We tested the hypothesis that oxygen delivery from the atmosphere to the tissues becomes more difficult as grasshoppers increase in body size throughout development due to increases in tracheal length. If this is true, then older, larger grasshoppers should have smaller safety margins [higher critical oxygen partial pressures (PO2s)] for oxygen delivery than younger, smaller grasshoppers. We exposed grasshoppers of first, third and fifth instars and adults to decreasing levels of atmospheric O2 and measured their ventilatory responses. Contrary to our prediction, we found that larger grasshoppers had critical PO2s eight times lower than juveniles due in part to their threefold lower mass-specific metabolic rates and their ability to quadruple convective gas exchange. Adults more than doubled abdominal pumping frequency and increased tidal volume by 25% as PO2 decreased fourfold, whereas the youngest juveniles showed no such responses. This study indicates that juveniles may be more susceptible to hypoxia in natural situations, such as exposure to high altitude or restricted burrows. Also, larger size is not necessarily correlated with a smaller safety margin for oxygen delivery in insects.
The development of physiological traits is a critical aspect of evolutionary and ecological physiology (Burggren, 1991). Most natural selection probably occurs during the juvenile stages, because young animals generally have much higher rates of mortality than fully developed, adult organisms (insects – Estevez and Gonzalez, 1991; reptiles – Iverson, 1991; arachnids– Tanaka, 1992; birds– Currie and Matthysen, 1998; fish – Molles, 1999; mammals – Durant, 2000). Therefore, knowledge of the physiology of juveniles and developmental patterns of physiological traits is important in understanding ecological success and evolution.
Adult and pupal insects tolerate low levels of oxygen (<5 kPa O2), at which most mammals would die (Wegener, 1993), and generally have resting critical oxygen partial pressures [Pc; the oxygen partial pressure (PO2) below which metabolism can no longer be maintained] well below those of vertebrates and perhaps below those of non-tracheate invertebrates (Table 1; Hoback and Stanley, 2001). Pc is the point at which an animal switches from being an oxy-regulator to an oxy-conformer (Yeager and Ultsch, 1989; Portner and Grieshaber, 1993), and thus it defines the safety margin for O2 delivery (the range of PO2s across which metabolism is constant; Kam and Lillywhite, 1994). For this reason, organisms with low Pcs tend to tolerate hypoxia better than animals with higher Pcs (Ultsch, 1973, 1974). To our knowledge, Pc has not yet been compared across the developmental stages of an insect.
One prominent hypothesis suggests that as insects become larger, gas exchange becomes more difficult due to increases in tracheal system lengths (Graham et al., 1995; Dudley, 1998). This logic is the basis for the hypothesis that increases in atmospheric O2 during the Paleozoic era facilitated insect gigantism (Graham et al., 1995; Dudley, 1998). Alternatively, larger insects could have greater tracheal diameters or could increase the use of ventilation to compensate for increased tracheal lengths. We tested this hypothesis using developing grasshoppers. If older, larger grasshoppers have smaller safety margins (higher Pcs) for oxygen delivery than younger, smaller grasshoppers, this would support the hypothesis of Graham et al. (1995).
Development of the respiratory system is particularly interesting, because all organisms grow in size and increase total oxygen demand with age. Pc changes during ontogeny will depend on whether the ratio of maximal tracheal conductance to metabolic rate changes with age. Maximal tracheal system conductance (Gmax; μmol g–1 h–1 kPa–1) will depend on tracheal system structure (e.g. spiracle size and dimensions and density of tracheae and tracheoles) and function (e.g. the magnitude of convective gas exchange). The scaling exponents for metabolic rates during insect development vary widely and may exceed 1.0 (Casey and Knapp, 1987; Vogt and Appel, 1999).
In the present study, we quantify developmental changes in metabolic rate, maximal tracheal system conductance and several aspects of tracheal system function (ventilation frequency and abdominal pumping height, an index of tidal volume) for the American locust Schistocerca americana. We also compare the Pc for oxygen consumption and carbon dioxide emission to verify that our Pc for CO2 emission truly reflected the Pc for oxygen delivery. In addition, we examine the effect of hypoxia exposure time on the Pc for CO2 emission, since we wanted to be sure that our calculated Pc values were not strongly affected by the duration of hypoxia exposure in our major experiment.
