Many insects ventilate discontinuously when quiescent, exhibiting prolonged periods during which little or no gas exchange occurs. We investigated the consequences of discontinuous ventilation (DV) on haemolymph acid–base status and tested whether spiracular opening during DV is due to changes in internal gas tensions in the western lubber grasshopper Taeniopoda eques. At 15 °C, resting T. eques exhibited interburst periods of about 40 min. During the interburst period, haemolymph rose from 1.8 to 2.26 kPa, with minimal acidification of haemolymph. Animals in atmospheres in which was 2 kPa or below continued to exhibit DV, while atmospheres in which was 2.9 kPa or above caused cessation of DV. These data indicate that accumulation of internal CO2 to threshold levels between 2 and 2.9 kPa induces spiracular opening in grasshoppers. In contrast to the situation in lepidopteran pupae, variation in atmospheric had no effect on interburst duration. Relative to lepidopteran pupae, the internal of grasshoppers during DV is threefold lower, the required for triggering spiracular opening is also threefold lower, and the open phase spiracular conductance is at least tenfold higher, demonstrating that considerable diversity exists in these aspects of insect respiratory physiology.

It is becoming increasingly clear that many, if not most, terrestrial adult insects exhibit discontinuous ventilation (DV) when quiescent similar to that first documented for lepidopteran pupae (Kestler, 1985; Slama, 1988; Lighton, 1988, 1994; Lighton and Lovegrove, 1990; Lighton and Wehner, 1993). Similar patterns of DV have also been observed in ixodid ticks (Lighton et al. 1993). During DV, episodes of gas exchange alternate with interburst periods characterized by greatly reduced CO2 emission, which may last for periods from minutes to hours (Lighton, 1994). In this study, we examined the effect of DV on haemolymph acid–base status and investigated the possible role of carbon dioxide accumulation or oxygen depletion in the control of spiracular opening during DV, in the western lubber grasshopper Taeniopoda eques.

The general physiology of DV in insects has recently been reviewed (Lighton, 1994). Briefly, the DV cycle consists of a closed (C) phase, in which the spiracles are sealed, followed by a flutter (F) phase, in which the spiracular valve opens and closes slightly at high frequency and finally an open (O) phase, in which the spiracular valves remain open. During the C phase, gas exchange is minimal, internal oxygen stores are depleted and carbon dioxide accumulates. During the F phase, at least in lepidopteran pupae, bulk inward flow of air enhances oxygen uptake relative to water and carbon dioxide emission (Buck, 1958; Levy and Schneiderman, 1958; Slama, 1988). In other insects, gas exchange during the F phase may be predominantly driven by abdominal pulsations (Lighton, 1991) or by diffusion (Lighton, 1994). In most insects, the bulk of carbon dioxide emission occurs during the O phase, during which gas exchange may be completely diffusive or associated with convection (Kestler, 1985; Lighton, 1991, 1994).

The control of DV has been most thoroughly investigated in lepidopteran pupae, in which the duration of the interburst period is positively correlated with atmospheric oxygen levels and the C phase is eliminated at an atmospheric below 6 kPa (Schneiderman and Williams, 1955; Buck and Keister, 1955). In addition to increasing the duration of the C phase, elevation of atmospheric oxygen concentration also eliminates the F phase, implying that initiation of the F phase is due to the depression of internal to a threshold level in normoxic pupae (Schneiderman and Williams, 1955; Levy and Schneiderman, 1966). Manipulation of tracheal gas levels indicates that spiracles begin to flutter at a tracheal below 5–12 kPa and open fully at a below 3 kPa (Schneiderman, 1960). Elevation of atmospheric or tracheal to 8 kPa or above causes the spiracles to open (Schneiderman and Williams, 1955; Schneiderman, 1960). Together, these results suggest that, in lepidopteran pupae, cessation of the C phase (coincident with initiation of the F phase) is triggered by a fall in internal to a threshold value, while cessation of the F phase (and, therefore, initiation of the O phase) is triggered by a rise in internal to a threshold value (Levy and Schneiderman, 1966). The effects of on spiracular opening appear to be primarily mediated by the responses of the metathoracic ganglia, while the effects of are peripheral (Burkett and Schneiderman, 1974).

