To DGC or not to DGC: oxygen guarding in the termite Zootermopsis nevadensis (Isoptera: Termopsidae)
1 Department of Biology, University of Nevada at Las Vegas, NV 89154-4004,
USA
2 California Institute of Technology, Biology Division, Pasadena, CA 91125,
USA
* Author for correspondence (e-mail: john{at}johnlighton.org)
Accepted 17 October 2005
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Summary |
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Key words: termite, Zootermopsis nevadensis, trachea, spiracle, oxygen, symbiont
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Introduction |
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The Hetz-Bradley hypothesis is tenable if, in fact, the DGC creates a
milieu interieur with a lower mean PO2
than alternative means of gas exchange. But paradoxically, a delayed oxygen
exposure penalty is created while PO2 is
maintained at a low level of ca. 4 kPa during the DGC's F phase. During the F
phase, thanks to stringent spiracular control, CO2 escapes at
<25% of production rate (Lighton,
1988 and references therein). Periodic flushing of this
accumulated CO2 is therefore necessary, and in fact this flushing
known as the open-spiracle (O) or burst phase is in large part
the operational definition of the DGC. As a side-effect of flushing the
CO2 produced during the C and F phases, in the O phase internal
PO2 rises to near-atmospheric levels throughout
the tracheal system (Hetz and Bradley,
2005
and references therein).
Obtaining comparative data that bear on the oxygen-guarding hypothesis is
difficult. While it is interesting that Drosophila endowed with extra
superoxide dismutase and catalase copies show a small but significant increase
in lifespan (see review by Sohal et al.,
2002), (a) the effect is minimal if wild-type, rather than
genetically compromised, flies are used in the experiments (ibid),
(b) overexpression of antioxidants may actually have deleterious effects
(ibid), and (c) most oxygen toxicity experiments are short-term and
make the assumption that evolutionary fitness and individual longevity are
coterminous. Thus current oxygen toxicity data are not helpful in resolving
the evolutionary origins of the DGC. Other comparative data are not helpful
either. Most insects are capable of extremely high rates of oxidative
catabolism (Suarez et al.,
1997
and references therein), yet to our knowledge no evidence
suggests an interspecific lifespan penalty among the more metabolically active
insect taxa. Combined with the fact that the distribution of the DGC is
discontinuous across clades of tracheate arthropods
(Lighton, 1996
;
Klok et al., 2002
;
Chown et al., 2005
), we
currently lack any evidence, comparative or otherwise, of a relationship
between the DGC, longevity and oxygen guarding in an adaptive and operational
rather than an inferential context.
Compared to possible avoidance of long-term oxygen toxicity, respiratory adaptations to avoid short-term oxygen toxicity would constitute a powerful assay of oxygen-guarding gas-exchange strategies. Finding evidence of respiratory adaptations that mitigate short-term oxygen toxicity might then bear directly on the adaptive significance of the DGC in ameliorating longer-term oxygen stress. However, short-term oxygen toxicity in normoxia is not known to be a limiting factor for any insect.
The same is not true, however, of symbiotic microorganisms on which some
insect taxa depend for access to carbon. The lower termites (class isopoda,
families Kalo-, Masto-, Hodo-, Serri- and Rhinotermiditae) are a good example.
The guts of these insects contain symbiotic microbes, including
cellulose-digesting protists that they depend upon for survival
(Cleveland, 1924). These
protists, and many other gut microorganisms, are true anaerobes, dying when
termites are exposed to oxygen at high concentrations and/or partial pressures
(Cleveland, 1925
;
Messer and Lee, 1989
), or to
normoxia outside of the protection of the gut environment
(Trager, 1934
). Exact
measurements of the oxygen tolerance of protists in pure culture are not
available, but their culture requires an anaerobic medium supplemented with a
reducing agent (Yamin, 1978
,
1981
). Acetogenic spirochetes
isolated from the termite gut were able to tolerate transient exposure to low
concentrations of O2, but ceased growth in the presence of
1%
O2 (Graber and Breznak,
2004
).