Materials and methods
Schistocerca americana Drury were reared from eggs in culture at Arizona State University as previously described (Harrison and Kennedy, 1994). For these experiments, it was important to have grasshoppers of known age and body size. We took 200 grasshoppers that hatched on the same day and marked them with paint (Testors, Rockford, IL, USA). We monitored the colony each day, and, when an animal molted, we applied a new color of paint. All animals that molted on a given day had the same paint color, so we knew the age in days of each individual. Experimental animals were randomly selected from the animal care facility in the morning and kept in a lit, 35°C incubator with access to green leaf lettuce for 0.5–6 h before measurements were made. We recorded body mass (Mb) of all animals, weighing them to the nearest 0.001 g using an analytical balance (Mettler Instruments, Hightstown, NJ, USA). For measurements of adult insects, we used only males, but for measures of juveniles, sex was not determined.
Comparison of Pc values for O2 consumption and CO2 emission
Adult animals were placed in a 26 ml Plexiglas cylinder sealed with rubber stoppers. Inside the chamber, we placed cotton anterior and posterior to the grasshopper to prevent movement and around the animal's head to prevent visual stimulation. Test animals sat in the chamber for 20 min before measurements were made, a procedure that kept ventilatory frequencies similar to those of unrestrained grasshoppers behind one-way mirrors (Gulinson and Harrison, 1996). Each grasshopper was exposed to 11 different levels of PO2 (21, 16, 13, 9, 7, 5, 3, 2, 1, 0.5 and 0 kPa O2) for at least 15 min in descending order to prevent increases in metabolic rate associated with prior hypoxic exposure (Greenlee and Harrison, 1998).
Gas mixtures were made by diluting dry, CO2-free air with N2 (Balston purge-gas generator; Havervill, MA, USA). The ratio of air to N2 was controlled by a Brooks 5878 mass flow controller and mass flow meters (Brooks Instruments, Hatfield, PA, USA). Gas mixtures were pushed through three identical chambers at 223–326 ml min–1. Flow from one of these chambers was directed to the reference cells of the gas analyzers. The sample cells of the gas analyzers received output either from the animal chamber or an empty baseline chamber. All three chambers emptied into 60 ml syringes from which excurrent gases were subsampled. The reference stream and the sample stream were pulled through the CO2 analyzer (LI-6252; Li-Cor, Lincoln, NE, USA) and then the O2 analyzer (Sable Systems Oxzilla, Las Vegas, NV, USA) by an AMETEK R-1 flow controller (Pittsburgh, PA, USA). Water and CO2 were removed between the two analyzers using columns of drierite and ascarite. The output of both gas analyzers was digitized and recorded (Sable Systems).
We calculated the rates of CO2 emission and O2 consumption (ṀCO2 and ṀO2, respectively; μmol g–1 h–1) using the following equations: 1 2 where V̇in is the flow rate measured upstream of the animal chamber (ml min–1 STP; Omega mass flow meter, Stamford, CT, USA), V̇O2 is the rate of O2 consumption, FiCO2 and FiO2 are the incurrent CO2 and O2 fractions, respectively, and FeCO2 and FeO2 are the excurrent CO2 and O2 fractions, respectively. Additionally, 2678.57 is the conversion factor used to convert ml g–1 min–1 to μmol g–1 h–1 (1000 μlml–1, 60 min h–1 and 22.4 μlμ mol–1). Although flow rate was measured upstream from the chamber, the added volume flow due to water production by the animal was negligible, introducing an error of less than 0.08% (Greenlee and Harrison, 1998).
Effect of duration of hypoxic exposure on ṀCO2
We wanted to be sure that our calculated Pc values were not strongly affected by the short-duration hypoxia exposure in our major experiment. Therefore, we exposed first-instar juvenile and adult grasshoppers for 1 h to each of seven levels of PO2 (adults– 21, 5, 3, 2, 1, 0.5 and 0 kPa O2; juveniles – 21, 16, 13, 9, 7, 3, 1 and 0 kPa O2) and measured CO2 emission rates. Animals were treated as described above, except that small animals were placed in glass cylinders and larger animals were in Plexiglas cylinders. In all cases, the inner diameter of the chamber was just large enough to insert the animal but small enough to inhibit movement. Gas mixtures were made as described above and were sub-sampled from a 60 ml syringe using an AMETEK R-1 flow controller, which pushed the mixture through the respirometry chamber and then through the CO2 analyzer only. Flow rates ranged from 30 ml min–1 to 300 ml min–1, increasing with the size of the animal. The weakest signal-to-noise ratio (average p.p.m. CO2 in excurrent air relative to peak-to-peak system noise) was 13:1 during anoxia for first-instar grasshoppers. The signal-to-noise ratio improved as oxygen level and animal body sizes increased (e.g. signal-to-noise ratio was 84:1 in normoxia for first-instar grasshoppers and 127:1 for adults in anoxia). ṀCO2 was calculated at various time periods ranging from 3 min to60 min after exposure to a new gas mix using the following equation: 3 For these calculations, we assumed that the respiratory exchange ratio (RER) was 1; the misestimation of flow due to possible variations in RER was negligible, as variation between 1.0 and 0.7 would have produced a variation in calculated ṀCO2 of less than 0.026%.