There are few data available to determine whether the control mechanisms documented for lepidopteran pupae are generally applicable (Lighton, 1994). Spiracular movements are affected by local concentrations of CO2 and O2 in adult cockroaches, fleas, flies and locusts (Hazelhoff, 1927; Wigglesworth, 1935; Case, 1956; Hoyle, 1960). However, to demonstrate that spiracular opening during DV is triggered by changes in internal gas tensions, it is necessary to demonstrate that the quantitative effects of or on spiracular opening are consistent with the measured internal changes in gas tensions, as has been shown in pupae (Levy and Schneiderman, 1966; Burkett and Schneiderman, 1974).

The DV cycle also has potentially important implications for understanding the acid–base physiology of insects. Specifically, does DV impose large cyclic variation in extra- and intracellular pH on insects? There have been no direct measurements of the effect of DV on the acid–base status of any insect, although comparisons of the volume of CO2 emitted during the burst with the whole-animal non-bicarbonate buffer value suggest that pH changes associated with DV in pupae should be small (Buck and Friedman, 1958; Bridges and Scheid, 1982).

The grasshoppers T. eques and Romalea guttata exhibit DV when quiescent (Quinlan and Hadley, 1993; Hadley and Quinlan, 1993). In these species, gas exchange during the interburst period cannot always be clearly separated into C and F phases because individuals vary considerably in their patterns of interburst gas exchange (Quinlan and Hadley, 1993; Hadley and Quinlan, 1993). Since we could not reliably distinguish the C and F phases in T. eques, for the purpose of the present study we defined the interburst period as the C+F phases, and we examined only the control of the initiation of the O phase (i.e. the end of the interburst period). We studied DV in T. eques at 15 °C partly because, at this low temperature, the durations of the interburst and O phases were long relative to handling and sampling times. The low experimental temperature, large body size and docile nature of this aposematic species all reduced the magnitude and speed of handling effects on their acid–base status.

Animals

Taeniopoda eques (Burmeister) were collected from sites near Portal, Arizona, USA, and kept in culture at Arizona State University on a 14 h:10 h L:D cycle, with cycling daily temperatures (approximately 20–35 °C), and access to a heat bulb for thermoregulation. Animals were fed a diet of Romaine lettuce, kale and trout chow. Animals were kept in culture for 1–2 months before experiments. All animals used were adult males. Grasshoppers were kept individually overnight at 30 °C with lettuce and for 1 h without food prior to experiments. All experiments were carried out at 15 °C in the dark, as these conditions maximized the probability that T. eques would exhibit DV and lengthened the interburst period (Quinlan and Hadley, 1993). Barometric pressure averaged 97 kPa during the experiments.

Effect of handling on CO2 emission

Carbon dioxide emission (; μmol g−1 h−1) was measured using a flow-through respirometry system optimized for speed of response and a LICOR LI-5252 CO2 analyzer. Animals were placed in Lucite respirometry chambers (20 ml) in the dark, within a 15 ° C temperature-controlled cabinet. Flow rates were regulated with a Brooks (model 5850) mass flow meter and Brooks (model 5878) controller and averaged 1.45±0.02 l min−1 (mean ± S.E.M.). Under these high-flow, low-volume conditions, 98 % equilibration time for the chamber was approximately 7 s. By minimizing the length of tubing between the respirometry chamber and the CO2 analyzer, the response time of the analyzer to an injection of CO2 into the chamber was reduced to less than 3 s.

For measurements of haemolymph acid–base status, we obtained the haemolymph sample within 10 s of opening the respirometry chamber. We conducted preliminary tests to determine whether our sampling technique was rapid enough to measure in vivo haemolymph acid–base status accurately. To mimic the disturbance of sampling, we opened the temperature cabinet and shook the respirometry chamber while measuring

We calculated during the 10 s following disturbance from the measured corrected for the time lag between analyzer and chamber. We compared the amount of CO2 emitted with the normal quantity of CO2 measured during the O phase, to determine the fraction of CO2 lost from the animal during the sampling procedure.

Experiment 1: effect of DV cycle on haemolymph acid–base status

The goal of these experiments was to compare haemolymph acid–base status before and after a burst of CO2 emission (pre- and post-O phase) for T. eques breathing normoxic, dry, CO2-free air. Animals were placed in the flow-through respirometry chambers at 15 °C and the DV cycle was monitored by measuring . For measurement of haemolymph acid–base status at the end of the O phase, we allowed animals to perform at least three DV cycles. As carbon dioxide emission declined towards zero at the end of the O phase, we quickly removed the grasshopper from the chamber and sampled haemolymph from the ventral neck region as previously described (Harrison, 1988a). For measurement of haemolymph acid–base status at the initiation of the O phase, we removed the animal from the chamber for haemolymph sampling just as a rise in CO2 emission at the end of the interburst period was observed. Haemolymph pH was measured using a Radiometer capillary electrode thermostatically controlled to 15 °C, and total CO2 (, mmol l−1) was measured by gas chromatography as previously described (Harrison and Kennedy, 1994). Haemolymph and [HCO3] were calculated from pH and values using CO2 solubility coefficients and carbonic acid dissociation constants for locust haemolymph (Harrison, 1988a).