It has been demonstrated that oxygen penetrates up to 200 µm into the
lumen of hindguts removed from the termite, and can significantly impact
carbon and electron flow in this system
(Brune et al., 1995;
Tholen and Brune, 2000
). The
ability of the insect host to impact oxygen concentrations in the gut has yet
to be explored, but may provide a demonstrable incentive for stringent oxygen
guarding in the short term (minutes to hours), as well as a putative
requirement for longer-term oxygen guarding sensu Hetz and Bradley
(2005
). Their gas-exchange
strategies are therefore arguably more relevant to the possible evolution of
oxygen-guarding respiratory strategies than are those of insects without
short- as well as long-term requirements for oxygen guarding.
Few studies have been undertaken on termite gas-exchange kinetics, and
those that exist (Shelton and Appel,
2000b,
2001a
,b
,c
)
show no evidence of a DGC. Rather, they show continuous gas exchange,
sometimes with brief, more or less regular, increases in CO2
output. In no case does the CO2 output fall to the near-zero or
very low levels diagnostic of the DGC's C or F phases, and the occasional
CO2 peaks are far smaller than those of the DGC's O phase.
Expressing termite gas exchange in terms of coefficient of variation
(CV=standard deviation/mean), typical termite CVs are 1550% (Shelton
and Appel, 2000b
,
2001a
,b
,c
)
vs >200% for a typical DGC
(Lighton, 1990
). Thus the
gas-exchange kinetics of termites are nearly flat-line and the occasional
periodicities do not reflect discontinuities of the kind that characterize the
DGC. This neither strictly continuous nor even remotely discontinuous form of
gas exchange has been widely documented in other insects
(Lighton, 1996
;
Williams and Bradley, 1998
;
Lighton et al., 2004
; see
especially Klok and Chown,
2005
, who refer to it as `Cyclic Gas Exchange' or CGE). CGE has
also been found in non-hexapod tracheate arthropods
(Lighton, 2002
;
Lighton and Joos, 2002
).
We tested two hypotheses regarding oxygen guarding in individual dampwood termites, Zootermopsis sp.
First, we hypothesized that if the DGC exerts a significant overall
oxyprotective effect, the termites would switch from CGE to the DGC under
hyperoxic conditions. This hypothesis assumes that termites are
phylogenetically and physiologically capable of generating the DGC.
Phylogenetically, we infer that this is the case from the fact that the
closely related clade of the roaches (dictyoptera) are capable of expressing
the DGC (Kestler, 1985;
Marais and Chown, 2003
), as
are other closely related clades such as the orthoptera
(Hadley and Quinlan, 1993
).
Physiologically, the DGC is very widespread, if patchily distributed, among
tracheate arthropods. This argues that most such arthropods are capable of the
necessary spiracular control and of responding directly to changes in tracheal
oxygen levels (see also Chown and Holter,
2000
).
Our second hypothesis addressed the question of oxygen guarding in CGE
specifically. It has recently been discovered that insects expressing CGE
initially react to elevated ambient oxygen levels by constricting their
spiracles (Lighton et al.,
2004), suggesting that even in insects expressing CGE, internal
PO2 is controlled in a manner analogous to that
F phase of the DGC, though at an unknown level. The resulting spiracular
constriction causes external CO2 output to decline briefly on
exposure to hyperoxia (Lighton et al.,
2004
). At this juncture two mutually exclusive oxygen regulation
strategies exist. Tracheal PO2 may rise (no
oxygen guarding), or it may be guarded at or close to its normoxic level. We
hypothesized that if oxygen guarding took place in the termites, prolonged
hyperoxia would elevate tracheal PO2 until
sufficient outward CO2 flux occurred in spite of the spiracular
constriction required to control tracheal PO2
at its regulated, low level. Because of the resulting higher tracheal
PCO2, oxygen guarding would result in a transient efflux
of CO2 following re-establishment of normoxia. The regulatory
alternative that tracheal PCO2 would be controlled
at normal levels and that tracheal PO2 would
therefore be permitted to rise would cause no compensatory rise in
CO2 emission levels after normoxia was re-established. Lighton et
al. (2004
) did not distinguish
between these post-hyperoxic strategies because their experiment terminated
hyperoxic exposure with exposure to anoxia in order to determine maximal
spiracular throughput.