Ontogenetic change in the responses of ṀCO2 and ventilation frequency to progressive hypoxia
Respirometry chambers, as described above, were placed in a 35°C water bath, the preferred field body temperature for most grasshoppers (Uvarov, 1966). First-, third- and fifth-instar and adult grasshoppers were each exposed to 12 different levels of PO2 (21, 16.2, 13.2, 9.1, 7.5, 6.2, 5.1, 3.8, 2.4, 1.3, 0.7 and 0 kPa O2) for 3–4 min at each PO2. We counted ventilatory frequency at each PO2 while viewing the animals through a dissecting microscope. We calculated CO2 emission rate using equation 3 above.
Determination of Pc for gas exchange and abdominal pumping
We determined individual Pc values for gas exchange by statistically identifying the PO2 at which CO2 emission or O2 consumption dropped (Fig. 1, box A). We compared 95% confidence intervals (CI) created using the mean ± (t×s.e.m.) where t=1.96 (Zar, 1999). There were three criteria for designating a PO2 as critical: the 95% CI at the Pc could not overlap and had to be (1) less than the CI of the previous PO2 (Fig. 1, box B), (2) less than the CI of all the previous PO2s combined (Fig. 1, box C) and (3) greater than or equal to the CI of all subsequent PO2s. The mean PO2 between the PO2 at which CO2 emission dropped significantly and the next higher PO2 was designated as the Pc (Fig. 1, box B; 1.3% O2). The Pc for abdominal pumping frequency was determined for each individual as the first PO2 at which abdominal pumping frequency dropped below that in normoxia and continued to decrease.
Magnitude of abdominal pumping
We obtained indices of tidal volume for first-instar juvenile and adult grasshoppers by measuring their abdominal height changes in response to hypoxia using videography. Each grasshopper was exposed to six levels of PO2 (adults – 21, 13, 5, 2, 1 and 0.5 kPa O2; juveniles – 21, 16, 13, 9, 5 and 1 kPa O2) at flow rates of 500 ml min–1. The grasshopper's abdomen was magnified with a dissecting microscope and the image was digitized with a Hitachi 3CCD camera (Tokyo, Japan) and displayed on a television monitor. We adjusted the magnification so that the abdomen nearly filled the monitor. Abdominal pumping was then video-recorded with a Panasonic SVHS (Desktop Editor Pro-Line, Secaucus, NJ, USA). Afterwards, the recording was analyzed frame by frame, and the height of the abdomen was measured at the fourth abdominal segment using a digital micrometer (Mitutoyo Corporation, Kawasaki, Japan). We recorded maximal height (inspiration) and minimal height (expiration) and calculated the percent difference using the following equation: 4 This measure provided an index of tidal volume, where larger percentage differences indicated higher tidal volumes. We calculated a mean value at each PO2 for each animal from three breaths and used this mean as a data point. Since we measured this index of tidal volume at relatively few PO2s, we did not calculate Pc values for this parameter.
Calculation of maximal tracheal system conductance
We calculated mass-specific tracheal system conductance for oxygen delivery (Gmax; μmol g–1 h–1 kPa–1; from air to mitochondria, incorporating both diffusive and convective conductances) as: 5 We assumed that at the Pc, tracheal system conductance would be maximized, since animals should have their spiracles opened wide (Case, 1956; Miller, 1960), ventilation maximized (Greenlee and Harrison, 1998) and tracheolar fluid removed (Wigglesworth, 1983). This calculation also assumes that mitochondrial PO2 is near zero at the Pc (Richmond et al., 1999) and that RER is 1 (see Results). Since Pc was calculated as an average of two PO2s, we used the value of ṀCO2 at the PO2 just above the Pc.
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%. For our analyses, the adult instar was designated as instar 7. We used repeated-measures analysis of variance (ANOVA) to compare the hypoxia responses of each instar, since individuals were measured at multiple levels of PO2; N=8 for all treatment groups. All Pc values 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). To determine whether there was a linear effect of instar on Pc, we also compared those values using nonparametric regression, Kendall's robust line-fit method and rank correlation coefficient (Sokal and Rohlf, 1995).