Experiment 2: effect of atmospheric CO2 level on DV

To test the effect of atmospheric CO2 on DV, we allowed animals to perform three complete DV cycles in normoxic, dry, CO2-free air. The DV cycle was monitored by measuring changes in excurrent oxygen fraction using an Ametek S3A oxygen analyzer, with the analog output filtered and signal-averaged so that the oxygen content of the excurrent air was measured to an accuracy of ±0.003 %. Flow rates were set at 0.1 l min−1, using the mass flow meter. At the beginning of the fourth interburst period, we switched the airstream to one of several dry gas mixtures that varied in CO2 level ( was 0.98, 1.95, 2.93 or 3.90 kPa, was 21 kPa, balance N2). We monitored the oxygen content of the excurrent air for the next two DV cycles (if present). When the animal continued to exhibit DV with the test gas, we examined the effect of the test gas on the interburst duration.

Experiment 3: effect of atmospheric O2 level on DV

The protocol for experiment 3 was similar to that of experiment 2, except that atmospheric O2 rather than CO2 level was varied and DV was monitored by measuring using an ANARAD AR-411 CO2 analyzer (Quinlan and Hadley, 1993). Flow rate was set at 0.15 l min−1 using a Brooks (model 5878) mass flow controller. The values of the test gases used were 10.7, 21 or 31 kPa (balance nitrogen). Because all animals used in these experiments continued to exhibit DV in these test gases (see below), we designed our experimental protocol to detect changes in the duration of the interburst period. Grasshoppers were exposed to normoxic atmospheres for at least three DV cycles, then to the test gas for at least three DV cycles and finally to normoxic conditions again for a further three DV cycles. This procedure allowed us to control for the tendency of the interburst period to increase with time. All inspiratory gases used were water- and CO2-free.

Haemolymph and whole-body non-bicarbonate buffer values

Buffer values of the haemolymph and whole-body fluids of T. eques were measured by titration of diluted samples over the pH range 7.0–7.5. Haemolymph samples were collected from animals kept as described above and immediately transferred to a pre-weighed Eppendorf vial containing a simple, unbuffered locust saline designed to inhibit cellular metabolism (NaF, 100 mmol l−1; K2SO4, 5 mmol l−1; MgSO4, 10 mmol l−1; nitriloacetic acid, 5 mmol l−1; Hanrahan et al. 1984; Pörtner et al. 1990). The vial and sample were weighed for calculation of the amount of sample added (dilutions were approximately ninefold); the vials were frozen and kept at −20 °C until analysis.

Whole-body samples were obtained by pulverizing T. eques, kept as described above, in liquid nitrogen. The pulversate was transferred to a pre-weighed test tube containing locust saline. The saline used was the same as that described above, except that KF was substituted for NaF, as most buffers titrated in the whole-body sample are intracellular. The test tube was weighed after pulversate addition for calculation of sample dilution (six- to tenfold). Samples were frozen and kept at −20 °C until analysis.

Samples of the diluted haemolymph or whole-body pulversates (3 ml) were first acidified to below pH 4.0 by adding a small amount (approximately 40 μl) of 0.5 mol l−1 HCl and stirred for 2 h to remove bicarbonate. The pH of the samples was then adjusted to approximately 7.0 with 0.5 mol l−1 NaOH. Titrations were carried out by adding 10 μl samples of 0.05 mol l−1 NaOH, and measuring pH using a Radiometer GK2401C electrode and a Radiometer pHM 84 pH meter. Non-bicarbonate buffer value (mequiv pH unit−1 kg−1) was calculated from the number of moles of titrant added, the change in pH and the solution volume, correcting for dilution and for the buffer value of the saline. Each whole-body sample was split into at least eight aliquots, which were separately titrated (the coefficient of variation within samples was less than 20 %), and the values were averaged. There was only sufficient fluid to titrate each haemolymph solution once. A separate sample of animals was dried to constant mass at 55 °C to determine body water content.