Testing the two hypotheses outlined above therefore addresses two issues. First, the capability of termites to switch to the DGC under oxygen stress is evaluated. If they do so, then the DGC is likely related at least in part to minimizing internal oxygen exposure. Second, the ability of termites to control their gas-exchange characteristics in a manner compatible with oxygen guarding is evaluated. If they do so, and if they do not engage in the DGC while doing so, it can be argued that other gas-exchange strategies may guard against oxygen toxicity at least as effectively as the DGC especially when it is recalled that the DGC periodically flushes the tracheal system with normoxic air.
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Materials and methods |
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Respirometry
We used a Sable Systems International (SSI;
www.sablesystems.com;
Las Vegas, NV, USA) TR-2 respirometry system, modified to allow the exchange
of ambient air with pure oxygen under computer control. The outline of the
system is shown in Fig. 1.
Briefly, we exchanged moist, CO2-free air (21% O2: 79%
N2) for moist, CO2-free O2 for the middle 50
min of a 3 h recording of CO2 emission rate
(CO2).
|
The temperature of the termite and its surroundings was controlled at 15°C by a SSI PELT-5 temperature controller. Data were acquired and analyzed with SSI's ExpeData data acquisition and analysis software, which also controlled baselining and gas switching. In a typical run, the termite was selected at random from the colony, weighed to 0.1 mg (Mettler AG245, Columbus, OH, USA) and placed in a 5 cm3 respirometer chamber. The chamber was placed in the temperature controlled cabinet, and water saturated, CO2-free air was diverted past it to establish the zero-CO2 baseline of the system, using an SSI RM-8 gas flow multiplexer. The ca. 30 ml water reservoir required about 12 h to equilibrate prior to use. A push flow system was used, with a flow rate of 40 ml m1 controlled by a mass flow controller (SSI FC1 controller and Tylan FC-260 mass flow control valve). The mass flow rate at the exit of the system after the CO2 analyzer was monitored and recorded (SSI SS-3 integrated subsampler and mass flow meter, which also provided the sealed diaphragm pump that pushed the system's gas flow) to detect any leaks or plumbing problems. After 2 min of baseline measurement the air was diverted through the respirometer chamber containing the termite, and the recording was paused for 2 min while the accumulated CO2 washed out of the system. The recording was restarted, and lasted for a total of just under 3 h including a second baseline at the end. At 65 min into the recording, the air pushed through the system was replaced by water saturated, CO2-free O2. After a further 50 min the O2 was replaced again by water saturated, CO2-free air. As determined by blank recordings, no change in CO2 baseline concentration occurred during the gas changes. After the final baseline was taken the recording was repeated, 24 times per termite.
Data were analyzed using ExpeData. Respirometry equations were as described
previously (Lighton and Turner,
2004). Prior to introduction of O2, the lowest 30 min
of CO2 emission was located and its mean was used for calculating
pre-treatment metabolic rate, expressed as rate of CO2 production
or
CO2. After
introduction of O2, the lowest 60 s of CO2 production
was located, from which the low O2 treatment
CO2 was
calculated. Next, the lowest section of 15 min, starting at 15 min into the
O2 treatment, was located and the equilibrium O2
treatment
CO2
was calculated. Next, after air was reintroduced, the highest 60 s of
CO2 production within the next 15 min was located, from which the
high post-treatment
CO2 was
calculated. Finally, the lowest section of 15 min, starting 15 min after the
introduction of air, was located and the equilibrium post-treatment
CO2 was
calculated.
Activity was constantly monitored using an SSI AD-1 optical activity
detector, and calculated for each of the sections noted above as described in
detail elsewhere (Lighton and Turner,
2004). Briefly, any movement by the termite caused fluctuations in
detected light levels within the detector. In ExpeData, the sign-insensitive
sum of differences between adjacent data points was calculated. If light
levels did not change, this absolute difference sum (ADS) increased very
slowly over time owing to random electrical noise in the detection circuitry.
If the light level fluctuated significantly because of the termite's
movements, the sum increased more rapidly. We therefore employed the rate of
change of the ADS vs time, i.e. the slope of the linear regression
over the selected section of ADS data, as an operational index of
activity.
