body size (Graham et al.,
1995; Dudley,
1998
). In the present study, we first tested the hypothesis that
larger caterpillars are unable to match tracheal O2 delivery
capacity to tissue O2 needs during ontogeny. If larger insects have
problems with oxygen delivery, then we would predict that larger caterpillars
would have higher critical PO2 values
(Pc, the PO2 below which
metabolism and feeding can no longer be sustained). Dimensional differences
are most prominent across instars, so to test this hypothesis we compared
caterpillars at the beginning of different instars. In addition, we tested the
hypothesis that as caterpillars grow throughout an instar, their oxygen
delivery capacity does not match increases in tissue oxygen demand due to lack
of plasticity in tracheal structure within an intermolt period. If animals
nearing the molt have problems with oxygen delivery, we would predict that
caterpillars near the end of an instar would have higher
Pc values compared with animals that have recently
molted.
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Materials and methods |
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Respirometry
We measured CO2 emission of caterpillars during feeding to
maximize metabolic rates and to minimize struggling in our respirometry
system. Food was placed in a small dish and weighed using an analytical
balance to the nearest 0.0001 g (Mettler AE-240, Columbus, OH, USA). The dish
and food were then placed in the respirometry chamber and flushed with
CO2-free air (approximately 100 ml min-1) for 20 min to
remove any CO2. Food treated in this manner had no measurable
CO2 emission when placed in the chamber alone. Additionally, water
loss from the food was negligible (<0.7% of the initial food mass) as
estimated by placing food alone in the chamber for approximately 2 h, a period
longer than any experimental trial. The experimental animal was then weighed
in the same manner, placed on the food in the chamber, and allowed to
acclimate to the chamber for 20 min. During each trial, caterpillars were
exposed to 10 min each of five different gas mixes (21, 15, 10, 5, 3 and 0 kPa
O2) generated with a Brooks 5878 mass flow controller and Brooks
mass flow meters (Brooks Instruments, Hatfield, PA, USA). Gas mixes were
subsampled from a 60 ml syringe and pushed through the chamber by an Ametek
R-1 flow controller (Ametek, Pittsburgh, PA, USA) at 18160 ml
min-1 STP. The excurrent air stream was directed through
a CO2 analyzer (Li-6252 Li-Cor, Lincoln, NE, USA) to measure
CO2 fraction in the air and then through an Ametek S3-A oxygen
analyzer to verify the gas mixture. Signals from the CO2 and
O2 analyzers were digitized and recorded using Sable Systems
DataCan software and hardware (Salde Systems International, Las Vegas, NV,
USA). We calculated
CO2 (µmol
g-1 h-1) as:
![]() | (1) |
In addition to determining metabolic rates, we calculated critical
PO2 values
(Pc-CO2)
(Greenlee and Harrison,
2004a). Briefly, we compared 95% confidence intervals around the
mean CO2 emission rates at each PO2.
A PO2 was considered to be the
Pc-CO2 if the mean
CO2 at the next
higher and across all higher PO2s were
significantly higher.
We then calculated maximal mass-specific tracheal system conductance,
Gmax (µmol g-1 h-1
kPa-1, Greenlee and Harrison,
2004a). Briefly, we assumed that at the
PO2 just higher than the
Pc-CO2 for
CO2, the animals
were maximally conducting gases through wide-open spiracles and a fluid-free
tracheal system, and that mitochondrial PO2 at
the Pc-CO2 is near zero. We converted
CO2 to
O2 assuming a
RER of 0.88 and calculated:
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Feeding behavior
Animals began feeding as soon as they were placed on food in the
respirometry chamber. We observed them during each hypoxic exposure to
determine whether they were continuing to feed, and considered total feeding
time to be the acclimation time in the chamber plus the time until the animal
stopped feeding. The PO2 at which animals
ceased feeding was identified as critical point for feeding
(Pc-feeding). Food was weighed after the last hypoxic
exposure, and total feeding rate (g h-1) was calculated as the
difference in food mass from the beginning to the end of the trial divided by
the time that the animals were observed feeding. Animals ate on average 25% of
the food offered (range 271%).
Statistics
Mean values ± S.E.M. are presented for parametric data,
and median values are shown for nonparametric data. Statistical analyses were
performed using SYSTAT 10.2, with our within-experiment type I error less than
or equal to 5%. We analyzed
CO2 data using
repeated measures analysis of variance (ANOVA), since individual caterpillars
were exposed to multiple PO2 values. Feeding
rates were compared using ANOVAs with the independent variables being instar
and stage. Pc and Pc-feeding were
statistically analyzed as nonparametric data, since these are discrete
variables. We used the Kruskal-Wallis test, a single-factor analysis of
variance in SYSTAT 10.2 and also calculated nonparametric multiple comparisons
as described in Zar
(1999
).