Developmental effects on mass and normoxic CO2 emission
Body mass (Mb) increased over 50-fold from hatching to early adulthood (Fig. 2). Metabolic rates increased with body mass (metabolic rate=0.005×Mb0.77; r2=0.95). Third instar CO2 emission rate was higher than predicted by the general scaling relationship for ṀCO2 and Mb (Fig. 3).
Comparison of the Pc for ṀCO2 and ṀO2
In adult grasshoppers, the Pc for ṀO2 and the Pc for ṀCO2 did not differ significantly (Table 2; Fig. 4; Mann–Whitney U test, U=32, P=1.0). Respiratory exchange ratios remained near one until the Pc was reached, below which they increased dramatically (Fig. 4 inset; repeated-measures ANOVA, F7,49=6.1, P<0.001).
Effect of the duration of hypoxia exposure on the Pc for ṀCO2
Duration of exposure (3 min or 1 h) had no significant effect on the Pc of first-instar grasshoppers (Table 2; Fig. 5). Adults exposed for 1 h at each PO2 had lower CO2 emission rates than those animals exposed for 3 min, and longer exposures slightly (but significantly) increased the Pc for ṀCO2 (Table 2; Fig. 5; Mann–Whitney U test, U=4.5, P=0.002).
Across-instar developmental effects on the Pc for ṀCO2
There was a significant interaction between PO2 and instar in the repeated-measures ANOVA, indicating that the decrease in ṀCO2 in response to hypoxia differed in different instars (F27,252=10.5, P<0.001). As has been previously shown (Greenlee and Harrison, 1998), adult grasshoppers exposed to hypoxia were able to maintain constant CO2 emission down to a median PO2 of 1.8 kPa (Fig. 6). However, first-instar grasshoppers appeared to be oxygen limited across PO2 levels lower than the median Pc of 14 kPa, as metabolic rate dropped virtually continuously with PO2 below 14 kPa (18 kPa for 1 h measures). Third and fifth instars showed intermediate responses, with median Pc values of 9.7 kPa and 4.5 kPa, respectively (Figs 6, 7). Overall, Pc decreased linearly over sevenfold from hatchling to adult grasshopper (Fig. 7; Kendall's rank correlation coefficient, τ=–0.58, P<0.01).
Across-instar developmental effects on the response of ventilatory frequency to hypoxia
For all instars, breathing frequency plummeted at some low level of ambient oxygen (Fig. 8). In contrast to the Pc for ṀCO2, the Pc for abdominal pumping was very low across all instars (Table 2; Fig. 7). However, there was still a significant effect of instar on the Pc for abdominal pumping (Kruskal–Wallis test statistic=14.3, P<0.01). Nonparametric multiple comparisons revealed that only third instars and adults differed in their Pc for abdominal pumping, with the third-instar Pc threefold higher than that of adults (q=4.7, P<0.05).
In response to hypoxia, adults more than doubled their ventilatory frequencies, while the first instars showed no change (Fig. 8). The response of abdominal pumping frequency to hypoxia varied significantly with instar, as indicated by a significant interaction between PO2 and instar (repeated-measures ANOVA, F27,252=10.5, P<0.001). We used analysis of covariance (ANCOVA) to determine which instars had different responses to hypoxia based on the slope of the line created when abdominal pumping was regressed on PO2 at all PO2s above the Pc for abdominal pumping (21 kPa to 3 kPa). We limited our test to these PO2s because, below the Pc, abdominal pumping decreased dramatically. This analysis revealed that the first-instar grasshoppers had no response to hypoxia above the Pc (slope=–0.114, P=0.9; Fig. 8). Pair-wise comparisons of the slopes of the other instars indicated that all were different from the first-instar slope but not different from each other (first instar versus other instars: all P≤0.011; comparisons between third, fifth and adults: all P>0.6). Thus, only the first-instar grasshoppers did not increase breathing frequency in response to hypoxia, and the responsiveness of abdominal pumping frequency to hypoxia was similar from the third instar to the adult stage [abdominal pumping frequency=2.2(PO2)+42.2; r2=0.12; Fig. 8].
Across-instar effects on the responsiveness of tidal volume to hypoxia
Our index of tidal volume, percent change in abdominal height, was 35 times higher in normoxia for adults compared with first instars (Fig. 9). Juveniles compressed their abdomens less than 1% of maximal height, compared with a 20% abdominal compression in adults (Fig. 9). Because we measured tidal volume at different PO2s for each instar, we were able to statistically compare only the responses at common PO2s (21, 5 and 1 kPa). There was a significant interaction effect of instar and PO2 on tidal volume (repeated-measures ANOVA, PO2 × instar, F2,28=64.8, P<0.001). Adults increased tidal volume almost 50% in response to hypoxia, while juveniles showed no significant change in tidal volume (repeated-measures ANOVA, adults – F4,28=21.8, P<0.0001; first instar – F5,35=1.4, P=0.24).