Means are accompanied by standard deviations (S.D.) and sample sizes (N). Differences between means were assayed using Student's t-test, or by analysis of variance (ANOVA), with significance set at P<0.05. Where needed for statistical comparisons, points were digitized from published graphs with ExpeData's DigitEyes image analysis utility. Regressions were calculated by the least-squares method and compared by analysis of covariance (ANCOVA). Regression significance was evaluated with the F test. Regression slopes are accompanied by standard errors (S.E.M.).
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Results |
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Discussion |
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We were surprised that our sample of termites remained active despite being
placed in water-saturated air at a relatively low temperature. It is possible
that placing our termites in water-saturated air may have stimulated a higher
level of activity than would have been the case in dry air, analogous to the
findings of Gibbs et al. (2003)
in hygrophilic drosophilids. Dampwood termites are never naturally exposed to
completely dry air (as used by Shelton and
Appel, 2000b
), and exposure to it may have elicited a `freeze'
response equivalent to that reported by Gibbs et al.
(2003
) in xerophilic
drosophilids. This argument is reinforced by the fact that Shelton and Appel
(2000b
) report almost exactly
the consensus
CO2 expected for
a motionless, flightless insect of that mean mass (5.56 µl
h1, assuming a Q10 of 2.0, mean mass 0.0334 g;
Lighton et al., 2001
).
Exposure to dry air may also explain the semi-cyclic gas exchange reported
by Shelton and Appel (2000b)
in 60% of their sample of dampwood termites at 15°C. We did not observe
consistent CO2 emission cyclicity in our sample of termites. A
useful index of the degree of spiracular control displayed by an insect at
rest is the coefficient of variation or CV of
CO2, defined as
CO2
S.D./mean. The CV of
CO2 can vary
from near 0% for continuous gas exchange to >200% in insects displaying the
DGC (Lighton, 1990
). The CV of
CO2 in our
sample of termites was 15.7±4.5%, which is equivalent to the `acyclic'
Z. nevadensis termites described by Shelton and Appel (2000; 18.1% at
15°C, digitized from their Fig.
3, t=0.5, P>0.4). The two laboratories used
similar SSI TR-2 systems with similar flow rates, making their CV figures
directly comparable. Shelton and Appel
(2000b
) did not distinguish
between the
CO2
values of dampwood termites undergoing cyclic and acyclic gas exchange; it is
possible that their acyclic termites displayed
CO2 values more
similar to those of our sample of termites.
Clearly, the possible interactions between ambient gas hydration and gas exchange in termites merit further study. We nevertheless consider water-saturated gases and cool temperatures to have been the ideal ambient conditions for our experiments. They more closely approximated the presumptive natural conditions within the galleries of dampwood termite colonies, and eliminated osmotic stress as a possible complicating factor when interpreting our results.
Oxygen guarding: Mechanisms
We have shown that termites undergoing continuous gas exchange actively
reduce spiracular area under hyperoxic challenge, as has been shown in other
insects (Lighton et al.,
2004). New to this investigation, we have demonstrated that
maintaining internal hypoxia on a prolonged basis (tens of minutes) is
accompanied by extended internal hypercapnia, and thus that the homeostatic
imperative of maintaining internal hypoxia outweighs that of maintaining
elevated levels of internal CO2. The internal hypercapnia to which
we refer is inferred both from the transient depression of
CO2 following
exposure to hyperoxia, and from the transient elevation of
CO2 following
the re-establishment of ambient normoxia. It is especially clearly shown by
the hypercapnic load, which is released in its entirety on re-exposure to
normoxia (Fig. 7). Thus,
insects that do not engage in the DGC defend internal hypoxia, and not
internal PCO2, against changes in ambient gas
concentrations.