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Results |
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Whole-animal, normoxic CO2 emission rates increased four orders
of magnitude from the youngest to the oldest animals in our study
(Fig. 2; 0.229±0.05 to
317±14.9 µmol h-1; ANOVA, F1,46=52.4,
P<0.001). CO2 emission scaled with body mass
(Mb) to the 0.98 power (log
CO2 µmol
h-1=0.98 [log Mb] + 1.66,
r2=0.97, P<0.001;
Fig. 2), and metabolic energy
scaled with body mass to the 0.96 power
[Watts=0.0071(mass)0.96].
|
Mass-specific, normoxic
CO2 varied
differently with stage depending upon the instar (ANOVA, stage x instar
interaction, F1,46=17.7, P<0.0001). For
instance, first and third instar caterpillars' CO2 emission rates
decreased from early to late stage, while late-stage fifth instar caterpillars
increased mass-specific
CO2 to 60%
higher than the early-stage value (Fig.
3).
|
Hypoxic CO2 emission, Pc, maximal tracheal system conductance
In response to decreasing levels of atmospheric O2, caterpillars
generally maintained CO2 emission rates at their normoxic levels,
until a critical point was reached. However, depending on the instar, early-
and late-stage animals responded differently to the decreased
PO2 (Fig.
3; repeated measures ANOVA, instar x stage x
PO2 interaction,
F5,230=7.9, P<0.001). Analyzing each instar
separately, we found that the CO2 emission rate response to hypoxia
depended strongly on stage (Fig.
3; repeated measures ANOVA, PO2
x stage interaction; first instar: F5,75=4.7,
P<0.001; third instar: F5,75=5.8,
P<0.0001; fifth instar: F5,70=21.4,
P<0.0001).
Pc-CO2 also varied with stage depending on the caterpillar's instar (Fig. 4). When we analyzed each instar separately, we found that late-stage third and fifth instar caterpillars had significantly higher Pc values than early stages (Fig. 4, Mann-Whitney U test, third instar: U=8, P<0.01; fifth instar: U=2, P=0.001), while there was no stage effect in the first instars (Fig. 4).
|
Mass-specific maximal tracheal system conductance, Gmax, decreased with instar (Fig. 5; ANOVA, effect of instar, F1,46=11.1, P<0.01). Within each instar, mass-specific Gmax decreased on average 49% from early to late stage (Fig. 5; ANOVA, effect of stage, F1,46=8.1, P<0.01).
|
Feeding behavior
Three animals did not eat during the experiment and were therefore excluded
from these analyses. The remaining 47 animals ate continuously during the
experiment until the PO2 became limiting, at
which point they raised their heads off the food. Some animals even moved away
from their food. Food eaten (g h-1) varied with instar
(Fig. 6, ANOVA, effect of
instar, F1,43=14.2, P<0.001).
|
When we corrected the amount of food eaten for caterpillar body mass, [food eaten (g) g caterpillar-1 h-1], we found that the amount of food eaten varied with instar depending on the animal's stage (Fig. 6, ANOVA, instar x stage interaction, F1,43=5.6, P<0.03). All late-stage animals ate less food (g g caterpillar-1 h-1) compared with early-stage-animals; however these decreases were not statistically significant (ANOVAs, effect of stage, first instar: F1,13=4.7, P=0.05; third instar: F1,15=4.3, P=0.056; fifth instar: F1,13=3.2, P=0.099).
Pc-feeding varied with instar (Fig. 7; Scheirer-Ray-Hare extension of the Kruskal-Wallis test, effect of instar, H1,43=11.0, P<0.05). In addition, Pc-feeding decreased across instar for early-stage animals (Kruskal-Wallis test statistic=13.9, P<0.01). When we analyzed each instar separately to test for stage effects, we found that early-stage fifth instars continued to feed at significantly lower PO2 values than late-stage animals (Mann-Whitney U test, U=14, P=0.03).
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Discussion |
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Across instars, larger caterpillars did not appear to have more difficulty than smaller caterpillars in responding to hypoxia. There was no significant effect of instar on Pc-CO2, while Pc-feeding actually decreased in the older/larger instars (Figs 4, 7). Thus tracheal conductances matched or exceeded changes in oxygen consumption rates across instars.