Throughout development, the grasshopper respiratory system improved in its ability to respond to hypoxia, as indicated by the increased responsiveness of ventilation frequency (Fig. 8) and tidal volume (Fig. 9) to hypoxia; together, these lead to a fourfold increase in the mass-specific capacity of the tracheal system to conduct oxygen (Gmax; Fig. 10). Older grasshoppers were also much more able to tolerate hypoxia, as the Pc for metabolic rate decreased sevenfold during ontogeny (Fig. 7). The increased tracheal system conductance with age may have been in preparation for a switch in the principal mode of locomotion from hopping to flight. In addition, the increased responsiveness to hypoxia of the tracheal system of older grasshoppers may be related to a general increased use of convective gas exchange in larger insects. Furthermore, our finding that development strongly affected the ability of grasshoppers to respond to ventilatory system challenges may mean that juveniles would be more sensitive to hypoxic environments encountered within restricted burrows, at high altitudes or during discontinuous gas exchange (Lighton, 1996).
Validation of methods
CO2 emission as an index of aerobic metabolic rate
We used CO2 emission as our index of metabolic rate in the across-instar comparisons because we could measure fractional CO2 content of the excurrent air stream quickly and more accurately than if we measured O2, especially for the smallest grasshoppers. This approach raises the concern that CO2 emission might not reflect the Pc for aerobic metabolism. For example, the Pc for ṀCO2 might be lower than the Pc for ṀO2 if ṀCO2 was elevated at low PO2s due to anaerobiosis or CO2 washout from body tissues due to hyperventilation. In our study, despite persistent elevation of ṀCO2 at very low PO2s, adult grasshoppers had identical Pc values for ṀCO2 and ṀO2 (Table 2; Fig. 4). The Pc for ṀCO2 is probably also close to the Pc for ṀO2 in juveniles, because juveniles exhibited much lower ratios of CO2 emission in anoxia to CO2 emission in normoxia (Fig. 11; ANOVA F3,28=15.2, P<0.001). Therefore, we conclude that the Pc for ṀCO2 provided a reasonable approximation of the Pc for aerobic metabolism.
Interestingly, the RERs that we measured (Fig. 4 inset) were higher than those measured for resting Schistocerca gregaria (0.82; Krogh and Weis-Fogh, 1951). One possible explanation for the discrepancy is that fuel use was different. Our grasshoppers were fed on green leaf lettuce, kale and dried wheat germ, whereas the grasshoppers in the prior study were fed green cabbage and dried grass. Alternatively, our grasshoppers could have been in a stressed state. However, the normoxic, acute metabolic rates we measured (after 20 min chamber acclimation time) were similar to those measured over 1 h, indicating that grasshoppers either were still stressed after 1 h or were settled within 20 min. Typically, grasshopper ventilation frequencies return to normal 20 min after a disturbance (Gulinson and Harrison, 1996). Perhaps the difference was due to inherent differences between the two species.
Acute exposure to hypoxia
We were also concerned that the use of short-term exposures to hypoxia would not reflect steady-state conditions. However, calculated Pc values for ṀCO2 did not differ between first instar animals exposed for three minutes or one hour to the test PO2s (Table 2; Fig. 5). Adult grasshoppers exposed to hypoxia for 1 h did have much lower CO2 emission rates and a slightly higher Pc compared with acutely exposed animals (Fig. 5;Table 2). We attribute the elevated ṀCO2 for the 3 min exposures in adults to transient increases in CO2 emission that occurred in response to each drop in atmospheric PO2 (Fig. 1). The increased CO2 emission may be attributed to two events. First, it has been noted that grasshoppers may physically struggle to escape when exposed to lower oxygen (Hochachka et al., 1993; Wegener, 1993). Secondly, exposure to hypoxia causes an increase in ventilation (enhancing CO2 loss) relative to metabolic rate (CO2 production), which secondarily causes a drop in hemolymph PCO2 and total CO2 content (Greenlee and Harrison, 1998). An alternative explanation for the transient increase in CO2 emission is that acid metabolites, such as lactate, accumulated in the tissues as PO2 decreased (Hochachka et al., 1993). Acid metabolite accumulation would decrease hemolymph pH and hence increase internal PCO2, enhancing CO2 elimination. However, it is unlikely that acid metabolites accumulated in the hemolymph because we have direct evidence that hemolymph pH increases during hypoxia and bicarbonate concentration is higher than expected at lower PO2s (Greenlee and Harrison, 1998). Thus, the washout of CO2 from the hemolymph and tissues as the animal moved towards a new, lower, steady-state internal PCO2 is likely to be the major cause of the transient increase in ṀCO2 upon exposure to hypoxia. It is worth noting that juveniles showed no transient increase in ṀCO2 upon exposure to hypoxia (Fig. 5), again consistent with our conclusion that they lack a ventilatory response to hypoxia.
