The discontinuous gas exchange cycle of the pseudoscorpion Garypus californicus, mean mass 5.9 mg, is rudimentary and is characterized by bursts of CO2 at frequencies ranging from 3.6 mHz at 15°C to 13.3 mHz at 35°C. The mean volume of CO2 emitted per burst is 3.6 µl g−1 at 25°C, about a tenth of the amount emitted by tracheate arthropods with a well developed discontinuous gas exchange cycle. Interburst CO2 emission is high and increases with temperature, reaching near 45% of total CO2 production rate at 35°C. No fluttering spiracle phase is evident. The metabolic rate of G. californicus at 25°C (8.4 µW) is typical of other arthropods. We infer from the high rate of interburst CO2 emission in G. californicus that trans-spiracular O2 partial pressure gradients are small and that spiracular conductance is correspondingly high, which may lead to high rates of respiratory water loss relative to arthropods with more stringent spiracular control and higher CO2 buffering capacity. The typical moist, hypogeal environments and small body sizes of pseudoscorpions correlate well with their respiratory physiology.
The phylogeny of the arthropods is interesting in many respects, but the independent evolution of tracheation in several distinct clades is particularly noteworthy. Hexapods are the best known tracheate arthropods, and have developed a fairly complex spiracular control strategy for managing CO2 and O2 gas exchange in their tracheal system (see reviews by Kestler, 1985, Sláma, 1994, Lighton, 1994, 1996, 1998). This system is often referred to as the discontinuous gas exchange cycle, or DGC (sensu Lighton, 1996).
Recent evidence suggests that the DGC evolved independently in several non-hexapod clades of tracheate arthropods, including the ticks (Lighton et al., 1993a; Lighton and Fielden, 1995), solphugids (Lighton and Fielden, 1996) and diplopods (Klok et al., 2002). Karel Sláma has described respiratory cyclicity in a pseudoscorpion (Sláma, 1995); however, analysis of the DGC requires recordings of rates of CO2 emission (VCO2), which his microrespirometric system does not provide. The analysis of VCO2 has been shown by direct spiracular observation to be an excellent indicator of spiracular activity (Lighton et al., 1993b).
We here quantify the DGC of a pseudoscorpion, Garypus californicus, a native of seashore habitats on the western coast of the United States, as a function of temperature and thus metabolic flux rate. Pseudoscorpions possess two pairs of spiracles that are located laterally on the posterior margins of abdominal sternites 3 and 4. The spiracles open into four short tracheal trunks from which smaller branches ramify to supply the body. Pseudoscorpions are of particular interest because a clade of closely related tracheate arthropods, the solphugids (Shultz, 1990, Wheeler and Hayashi, 1998), have evolved a DGC that closely resembles that of insects (Lighton and Fielden, 1996).
Materials and Methods
Specimens of Garypus californicus were collected in the Northern hemisphere during Fall (September) from beneath dried kelp and rocks at the Hopkins Marine Station, Pacific Grove, California. They were maintained at 25 ± 3°C at an ambient L/D cycle in a 20 × 20 × 5 cm polyethylene container with a floor of moist sand and pleated filter paper. They were fed twice weekly on wingless fruit flies.
The DGC of Garypus californicus was quantified with a flow-through respirometry system (Sable Systems TR-2; www.sablesystems.com) as described elsewhere (Lighton, 1991a; Lighton and Berrigan, 1995). The mass flow rate of CO2-free air through the system was 20 ml min−1 regulated by a mass flow control valve (Sierra Instruments; http://www.sierrainstruments.com). For each run, we removed an individual from the pseudoscorpion colony, weighed it to 0.1 mg, and placed it in a glass and aluminum respirometry cuvette with Viton O-ring seals (Sable Systems RM-1). The animal's temperature was regulated (± 0.2°C) by a Peltier effect temperature cabinet (Sable Systems PELT-4 equivalent), and its activity level was monitored within the ca. 5 ml cuvette using a Sable Systems AD-1 activity detector. Only data from motionless animals were used. Data were acquired with DATACAN V software (Sable Systems), using a sampling interval of 2 seconds, finite impulse response digital filtration within each sample, and a typical recording duration of 5250 samples. Two to four successive recordings were typically made on a given animal at a given temperature, after which it was returned to the colony.
The effect of temperature on DGC parameters was tested with analysis of variance (ANOVA) supplemented by Studentized multiple range tests (Newman-Keuls) where indicated. Regressions were calculated by least squares with axis transformation where noted, and compared with analysis of covariance (ANCOVA). Statistical software was written by JRBL and validated against known data sets (Zar, 1996) and/or other programs (SYSTAT IV). Means are accompanied by standard deviations (SD) or standard errors (SE) and sample sizes (N).