DV at 15 °C in Taeniopoda eques

As previously reported, when T. eques are left undisturbed for a period of hours, they perform DV (Fig. 1). Peaks of oxygen consumption coincide with peaks of carbon dioxide production during the O phase (Fig. 1). The mean quantity of carbon dioxide released per burst was 2.71±0.276 μmol (N=24 individuals, 2–3 bursts averaged per individual). These animals averaged 2.30±0.073 g in mass (N=24). The duration of the O phase averaged 5.4±2.1 min (N=24). The average duration of the interburst period was 40.2±4.32 min (N=24). averaged over multiple whole cycles was 2.6±0.03 μmol g−1 h−1 (N=24).

Fig. 1.

Sample traces showing O2 consumption and CO2 production of an individual Taeniopoda eques at 15 °C.

Fig. 1.

Sample traces showing O2 consumption and CO2 production of an individual Taeniopoda eques at 15 °C.

Effect of handling on CO2 emission

Disturbance caused an increase in well above O phase and abolished DV in all animals tested (Fig. 2). However, in all cases (N=3) the amount of CO2 emitted during the 10 s following disturbance (representing the time necessary to obtain a haemolymph sample) represented less than 2 % of the amount of CO2 emitted during a normal O phase (<0.05 μmol). Therefore, it is unlikely that our measurements of haemolymph of animals collected at the end of the interburst period appreciably underestimated haemolymph of undisturbed locusts.

Fig. 2.

The effect of disturbance (shown by dashed vertical line) on in an individual Taeniopoda eques at 15 °C. Prior to disturbance, the animal emitted CO2 in O phase bursts lasting 2–8 min, separated by periods of very low M·CO2. Disturbance induced a large increase in CO2 emission for several minutes, followed by a prolonged cessation of DV. The inset shows M·CO2 over the 20 s following disturbance, corrected for the time lag between the animal chamber and the gas analyzer. Although M·CO2 following disturbance is large relative to O phase M·CO2, the quantity of CO2 (μmol) emitted during the 10 s following disturbance was only 2 % of the average quantity of CO2 emitted during the O phase for this individual.

Fig. 2.

The effect of disturbance (shown by dashed vertical line) on in an individual Taeniopoda eques at 15 °C. Prior to disturbance, the animal emitted CO2 in O phase bursts lasting 2–8 min, separated by periods of very low M·CO2. Disturbance induced a large increase in CO2 emission for several minutes, followed by a prolonged cessation of DV. The inset shows M·CO2 over the 20 s following disturbance, corrected for the time lag between the animal chamber and the gas analyzer. Although M·CO2 following disturbance is large relative to O phase M·CO2, the quantity of CO2 (μmol) emitted during the 10 s following disturbance was only 2 % of the average quantity of CO2 emitted during the O phase for this individual.

Experiment 1: effect of DV on haemolymph acid–base status in haemolymph samples from the end of the O phase was significantly lower than in samples taken at the initiation of the O phase (Fig. 3). Changes in haemolymph pH, and [HCO3] were minor and not significant (Fig. 3).

Fig. 3.

Haemolymph acid–base status of T. eques males sampled at the end of the interburst period (start of O phase) or at the end of the O phase (post-CO2 emission). Mean ± S.E.M., N=8 for each group. The asterisk indicates a significant difference between groups, t-test, P<0.05.

Fig. 3.

Haemolymph acid–base status of T. eques males sampled at the end of the interburst period (start of O phase) or at the end of the O phase (post-CO2 emission). Mean ± S.E.M., N=8 for each group. The asterisk indicates a significant difference between groups, t-test, P<0.05.

Experiment 2: effect of atmospheric CO2 level on DV

At the flow rates used, the O phase was easily discernible by a decrease in excurrent oxygen content of about 0.07 % (Fig. 4). However, we did not calculate oxygen consumption rates during these experiments, because the signal-to-noise ratio was too low. When T. eques were exposed to atmospheres containing a of 2 kPa or lower, all animals continued to ventilate discontinuously. Interburst duration increased significantly in 2 kPa but not in 1 kPa atmospheres relative to those in 0 kPa (0 kPa 35.9±5.55 min, N=8; 1 kPa , 38.8±4.48 min, N=4; 2 kPa 47.2±6.69 min, N=8; mean ± S.E.M., paired t-tests). Animals exposed to atmospheres containing 2.9 kPa or higher exhibited a single O phase, followed by continuous, erratic oxygen consumption (Figs 4, 5).