Interestingly, a transient rise in CO2 emission upon exposure to a lower PO2 is observable even at relatively high PO2s where ventilation rates were unaffected by hypoxia (Fig. 1). One possibility is that ventilation increased transiently in response to hypoxia and then lowered in response to reduced internal PCO2 levels by the time we measured it. Alternatively, the transient rise in ṀCO2 could be due to transient changes in nonventilatory mechanisms for increasing tracheal conductance (e.g. increasing spiracular opening or decreasing tracheolar fluid levels).
Method for determination of Pc
Critical points are commonly determined in physiological research, and a variety of methods have been proposed for statistical analysis of critical point data (Nickerson et al., 1989; Yeager and Ultsch, 1989). However, in our study, and many others, critical points were determined by exposing individuals to a range of environmental conditions, an experimental design that should be analyzed using repeated-measures approaches (Potvin et al., 1990). Unfortunately, published methods for determining Pcs have ignored the statistical problems with a repeated-measures design (Yeager and Ultsch, 1989). In the present study, we developed a method that allowed us to assign a Pc value to each animal and then use this as a data point for further statistical analysis. This method may be generally useful for investigators who wish to compare critical points across groups of animals in studies where the parameters are repeatedly measured for each animal.
Our technique could be criticized because the animal's resting ṀCO2 value has a strong effect on the Pc, and visual inspection of Fig. 6 shows a very large drop in ṀCO2 for all instars at ∼2–3 kPa. To address this issue and compare several methods of determining Pc, we first analyzed the individual data files and identified the PO2 at which ṀCO2 dropped below the 75th, 50th or 25th percentile of normoxic ṀCO2 (Fig. 7, lines C, D and E, respectively). Of these lines, the 75th and 50th percentiles result in a significant decrease of Pc with instar (Kendall's rank correlation coefficient, 75th – τ32=–0.65, P<0.01; 50th – τ31=–0.32, P<0.01). Only 14 animals had ṀCO2 that dropped below 25% of normoxic metabolic rate and, of these, eight were first-instar juveniles. There was no effect of instar on these Pc values (median=0 kPa). We also used paired t-tests to determine the first PO2 at which mean ṀCO2 decreased significantly below the mean ṀCO2 in normoxia (Fig. 7, line F). Pc determined in this way decreased approximately sixfold with instar. The fact that most of these methods yield linear decreases of Pc with age supports our conclusion that Pc falls with age/size in S. americana. However, the fact that there is no age effect on the PO2 at which ṀCO2 drops to 25% of its normoxic rate suggests that even the youngest grasshoppers may be able to sustain some aerobic metabolism at very low atmospheric PO2. Alternatively, the very low ṀCO2 measured at low air PO2 may represent anaerobic metabolism.
Ontogenetic variation in the ventilatory response to hypoxia
In general, as grasshoppers grew, their hypoxia tolerance increased, as shown by the decreased Pc for ṀCO2 with age. This pattern is similar to that seen in the lobster Nephrops norvegicus, where adults have a lower Pc for O2 consumption compared with larvae (Spicer, 1995). Adult and older juvenile crayfish, Procambarus clarkii, respond to hypoxia by increasing ventilation frequency, whereas larval ventilation frequency decreases in hypoxia (Reiber, 1997). To our knowledge, Pc has not been compared throughout ontogeny for any other insect species, so it is unclear whether this pattern will prove to be widespread throughout the arthropods.