CO2 emission rate in normoxia
CO2 emission rates scaled with body mass to the 0.98 power
(Fig. 2), a finding similar to
that for some lepidopterans throughout ontogeny (cecropia moths,
Hyalophora cecropia, 0.9,
Schroeder and Dunlap, 1970;
M. sexta, 1.0, Dahlman and Herald,
1971
; and brown-tailed moths, Euproctis chrysorrhoea,
1.0, Migula, 1974
) but not
others (corn earworms, Heliothis zea, 0.7,
Edwards, 1970
; tent
caterpillars, Malacosoma neustria, 0.8,
Migula, 1974
; and M.
sexta, 0.8, Alleyne et al.,
1997
). Our finding that
CO2 varies
strongly within an instar suggests that the variation among scaling
coefficients reported for caterpillars could be due to variation in the age
within an instar of the caterpillars studied.
Variation in age within an instar may contribute strongly to the variation in mass scaling of metabolic rate during ontogeny across insect species. For example, mass-specific metabolic rate was higher for late-stage fifth instar caterpillars than for early stage caterpillars of the same instar, while an opposite pattern was observed for the first and third instars (Fig. 3). Late-stage fifth instar caterpillars had increases in absolute CO2 emission (630% from early-stage) that were double the increases in body mass (360% from early-stage). A possible explanation is that preparation for wandering and pupation increase metabolic rate in fifth instars relative to other instars. The high late-instar metabolic rates of fifth instars contribute to the reduced safety margin for O2 delivery observed for this group. Our results indicate that future studies of the ontogenetic scaling of gas exchange and, probably physiological processes in general, must carefully control for age within instar.
Hypoxia response and Pc for CO2
Across-instar effects
Newly molted caterpillars were able to maintain CO2 emission
down to low PO2 levels (as low as 5 kPa). These
results are comparable to those found for other insects
(Keister and Buck, 1974;
Wegener and Moratzky, 1995
;
Greenlee and Harrison, 1998
;
Emekci et al., 2002
;
Greenlee and Harrison, 2004a
).
The constant Pc-CO2 across instars results from
a matching of maximal tracheal conductance to metabolic rate changes, as both
mass-specific Gmax and CO2 emission rate
decrease by about 50% from first to fifth instar
(Fig. 5B). These patterns are
not a general phenomenon in insects, as Pc in grasshoppers
decreases and absolute Gmax increases during ontogeny
(Greenlee and Harrison,
2004a
).
CO2 emission rates at low PO2
levels were (for a short time at least) maintained at relatively high levels.
In anoxia, all animals were able to maintain CO2 emission rates at
30% of their normoxic values for the 10 min trial
(Fig. 8). These high ratios of
anoxic
CO2/normoxic
CO2 suggest
possible use of anaerobic metabolism during extreme hypoxia/anoxia. In support
of this hypothesis, caterpillar intersegmental muscles contain considerable
quantities of lactate dehydrogenase (Gade,
1975
). The high ratios of anoxic
CO2/normoxic
CO2
(Fig. 8) are also likely due to
washout of CO2 from the tissues as hypoxia/anoxia promote maximal
spiracular opening (Case,
1956
), evacuation of fluid from the tracheoles
(Wigglesworth, 1931
) and
reduced tissue PCO2 values
(Greenlee and Harrison
1998
).
|
Within-instar effects
CO2 emission rates and feeding rates of caterpillars late within
each instar were clearly limited at higher PO2
values than their earlier counterparts as evidenced by the much higher
Pc-CO2 and Pc-feeding in
late-stage caterpillars. Early-stage caterpillars had lower
Pc-CO2 values compared with late-stage
caterpillars, despite the generally lower mass-specific CO2
emission rates for late-stage animals (Figs
3 and
4). These data support the
hypothesis that caterpillars within an instar experience a shrinking safety
margin, as changes in O2 delivery capacity do not parallel changes
in tissue oxygen needs. In fact, mass-specific Gmax
decreased by nearly 50% from early to late-stage within each instar
(Fig. 5B), whereas
within-instar variation in mass-specific CO2 emission rates was
different for each instar (Fig.
3). First instar animals showed a similar decrease in both
mass-specific
CO2 and
Gmax, while third instar animals only had a 10% decrease
in mass-specific
CO2, and fifth
instar animals actually had a 60% increase in mass-specific
CO2 from the
beginning to the end of an instar.