Fig. 4.

The effect of a switch (at the dashed line) from an atmosphere containing 0 kPa CO2 (21 kPa O2) to an atmosphere containing 3 kPa CO2 (21 kPa O2) on the O2 content of the excurrent air from a respirometry chamber containing a male Taeniopoda eques at 15 °C. In atmospheres lacking CO2, this animal exhibited DV with an interburst duration of approximately 45 min. When the atmosphere was switched to 3 kPa CO2 during the interburst period, animals exhibited a single O phase, followed by cessation of DV.

Fig. 4.

The effect of a switch (at the dashed line) from an atmosphere containing 0 kPa CO2 (21 kPa O2) to an atmosphere containing 3 kPa CO2 (21 kPa O2) on the O2 content of the excurrent air from a respirometry chamber containing a male Taeniopoda eques at 15 °C. In atmospheres lacking CO2, this animal exhibited DV with an interburst duration of approximately 45 min. When the atmosphere was switched to 3 kPa CO2 during the interburst period, animals exhibited a single O phase, followed by cessation of DV.

Fig. 5.

The effect of atmospheric PCO2 on the percentage of animals exhibiting DV. Animals were selected which exhibited DV in atmospheres lacking CO2. They were then exposed to atmospheres of various PCO2(PO2=21 k Pa). For PCO2=1.95 and 2.93 kPa, N=9; for PCO2=0.98, 3.90 and 4.95 kPa, N=4. The vertical line shows pre-O phase haemolymph PCO2

Fig. 5.

The effect of atmospheric PCO2 on the percentage of animals exhibiting DV. Animals were selected which exhibited DV in atmospheres lacking CO2. They were then exposed to atmospheres of various PCO2(PO2=21 k Pa). For PCO2=1.95 and 2.93 kPa, N=9; for PCO2=0.98, 3.90 and 4.95 kPa, N=4. The vertical line shows pre-O phase haemolymph PCO2

Experiment 3: effect of atmospheric O2 level on DV

All animals exposed to either 11 or 31 kPa continued to exhibit DV. Exposure to 11 or 31 kPa did not affect interburst duration, although interburst duration did tend to increase with time (Fig. 6). We also conducted a few experiments in which atmospheric O2 levels were increased to 60 kPa. In none of these cases did the interburst duration vary with atmospheric (Fig. 7).

Fig. 6.

Effect of atmospheric PO2 on the duration of the interburst period (min) during DV in Taeniopoda eques at 15 °C. All animals were monitored over a period of at least 3 h at 21 kPa PO2, followed by 3 h at the test PO2 (11 or 31 kPa), and finally 3 h at 21 kPa PO2. N=8 for each test gas. Interburst durations in the test gases did not differ from initial, final or averaged controls (paired t-tests, P>0.05). Values are means ± S.E.M.

Fig. 6.

Effect of atmospheric PO2 on the duration of the interburst period (min) during DV in Taeniopoda eques at 15 °C. All animals were monitored over a period of at least 3 h at 21 kPa PO2, followed by 3 h at the test PO2 (11 or 31 kPa), and finally 3 h at 21 kPa PO2. N=8 for each test gas. Interburst durations in the test gases did not differ from initial, final or averaged controls (paired t-tests, P>0.05). Values are means ± S.E.M.

Fig. 7.

The effect of atmospheric N2/O2 levels (indicated above trace) on M·CO2 in a single male Taeniopoda eques at 15 °C. Increasing atmospheric O2 level from 21 kPa to 61 kPa had no effect on interburst duration.

Fig. 7.

The effect of atmospheric N2/O2 levels (indicated above trace) on M·CO2 in a single male Taeniopoda eques at 15 °C. Increasing atmospheric O2 level from 21 kPa to 61 kPa had no effect on interburst duration.

Haemolymph and whole-body buffer values and the estimated increase in whole-body CO2 stores

Mean haemolymph non-bicarbonate buffer value (βh) was 16.6±2.88 mequiv pH unit−1 kg−1 (S.E.M., N=12). Mean whole-body non-bicarbonate buffer value was 24.8±2.13 mequiv pH unit−1 kg−1 (S.E.M., N=6). Animal water content averaged 72.2±0.069 % (S.E.M., N=9). Non-bicarbonate buffer value per kilogram animal water (βa) was 34.4±2.95 mequiv pH unit−1 kg−1 (S.E.M., N=6).