Adult grasshoppers exposed to hypoxia were able to maintain constant CO2 emission down to a PO2 of 1.8 kPa (Figs 6, 7). Adult grasshoppers had such a low Pc because they increased both breathing frequency and abdominal pumping height in response to hypoxia (Figs 6, 7, 8, 9). Together, the increases in abdominal pumping frequency and tidal volume resulted in a fourfold increase in Gmax from first-instar to adult grasshoppers (Fig. 10; ANOVA effect of instar, F1,30=31.6, P<0.001). Interestingly, while Gmax increased strongly with age when using ṀCO2 measures and Pc values from the 3 min exposures to hypoxia, Gmax was similar for first instars and adults when analyzed using 1 h exposures (Fig. 10). This pattern occurred because the 1 h exposure to hypoxia substantially decreased ṀCO2 in adults but not first instars (Fig. 5) and Pc increased more dramatically in adults (Table 2). The physiological bases to the disparity between the 1 h and 3 min patterns for Gmax are unknown without information on internal gas level variation during ontogeny. Based on our present data, the most likely explanation seems to be that ṀCO2 in adults exposed to short-term hypoxia was elevated by changes in internal PCO2 that did not occur in juveniles (Figs 5 and 11 suggest greater CO2 washout occurs during hypoxia for adults). When CO2 emission occurs at rates equivalent to metabolic CO2 production (as presumably happens when ṀCO2 is measured over 1 h), Gmax is size invariant. This suggests that first instars were able to achieve similar gas exchange rates despite lower tidal volumes and ventilatory frequencies, presumably because their smaller size enhances diffusive gas exchange.
The increased percent change in abdominal height (Fig. 8) is in contrast to our previous work (Greenlee and Harrison, 1998), which showed that tidal volume did not change in response to hypoxia in S. americana. The difference between the two studies may be due to population differences or the fact that we previously measured three dimensions of the abdomen; in the present study, we measured only the dorso-ventral dimension for a quicker index of tidal volume. However, prior research on Locusta migratoria, a closely related species, did show increases in both breathing frequency and tidal volume with decreased PO2 (Arieli and Lehrer, 1988). To further address this discrepancy, we calculated tidal volumes for adults at 2, 5, 10 and 21 kPa O2 using the equation: 6 where ṀCO2 is the whole animal CO2 emission rate in μmol min–1, 22.4 (μl μmol–1) is the ratio for converting ṀCO2 to a volume, and FCO2 is the fraction of CO2 in the tracheal air for adult S. americana from Greenlee and Harrison (1998). According to this calculation, tidal volume did not vary with PO2 (mean=29±1.9 μl breath–1), supporting our prior conclusion that tidal volume does not change during hypoxia exposure (Greenlee and Harrison, 1998). However, these calculated values were lower than those we measured optically (mean=44±3.7 μl breath–1), possibly due to approximately 40% lower ventilation frequencies measured in that study (Greenlee and Harrison, 1998).
In contrast to the pattern seen in older grasshoppers, first-instar animals had no apparent ventilatory response to hypoxia. They showed no change in ventilation frequency (Fig. 8) or tidal volume (Fig. 9), and ṀCO2 dropped, though not always significantly, with every decrease in atmospheric PO2 (Fig. 6). Conceivably, first instars could have responded to hypoxia by increasing diffusive gas exchange (e.g. opening spiracles or removing tracheolar fluid). To test this idea, we calculated the ratio of ṀCO2 (μmol min–1) to abdominal pumping frequency. If younger grasshoppers were using more diffusive gas exchange, then the μmol CO2 g–1 breath–1 should be higher in first instars and should increase during hypoxia. Indeed, in first-instar grasshoppers, this index of gas exchange per breath was higher than that in older instars (Fig. 12), supporting the hypothesis that first instars were more reliant on diffusion than older grasshoppers. Alternatively, first instars could simply have had higher internal and expired PCO2 levels. However, there is no evidence that first instars responded to hypoxia by opening spiracles or removing tracheolar fluid, as the μmol CO2 g–1 breath–1 was constant as air PO2 decreased until PO2s dropped below the Pc for abdominal pumping (Fig. 12).
Why do first-instar grasshoppers lack a ventilatory response? One hypothesis is that younger grasshoppers have not developed the complete neural circuitry for responding to hypoxia. This idea is supported by the findings of Miller and Mills (1976), who noted that first- and second-instar S. gregaria also lack synchronization between spiracular valve activity and abdominal pumping. The adult pattern of synchronization between abdominal pumping and spiracular valve activity appears sporadically in the third instars (Miller and Mills, 1976). In our study, third-instar animals demonstrated an adult-like ventilatory response to hypoxia (Fig. 8). Alternatively, first-instar grasshoppers may have not yet developed the capacity to sense oxygen. This hypothesis could be tested by looking for members of the HIF-1α oxygen-sensing cascade (Lavista-Llanos et al., 2002) and comparing the expression patterns between instars. Interestingly, expression of HIF-1α and HIF-1β has been found to decrease with juvenile development in mice, the opposite of what we would predict for grasshoppers (Madan et al., 2002). Finally, first-instar grasshoppers may lack the large air sacs found in older animals, reducing the capacity for convective gas exchange.
What causes Pc to decline throughout development?