We analyzed 771 DGCs by an approximately equal number (10–11) of haphazardly sampled animals at 15, 25 and 35°C, with a mean mass of 5.90 ± 1.28 SD mg. Larger numbers of DGCs were analyzed at higher temperatures because they occurred at a faster rate. We analyzed 137 DGCs from 11 animals at 15°C, 181 DGCs from 10 animals at 25°C, and 453 DGCs from 11 animals at 35°C. The data were analyzed by individual within each temperature treatment. Because the pseudoscorpion colony had approximately 19 inhabitants, and because of the random (or more precisely, haphazard) selection technique, i.e. selecting a new individual at random and then replacing the previously measured individual, some pseudoscorpions will have been measured more than once in different, but never consecutive, runs. This is an inevitable consequence of the restricted pool size of our experimental organisms. We have no reason to believe that our sampling technique introduced any bias, by virtue of its inherently random nature.
Typical CO2 emission data by a 6.02 mg G. californicus at 15°C are shown in Fig. 1. The DGC in our pseudoscorpions was generally characterized by incomplete spiracular closure during the constricted spiracle or “C” phase, which therefore is something of a misnomer in this group. We retain the term because spiracular closure was, on occasion in some individuals, close to or indistinguishable from zero at low temperatures. In addition, in no arthropod are the spiracles totally constricted. The reader may wish to think of the C phase as used here as an “interburst phase.” No evidence was found for an F (fluttering spiracle) phase. For purposes of analysis we divided the DGC of G. californicus into the C and O (Open-spiracle) phases; again, we remind the reader that these terms should be understood as shorthand references to the interburst and burst phases of the DGC, respectively, rather than as references to conventional C and O phases.
Overall metabolic flux rates
At 25°C, the mean VCO2 of our animals was 1.126 ± 0.567 SD µl hr−1, or 0.201 ± 0.101 SD ml g−1 hr−1, N = 181 DGCs by 10 animals, mean mass 5.65 ± 1.31 mg. Assuming aerobic catabolism of palmitate (respiratory quotient 0.72), this corresponds to a metabolic rate of 8.44 ± 4.25 SD µW, which does not differ significantly from the value expected for a consensus arthropod of that mean body mass and temperature (Lighton et al., 2001); 11.5 µW (t = 0.68; P = 0.4).
As might be expected because of the small range of body masses, there was no correlation between body mass and metabolic rate (ANCOVA of log body mass vs. log metabolic rate across the three temperature treatments, F[1, 28] = 1.43, P[no correlation] > 0.2).
By regressing the logarithm of metabolic rate against temperature, the slope of that relationship (0.0336) was found to correspond to the temperature sensitivity of metabolism. Expressed as a Q10, it is 100.336 or 2.16. Using this model, temperature explains 72% of metabolic rate variance (F[1, 30] = 76.9; P < 0.0001).
The DGC frequency increased exponentially with temperature, with a slope of 0.0261 for log-transformed frequency vs. temperature, corresponding to a Q10 of 100.261 or 1.82; temperature explained 57% of DGC frequency (F[1, 30] = 39.5; P < 0.0001). At 25°C mean DGC frequency was 5.19 ± 2.66 SD mHz, corresponding to a mean cycle length of 193 seconds or slightly over three minutes.
C phase duration increased with DGC duration (Fig. 2), with a dimensionless shared slope of 0.64 across temperatures (r2 = 0.71; F[1, 30] = 75.4; P < 0.001). The intercept is −36 ± 61 seconds, which does not differ significantly from zero. C phase length decreased significantly (P < 0.05) as temperature increased, reflecting the increase in DGC frequency, and thus the reduction in DGC duration, as temperature rose (see DGC frequency above).
C phase VCO2 increased steeply and significantly with temperature, with a Q10 of 2.75 (r2 = 0.70, F[1, 30] = 68.5, P < 0.0001). Because of the rapid increase of C phase VCO2 with temperature, its contribution to total CO2 release increased with temperature (Q10 = 2.75/2.16 = 1.27) until at 35°C it approached 45% of total CO2 emission.
O phase duration increased with DGC duration, with a dimensionless shared slope of 0.36 across temperatures (r2 = 0.45; F[1, 30] = 24.5; P < 0.001). The intercept is 36 ± 61 seconds, which does not differ significantly from zero. O phase duration was strongly affected by temperature, decreasing from 201 ± 24 SE seconds at 15°C to 106 ± 17 SE seconds at 25°C and 71 ± 8 SE seconds at 35°C (Fig 3). O phase volume rose modestly between 15 and 25°C, from 3.06 ± 0.41 SE µl mg−1 to 3.63 ± 0.52 SE µl mg−1. It then declined steeply at 35°C to 2.14 ± 0.24 SE µl mg−1. Thus the effect of temperature on O phase volume, though marginally significant by ANOVA (F[2, 29] = 3.48; P < 0.05), was not directional, reaching a peak value at the intermediate temperature of 25°C. O phase VCO2, in contrast, increased exponentially with temperature (F[1, 30] = 45.3; P < 0.0001) because of the rapid reduction of O phase duration with temperature. The Q10 of O phase VCO2 was 1.97.