The increase in dissolved CO2 stores in the animal during the interburst period was 0.3 μmol, calculated as:
where d is the increase in whole-body dissolved CO2 stores, is the difference in the measured in haemolymph samples taken before and after the O phase (0.46 kPa), s is the CO2 solubility coefficient in locust haemolymph at 15 °C (384.9 μmol kg−1 kPa−1, Harrison, 1988a), m is the mean animal mass (0.0023 kg) and w is the mean animal water content (0.72 kg kg−1 body mass). Gaseous (tracheal) CO2 stores increased by approximately 0.2 μmol, calculated as:
where g is the increase in tracheal CO2 stores, is as defined above, Pb is the mean barometric pressure during the experiments (97 kPa), t is the mean tracheal space of a grasshopper (750 μl, Harrison, 1988b), and x is a factor used to convert μl to μmol (0.0446 μmol per μl).

The increase in whole-body HCO3 stores will depend on βa and the average pH of the whole animal at the start of the interburst period. We made two estimates of the increase in [HCO3] during the interburst period using the graphical approach described by Davenport (1974). We plotted βa for the 1.8 and 2.26 kPa isopleths (Fig. 8) assuming that the average pH of the whole animal at the start of the interburst was either (1) equivalent to haemolymph pH, or (2) 0.12 units more alkaline than the haemolymph pH, as in Schistocerca nitens (Harrison, 1988b). On Fig. 8, the vertical distances between the points where βa crosses the 1.8 and 2.26 kPa isopleths are estimates of the increase in whole-body [HCO3] during the interburst period. These Δ[HCO3] values ranged between 1.2 and 1.3 mmol kg−1, depending on the initial pH chosen (Fig. 8). The increase in whole-body [HCO3] was calculated from:

Fig. 8.

Davenport diagram (Davenport, 1974) for Taeniopoda eques at 15 °C showing the pH and [HCO3] at the start and end of the O phase, haemolymph in vitro non-bicarbonate buffer value (βh) and whole-body non-bicarbonate buffer value (βa). βa is plotted either through the haemolymph pH and [HCO3] at the end of the O phase, or through the pH value that intersects the 1.8 kPa CO2 isopleth, assuming that average intracellular pH is 0.12 units more alkaline than haemolymph pH (as in Schistocerca nitens; Harrison, 1988b). The vertical distances between the points where βa crosses the 1.8 and 2.26 kPa PO2 isopleths are estimates of the increase in whole-body [HCO3-] during the interburst period.

Fig. 8.

Davenport diagram (Davenport, 1974) for Taeniopoda eques at 15 °C showing the pH and [HCO3] at the start and end of the O phase, haemolymph in vitro non-bicarbonate buffer value (βh) and whole-body non-bicarbonate buffer value (βa). βa is plotted either through the haemolymph pH and [HCO3] at the end of the O phase, or through the pH value that intersects the 1.8 kPa CO2 isopleth, assuming that average intracellular pH is 0.12 units more alkaline than haemolymph pH (as in Schistocerca nitens; Harrison, 1988b). The vertical distances between the points where βa crosses the 1.8 and 2.26 kPa PO2 isopleths are estimates of the increase in whole-body [HCO3-] during the interburst period.

where b is the increase in whole-body HCO3 stores, Δ [HCO3] is the increase in the whole-body [HCO3] during the interburst period, calculated as described above using Fig. 8 (1.2–1.3 mmol kg−1), and m and w are as defined above. The total increase in whole-body CO2 during the interburst period was 2.5–2.7 μmol, depending on the whole-body average pH chosen (Table 1).
Table 1.

Calculated increases in the whole-animal pools of CO2 during the interburst period in Taeniopoda eques at 15 °C

Calculated increases in the whole-animal pools of CO2 during the interburst period in Taeniopoda eques at 15 °C
Calculated increases in the whole-animal pools of CO2 during the interburst period in Taeniopoda eques at 15 °C

Our data demonstrate that spiracular closure during DV in T. eques results in very little change in the haemolymph acid–base status. We also show, for the first time in an adult insect, that opening of the spiracles at the beginning of the O phase is triggered by a rise in internal to a threshold value.

Although some of the control mechanisms of DV appear to be similar between pupal lepidopterans and adult grasshoppers, our data indicate that it cannot be assumed that either the quantitative patterns of tracheal gas changes or the mechanisms of spiracular control documented in pupae are generally applicable to adult insects.