Rearranging equation 2 to solve the Pc for ṀCO2, we can see that the Pc depends positively on ṀCO2 and negatively on conductance: 7 From first instar to adult, the Pc for ṀCO2 decreased eightfold (Fig. 7). Much of the decrease was accounted for by the approximately fourfold increase in Gmax (Fig. 10). However, the approximately threefold decrease in mass-specific ṀCO2 at the Pc with age also contributed strongly to the trend towards decreasing Pc with age (Fig. 13).
The decrease in mass-specific metabolic rate with size is well known among animals (Schmidt-Nielsen, 1984; West et al., 1997). However, the increase in mass-specific gas-exchange capacity with age was less expected (Fig. 10). One possibility is that the increased tracheal system conductance with age in these grasshoppers facilitates flight, an adult activity that requires a 40–50-fold increase in O2 consumption above basal rates (Krogh and Weis-Fogh, 1951). Additionally, larger insects may generally be more able to cope with hypoxia because they have evolved mechanisms, such as abdominal pumping, to facilitate convective gas exchange, as may be necessary for adequate oxygen delivery in large insects (Weis-Fogh, 1964).
Pc for abdominal pumping
In contrast to the decrease in Pc for ṀCO2 throughout ontogeny, the Pc for abdominal pumping was very low across all instars. At each instar, abdominal pumping continued even after ṀCO2 began to drop. The large disparity between the Pc for ṀCO2 and the Pc for ventilation frequency strongly suggests that insects are able to preferentially shut down certain body systems in response to hypoxia. Additionally, O2 delivery to all parts of the respiratory system may be better than to other parts of the body, since the tracheal supply to the pacemaker neuron is substantial.
Interestingly, when we calculated Pc as the PO2 at which ṀCO2 was lower than 50% of the normoxic rate, this line overlapped the Pc for abdominal pumping (Fig. 7). Does this correlation mean that the Pc for abdominal pumping represents the `true' critical point for oxygen delivery? This strongly depends on what is meant by `true' critical point. Any significant drop in ṀCO2 could be due to either a direct oxygen limitation of chemical reactions or to an oxygen-sensing mechanism that reduces oxygen-consuming behavioral or physiological functions (for example, hypoxia induces fatigue in humans under conditions in which oxygen is not believed to be limiting to muscle mitochondrial function). It is likely that animals have several Pc values that vary with the function being observed; an exciting test would be to determine the PO2 at which other functions, such as feeding, protein synthesis or locomotion, are affected.
Insect gigantism during the Paleozoic era
In this study, we partially tested the idea that larger insects have more difficulty with gas exchange. A negative scaling of the safety margin for oxygen delivery is one possible prediction derived from the hypothesis that atmospheric hyperoxia in the late Paleozoic facilitated insect gigantism (Graham et al., 1995; Dudley, 1998). Clearly, the results from this study provide no support for this prediction. In fact, our finding that smaller grasshoppers had a much higher Pc for gas exchange (Figs 6, 7) was exactly the opposite of what would be predicted if larger insects had decreased safety margins for gas exchange. However, our experiments did not distinguish between the effects of body size and development; to do so, the safety margin for oxygen delivery must be compared across individuals of different species at the same developmental stage. Additionally, a better test of whether gas exchange becomes more challenging for larger insects may be to examine the scaling of Pc for gas exchange for insects at their maximal ṀO2. Indeed, Erythemis simplicicollis dragonflies are oxygen limited during flight in normoxia, evidence that safety margins for oxygen delivery are small during activity (Harrison and Lighton, 1998).
It is generally thought that larger insects must use convective gas exchange to achieve adequate oxygen delivery (Weis-Fogh, 1964), and our data strongly suggest that this is true within the developmental stages of S. americana (Figs 8, 9, 13). Therefore, an alternative prediction derived from the hypothesis that larger insects have more difficulty with gas exchange is that larger insects may use compensatory mechanisms to overcome diffusion limitations. If so, we might expect the largest extant insects to exhibit maximal use of convective gas exchange. Increased use of convection might allow larger insects to have more responsive respiratory systems and control over diffusive water loss (Kestler, 1985). Examining the scaling of air sac volumes and use of convection across insects of various sizes would allow testing of this hypothesis.
Support for this project was provided by NSF IBN-9985857 to J.F.H., NSF IBN-0206678 to K.J.G. and J.F.H., and a Sigma Xi GIAR to K.J.G. We would also like to thank three anonymous reviewers, Michelle Fay, Jeffrey Hazel, Joanna Henry, Paul Kestler, Michael Quinlan, Brenda Rascón, Ron Rutowski, Glenn Walsberg, Art Woods and especially Scott Kirkton for helpful comments on this manuscript.
- © The Company of Biologists Limited 2004