It is clear from these results that although G. californicus does employ a DGC, it is a marginal one, lacking stringent spiracular closure. In fact the C phase contributed up to 45% of overall CO2 release at high temperatures, whereas the DGC of G. californicus took the form of small peaks of CO2 release “riding” on a large interburst baseline. Although solphugids, a clade of closely related tracheate arthropods (Shultz, 1990, Wheeler and Hayashi, 1998), have independently evolved a stringent, three-phase DGC that closely resembles that of insects (Lighton and Fielden, 1996), pseudoscorpions have apparently developed only a modest degree of spiracular control.
Some of this difference may be explained by total-animal CO2 buffering capacity. Pseudoscorpions emit only about a tenth of the volume of CO2 per unit mass in the O phase that hexapods and other tracheate arthropods do (see, e.g., Lighton et al., 1993; Quinlan and Lighton, 1999). For example, the fairly closely related clade of the solphugids release 20 µl mg−1 (Lighton and Fielden 1996), approximately six to eightfold more. The ability of pseudoscorpions to regulate CO2 release is therefore minimal compared with animals that employ the “classic” DGC. This is reflected in their high DGC frequency, about double to eight-fold that of similar sized insects (Lighton, 1991b, 1996). Investigation of CO2 buffering capacity in pseudoscorpions would be worthwhile; it is worth noting, in this context, that the primary trigger for the burst phase in pseudoscorpions has recently been shown to be hypoxia, rather than the more conventional hypercapnia (Lighton and Joos, 2002).
The temperature dependence of the pseudoscorpions' DGC revealed no surprises. While overall metabolic Q10 was 2.16, the Q10 of DGC frequency was 1.82. Thus DGC frequency increased with temperature more slowly than rate of CO2 emission, indicating that pseudoscorpions modulate both frequency and volume of gas exchange to accommodate changes in metabolic flux rates. The increase of CO2 volume per DGC with increasing metabolic flux rates was mostly confined to the interburst phase. Although the O phase VCO2 increased with temperature with a Q10 1.97, O phase volume decreased because of a steep decrease in O phase duration. The majority of insects studied so far modulate DGC frequency, rather than volume, to accommodate changes in metabolic flux rates (see Lighton 1998 and 1996 for discussions).
It is certainly in the magnitude of interburst CO2 emission that the DGC of G. californicus differs most radically from other tracheate arthropods that employ a DGC. The fact that interburst VCO2 increases rapidly with temperature demonstrates that G. californicus is unable to sustain sufficient oxygen flux rates without losing a large amount of CO2 and thus water vapor at the same time. This in turn suggests that the trans-spiracular gradient of oxygen partial pressure is minimal in this species. The likely presence of a minimal trans-spiracular oxygen partial pressure gradient, and an accompanying high rate of interburst CO2 release, suggests from respiratory water loss considerations that the O phase trigger in G. californicus may be predominantly hypoxic rather than hypercapnic. Examining the effects of ambient oxygen partial pressure on the DGC of this species can test this hypothesis. This work has now been performed and a hypoxic O phase trigger in G. californicus has been confirmed (Lighton and Joos, 2002).
If G. californicus is unable to maintain a protracted and steep oxygen partial pressure gradient across its spiracles, then its rate of respiratory water loss in its interburst phase will be higher than in comparable animals that employ a “classic” DGC with a fully constricted C phase and a long F phase marked by low VCO2 and endotracheal oxygen partial pressures as low as 4 kPa (see Lighton, 1998 and references therein). This is amenable to experimental testing. It is perhaps significant that pseudoscorpions are limited to marginal habitats in areas where water vapor pressure is likely to be high, such as in soil litter and under stones. Of course, the link (if any) between the DGC and respiratory water loss is somewhat controversial (see Lighton, 1998 for a discussion). Notwithstanding the above, the niche range of pseudoscorpions appears to be wider than that of another tracheate arthropod, the harvestperson (opiliones), in which spiracular control is minimal, and no cyclicity, let alone discontinuity, of external CO2 emission is evident (Lighton 2002).
We thank the National Science Foundation (IBN 9306537 and 9603873 to JRBL) and the Packard Foundation (fellowship to JRBL) for financial support, and Sable Systems International for the loan of instruments. We thank Anke Schmitz for helpful discussions, Michael Quinlan for technical assistance at an early stage of this study, and Robbin Turner for assistance in manuscript preparation. JRBL was also supported by personal funds.