Critique of methods

The haemolymph sampling method could have resulted in a loss of CO2 due to stress-associated spiracular opening, resulting in an underestimate of the rise in internal during the interburst period. However, measurements of during disturbance indicated that less than 2 % of the amount of CO2 normally emitted during the O phase was emitted during the first 10 s following disturbance, suggesting that any possible underestimate of at the end of the interburst period would be small. Since haemolymph lacks carbonic anhydrase in locusts (Levenbook and Clark, 1950), sampling-associated changes in haemolymph may have been even less than 2 %. However, Fig. 2 clearly illustrates that stress-associated changes in can be large relative to during DV; therefore, with a slower sampling protocol (or a faster-responding species), stress-associated loss of CO2 could preclude accurate measurement of internal gas levels during DV.

Handling of insects can also induce stress-associated accumulation of carbon dioxide in the haemolymph (Matthews et al. 1976). In the two-striped grasshopper Melanoplus bivittatus, activity is accompanied by an elevation of haemolymph of 2–3 kPa, which can be detected within 30 s at 20 °C (Harrison et al. 1991). This effect should be less rapid in the more sluggish T. eques. If handling increased the haemolymph in the present study, pre-and post-O phase measurements should be similarly affected, since the spiracles open upon disturbance (Fig. 2) and pre- and post-O phase animals were handled identically.

Another potential problem in assessing internal gas levels from haemolymph acid–base status is that haemolymph may not be in equilibrium with tracheal gases, owing to the lack of carbonic anhydrase in haemolymph. Potentially, non-equilibrium between haemolymph and tracheal gases could result in changes in haemolymph that are substantially smaller than those occurring in the tracheae. However, during the 40 min interburst period, prolonged spiracular closing should promote equilibrium between haemolymph and tracheal . It seems more likely that changes in haemolymph may lag behind changes in tracheal during the O phase and that tracheal (and perhaps cellular) may reach lower levels than we recorded in this study.

The most direct approach to assessing the validity of using our measures of haemolymph to estimate whole-animal average is to compare the average amount of CO2 released during the O phase, as measured by respirometry (2.7 μmol), with the amount of CO2 stored during the interburst period, estimated from the acid–base data (2.5–2.7 μmol; Table 1). The similarity of the values from these two independent techniques suggests that our sampling technique accurately measured haemolymph during DV and that disequilibrium between the haemolymph and the intracellular space must be small under these conditions.

The effect of DV on haemolymph acid–base status

In T. eques, DV has quite minor effects on haemolymph acid–base status. Changes in haemolymph pH (nonsignificant, 0.05 units) are small (and not significant) relative to those found during terrestrial locomotion in grasshoppers (0.1–0.3 units; Harrison et al. 1991) and during changes in body temperature (−0.017 pH units °C−1; Harrison 1988a,b). The changes in haemolymph acid–base status associated with DV are unlikely to be substantially greater at higher temperatures. In T. eques, a four- to fivefold rise in metabolic rate with temperature from 15 to 30 °C is accompanied by a tenfold decrease in the duration of the interburst period and an almost 50 % decrease in the volume of CO2 emitted during the O phase (Quinlan and Hadley, 1993). Over this temperature range, the solubility of CO2 in grasshopper haemolymph decreases by about 30 % (Harrison, 1988a), suggesting that DV at 30 °C should be associated with slightly smaller changes in internal and pH than those observed at 15 °C. Similarly, the change in average whole-animal pH during DV in pupae has been estimated to be less than 0.05 units (Buck and Friedman, 1958; Bridges and Scheid, 1982) and to be 0.030–0.037 units in T. eques (Fig. 8).

The changes in haemolymph pH, [HCO3] and are consistent with the hypothesis that all changes in extracellular pH during DV are due to CO2 accumulation and release. The Δ [HCO3]Δ),pH ratio during the O phase was 15.3 mmol l−1 pH unit−1, within the standard errors of the non-bicarbonate buffer value of 16.6 mequiv l−1 pH unit−1 measured on T. eques haemolymph in vitro (Fig. 8). This finding is of interest because Lighton and Gnaiger have shown, using microcalorimetry, that the initiation of the O phase in ants is accompanied by an endothermic event (Lighton, 1994). One possible explanation for this endothermic event is if the O phase were accompanied by active net acid transfer to the tissues or extracellular space. Such acidification could potentially increase the rate of CO2 removal during the O phase, decreasing O phase duration and respiratory water loss (Lighton, 1994). In grasshoppers, our data indicate that such a mechanism does not occur in the haemolymph. However, T. eques has a relatively long O phase (greater than 5 min) and respiratory water loss is a small component of their total water loss (Quinlan and Hadley, 1993), so mechanisms for increasing the rate of CO2 release during the O phase may be unimportant.