Why does mass-specific Gmax decrease within an instar? If the tracheal system was fixed within an instar (same spiracle and main tracheal dimensions, no tracheole sprouting), then absolute Gmax should be constant, but mass-specific Gmax would fall due to the rise in tissue mass with constant tracheal morphology. This pattern is observed for the first and third instars (Fig. 5), suggesting that the within-instar rise in Pc-CO2 for the third instars can be explained by rising animal oxygen needs without an increase in tracheal system oxygen delivery capacity. In the fifth instars, however, caterpillars increase absolute Gmax 192% (Fig. 5A) from early to late-stage, indicating a within-instar enhancement of oxygen delivery capacity. This increase in Gmax could be due to an increased use of convection or tracheole sprouting. The increase in absolute Gmax is far less than the 630% rise in absolute CO2 emission rate, leading to the rise in Pc-CO2 within the fifth instar.
Feeding behavior
Caterpillars were able to continue feeding, in most cases without
interruption, to very low PO2 values
(Fig. 7). The oldest
caterpillars in our study ate slightly more food (g g-1
h-1, Fig. 6)
compared to fifth instar M. sexta in another study using the same
diet and temperature (approximately 0.06 g g-1 h-1 over
a 4 h period, Kingsolver and Woods,
1997). However, the ontogenetic pattern of increasing feeding
rates with age that we observed follow those of previous researchers
(Fig. 6). For example, zebra
caterpillars (Melancha picta) feeding on sugarbeet leaves increased
feeding rates (leaf area/day) with age more than 70-fold
(Capinera, 1979
). Growing
armyworms (Mamestra configurata, Bailey,
1976
), saltmarsh caterpillars (Estigmene acrea,
Capinera, 1978
), and cotton
bollworms (Helicoverpa zea, Huffman and
Smith, 1979
) also increased leaf feeding area per day with age. In
addition, the mass-specific feeding rates of our caterpillars decreased
60-fold from hatching to fifth instar (Fig.
6), a finding similar to that observed in other lepidopteran
larvae (Slansky Jr, 1993
).
Possible mechanisms of hypoxia tolerance
The low and similar values of Pc-CO2 and
Pc-feeding suggest that caterpillars early in the
intermolt periods have very large safety margins for O2 delivery.
The existence of these large safety margins for O2 delivery in the
absence of any outward signs of increasing ventilation begs the question, what
is the mechanism for the large safety margin for O2 delivery? One
possible explanation for their hypoxia tolerance is that these insects have
`overbuilt" tracheal systems (larger diameters than needed) and simply
tolerate substantial variation in tissue PO2
during exposure to hypoxia. Another potential explanation is that the head
movements during feeding are also used for ventilation, possibly creating
convective gas exchange by compressing tracheae as the hydrostatic skeleton
changes shape with each rhythmic movement
(Westneat et al., 2003).
Hemolymph pressure changes could also be driven by contractions of
intersegmental (Slama, 1999
)
or heart (Smits et al., 2000
)
muscles that would generate convective gas flow and increase during hypoxia.
Lastly, caterpillars may increase spiracular opening and/or remove tracheolar
fluid to increase gas exchange
(Wigglesworth, 1981
).
Implications
The decreased Pc values and Gmax
within an instar support the hypothesis that O2 may play a role in
triggering larval to larval molting in insects (Figs
4 and
5). Also, since the late-stage
animals we measured were at least one day away from molting, the effect of
within-instar development on Pc and
Gmax may be even more striking as animals more closely
approach ecdysis. We hypothesize that as animals near the end of an instar,
internal PO2 decreases, providing a signal for
the initiation of the molting pathway. The signaling cascade is currently
thought to begin with a decline in ecdysteroids, which stimulates release of
pre-ecdysis-triggering hormone (PETH)
(Zitnan and Adams, 2000) and
ecdysis-triggering hormone (ETH) from epitracheal glands
(Zitnan et al., 1996
).
Eclosion hormone (EH) is then released by ETH and a positive feedback system
occurs between EH and ETH (Ewer et al.,
1997
). However, it is unclear what the initial trigger of the
pathway is. In one investigation, Nijhout
(1975
) found that in M.
sexta molting is initiated by achievement of a critical body size, a
finding potentially consistent with internal gas tensions being important as a
trigger. The involvement of O2 in the signal transduction pathway
to molting is supported by the findings of Greenberg and Ar
(1996
) who found that in
mealworms the number of molts was inversely proportional to
PO2. The Pc-CO2
in late-stage animals was approximately 15 kPa, indicating that a small safety
margin for O2 delivery still exists at this time. However, the
late-stage animals we measured were at least one day away from molting, with
approximately 50% more mass to be gained
(Goodman et al., 1985
). Thus,
it seems possible that the safety margin for O2 delivery may
disappear completely in the last hours before ecdysis, providing an ultimate
if not proximate explanation for the necessity of molting.
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Acknowledgments |
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