Our investigation raises some questions. Primarily, what are the actual
endotracheal PO2 and PCO2
levels during hyperoxia? Our results show that oxygen guarding exists, but do
not quantify its effectiveness. On a priori grounds it is logical to
assume that the limiting factor in oxygen guarding is the degree to which
internal hypercapnia can be tolerated. In this respect, quantifying responses
to graded hyperoxia would be interesting. We know from studies of the DGC (see
reviews mentioned in the Introduction) that in insects expressing the DGC,
internal PCO2 can rise to at least 45 kPa before
the O phase is initiated, yielding an equivalent partial pressure gradient
across the spiracles in normal air. Higher PCO2 values are
unlikely on physiological grounds. Even in normoxia the partial pressure
gradient of CO2 is dwarfed by that of O2, which is ca.
1618 kPa at sea level (ibid.). This imbalance means that
continuous gas exchange cannot be based on steady-state diffusion alone, even
in normoxia. Outward diffusion of CO2 must therefore be augmented
by periodic increases in spiracular area (as in CGE) or by bulk transfer
mechanisms such as intermittent convection. Although this field is starting to
attract attention (Lighton et al.,
2004; Klok and Chown,
2005
), it remains seriously understudied. The kinetics of the DGC
are far better understood than those that underlie less dramatic but arguably
more widespread gas-exchange strategies in tracheate arthropods.
Oxygen guarding and the DGC
Our findings are not incompatible with those of Hetz and Bradley
(2005). However, in the case
of our termites (and likely in the cases of other continuous gas exchangers;
Lighton et al., 2004
),
internal hypoxia presumably comparable to that of the F phase of the DGC is
maintained and actively guarded on a continuous basis, without periodically
flooding the tracheal system with O2 as occurs during the
open-spiracle or O phase of the DGC (Hetz
and Bradley, 2005
). Although the requisite long-term
PO2 measurements remain to be made in insects
with continuous gas exchange, it cannot be shown on the basis of present
knowledge that continuous gas exchange imposes a tissue O2 exposure
penalty relative to the DGC when taking into account periodic flushing with
normoxic air a definitional requirement of the DGC.
This begs the question of the ultimate selective pressures responsible for
maintaining a low internal PO2 in insects. The
Hetz-Bradley `oxidative damage hypothesis' (see
Chown et al., 2005 for
discussion) is certainly worthy of consideration and, though defined in terms
of the DGC, may be applicable sensu lato to continuous gas exchangers
as well in light of our findings. Hetz and Bradley
(2005
), however, reasonably
limit their consideration of oxidative damage to the insect's somatic
tissues.
We suggest that internal hypoxia in insects may have selective correlates
that have not previously been considered. These are brought into clear
conceptual relief by the subject animal of the present investigation. Termites
cannot survive without their symbiotic gut flora, which are therefore truly
symbiotic rather than merely commensal (Cleveland,
1924,
1925
). Termite gut flora,
especially the protists essential for cellulose digestion, are able to thrive
only in hypoxic environments (Yamin,
1978
,
1981
;
Trager, 1934
). Thus their host
animals confer not only the benefit of a continuous supply of cellulose, but
that of a hypoxic environment. Thus the question is whether internal hypoxia
preadapted termites for inhabitation by protists, or whether the maintenance
of internal hypoxia evolved as a response to the selective benefits created by
normoxia-sensitive symbionts among the termites, and perhaps also in
other insect taxa.
The success of termites throughout most of this planet's ecosystems is a
clear example of the importance of symbiotic microorganisms. Benefits
conferred by microorganisms in other insect taxa may not be as dramatic, but
are likely to be significant, as is the case with higher vertebrates
(Hooper and Gordon, 2001;
Fooks and Gibson, 2002
;
Guarner and Malagelada, 2003
).
We suggest that the selective advantages of gut bug guarding, or supplying an
hypoxic environment to gut symbionts, may in part explain tracheal oxygen
guarding, and may be a hitherto unrecognized adaptive factor in the evolution
of insect gas exchange. For example, it is possible that an initial selective
pressure for gut bug guarding maintaining internal hypoxia in order to
facilitate the growth of anaerobic gut microorganisms may have been
among the selective factors that allowed the evolution of the degree of
spiracular control required for the DGC. It is even possible that the periodic
flushing with outside air characteristic of the DGC might, far from
maintaining strict internal hypoxia, actually constitute a mechanism in some
insect taxa for modulating the growth of oxygen-sensitive micro-organisms,
whether intra- or extracellular.
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Acknowledgments |
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