Control of spiracular opening

Our data strongly suggest that spiracular opening at the end of the interburst period in T. eques is due to the accumulation of internal CO2 to a threshold between 2 and 2.9 kPa. The haemolymph of animals sampled at the initiation of the O phase averaged 2.3 kPa. Animals exposed to an atmospheric of 1 or 2 kPa continued to exhibit DV. However, animals exposed to an atmospheric of greater than 2.9 kPa ceased DV and began continuous gas exchange (Figs 4, 5). There is no evidence that reduction of internal to a threshold level is responsible for triggering the end of the interburst period in T. eques. Halving and doubling atmospheric had no effect on interburst duration, suggesting that depletion of tracheal O2 stores was insufficient to induce spiracular opening.

Surprisingly, exposure to a of 2 kPa increased the duration of the interburst period. We expected that exposure of animals to atmospheres containing 1–2 kPa CO2 would result in faster accumulation of CO2 to threshold values. However, the haemolymph of animals sampled at the end of the O phase was approximately 1.8 kPa, indicating that T. eques closed their spiracles well before their haemolymph was in equilibrium with atmospheric conditions. Thus, exposure to atmospheres containing 1 or 2 kPa would be expected to have little effect on animal CO2 content at the end of the O phase. Perhaps exposure to atmospheres containing 2 kPa caused a slight opening of the spiracles during the interburst period, decreasing the rate of accumulation of internal CO2. In pupae, exposure to subthreshold atmospheric does increase interburst (Schneiderman and Williams, 1955).

Comparison of the control of DV in locusts and lepidopteran pupae

Atmospheric is positively correlated with interburst duration in lepidopteran pupae (Schneiderman and Williams, 1955; Levy and Schneiderman, 1966) but not in T. eques. In pupae, tracheal drops to 4 kPa during interburst intervals (Levy and Schneiderman, 1966), while tracheal in grasshoppers must decline by, at most, a few kilopascals during the interburst period since increases by only 0.5 kPa. It seems likely that there is a minimum that triggers spiracular opening in T. eques, but that the minimum is not reached during the interburst period in this species. It is possible that oxygen depletion plays a role in the initiation of the F phase in grasshoppers, as in pupae; however, since we could not reliably distinguish between F and C phases in these animals, it was impossible to investigate this hypothesis.

Pupae and grasshoppers differ in their O phase tracheal conductance. Since most of the carbon dioxide emission in both pupae and grasshoppers occurs during the O phase, the O phase spiracular conductance (diffusive plus convective) can be approximated from , where is the average driving force for CO2 diffusion through the spiracle. Since is threefold greater in pupae, and is four- to tenfold lower in pupae (Buck and Keister, 1955;

Schneiderman and Williams, 1955; Quinlan and Hadley, 1993), O phase mass-specific spiracular conductance in pupae must be at least an order of magnitude lower than in grasshoppers. Such intraspecific variation in tracheal conductance could be due to morphological differences in the spiracles and trachea, or to greater O phase convective ventilation in T. eques. The high spiracular conductance in grasshoppers is consistent with their higher metabolic rates and scope for activity and lower vulnerability to desiccation relative to pupae. Although T. eques are desert grasshoppers, their diet has a relatively high water content and their cuticular permeability is moderate (Quinlan and Hadley, 1993).

Both internal and the change in associated with DV appear to be lower in lubber grasshoppers than in lepidopteran pupae. Peak tracheal during DV in grasshoppers is threefold lower than in pupae, in which tracheal increases to about 7 kPa (Burkett and Schneiderman, 1974). The lowest tracheal reported for pupae during DV (3.6 kPa; Levy and Schneiderman, 1966) is well above the peak values we observed for grasshoppers. Since tracheal gases are likely to be water-saturated, a threefold increase in the gradient for oxygen and carbon dioxide diffusion as in pupae should reduce respiratory water loss during the O phase by threefold. While more comparative data are clearly needed, it seems reasonable to predict that insects with low metabolic rates and high susceptibility to desiccation may have spiracles of low conductance and tolerate a higher internal and lower . These intraspecific differences in internal gas levels appear to be, at least partially, due to variations in the threshold for triggering spiracular opening.

This research was partially supported by grants DCB-9020284 and IBN-9317784 from the National Science Foundation to J.F.H.

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