The mangrove ant, Camponotus anderseni

ARTICLE IN PRESS
Journal of Insect Physiology 53 (2007) 505–508
www.elsevier.com/locate/jinsphys
The mangrove ant, Camponotus anderseni, switches to anaerobic
respiration in response to elevated CO2 levels
M.G. Nielsena,, K.A. Christianb
a
Department of Biological Science, University of Aarhus, Denmark
School of Science, Charles Darwin University, Darwin, NT Australia
b
Received 6 December 2006; received in revised form 1 February 2007; accepted 1 February 2007
Abstract
The small tree-living mangrove ant Camponotus anderseni is remarkably adapted for surviving tidal inundation. By blocking the nest
entrance with a soldier’s head, water intrusion into the nest cavity can be effectively prevented, but lack of gas-exchange caused extremely
high concentrations of CO2 ð430%Þ and very low O2 concentrations ðo1%Þ.
The O2 uptake in experiments with CO2 absorption showed a linear decrease until about 4%, whereas the O2 uptake in chambers
without absorbent showed a decrease with a different pattern, consisting of three parts. The first component of this decrease is a linear
decrease to about 18%, which is the normal O2 concentration in open natural nests. The second phase is an exponential decrease
continuing to about 4% O2 , showing that the CO2 concentrations have influence on the O2 uptake. The final component is also
exponential, but with a much smaller slope.
The respiratory quotient (RQ) was 0.92 until CO2 concentration increased to about 15–17%, and after that it showed a strong
increase, which is due to the initiation of anaerobic respiration.
Anaerobic respiration has not been demonstrated for social insects before, but it is not surprising that it is found in this ant species,
which lives in the extreme conditions of a hollow twig in an inundated mangrove.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Camponotus anderseni; Anaerobic respiration; Respiratory quotient; Oxygen concentrations; Mangrove
1. Introduction
Insects inhabit most terrestrial and aquatic habitats, and
their success is partly due to their remarkable ability to
adapt to extreme conditions, including hypoxic or anoxic
conditions (Hoback and Stanley, 2001). Insect larvae living
in dung, soil or other environments with hypoxic and
hypercapnic conditions can either extract O2 at very low
concentrations or switch to anaerobic respiration (Holter
and Spangenberg, 1997; Hoback et al., 2002; Zerm and
Adis, 2003). Insects produce several end products besides
L-lactate during anaerobic respiration (Hochachka et al.,
1993; Hoback and Stanley, 2001; Zerm et al., 2004; Chown
and Nicolson, 2004), and they also have a greater ability to
Corresponding author. Tel.: +45 8942 2723; fax: +45 8619 4186.
E-mail address: biomgn@biology.au.dk (M.G. Nielsen).
0022-1910/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinsphys.2007.02.002
recover from anoxia than vertebrates (Schmitz and
Harrison, 2004).
For soil living insects, anoxic tolerance may be an
adaptation to the risk of being submerged after heavy
rainfall (Ko¨lsch, 2001). The subterranean termites, Reticulitermes spp., can tolerate complete inundation for many
hours (LT50 of 20 h), which is sufficient to survive normal
rainfall conditions (Forschler and Henderson, 1995). The
small ant, Lasius flavus, which occur on tidal meadows in
Denmark can survive several days of total inundation
during the winter (Nielsen, 1981). Insects living in the tidal
zone, including mangroves, are exposed to twice daily
inundations and must be able to avoid drowning to survive
in this habitat. For example, the ant Polyrhachis sokolova
Forel survives inundation in air pockets in the nest
chambers in the mud (Nielsen, 1997a,b; Nielsen et al.,
2003) in tropical Australia.
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Another Australian mangrove ant is the small Camponotus anderseni McArthur & Shattuck which lives exclusively in hollow twigs of the tree Sonneratia alba J. Smith in
a mutualistic relationship with the Coccid, Myzolecanium
sp. 1. These ants avoid drowning during high tide by
blocking the entrance hole of the nest with a soldier’s head
(Nielsen, 2000). The nest cavities are quite small, normally
only one internode, and up to 50% of the volume can be
filled with ants and coccids (Nielsen, 2000). The inundation
of C. anderseni nests can last up to 3 h, and during that
period, the respiration of the ants and coccids can make the
nest atmosphere hypercarbic and nearly anoxic (Nielsen
et al., 2006).
The aim of this study was to investigate the influence of
high concentrations of carbon dioxide on oxygen consumption in artificial, sealed nests in the laboratory. A
further aim was to determine the conditions under which
the ants change to anaerobic respiration by measuring the
respiratory quotient (RQ) under increasing CO2 concentrations.
2. Methods
The ants used in this study were collected in the
mangrove near Darwin, Northern Territory, Australia
(12130S 1311E). Twigs of Sonneratia alba trees containing
nests of C. anderseni were collected from a boat at
intermediate tides and placed in an insulated box with ice
after the nest entrances had been covered with gauze, to
prevent the ants from escaping.
In the laboratory, the nests were opened and 100–150
worker ants were sampled and cooled some minutes in a
refrigerator to reduce their mobility before being placed in
an experimental chamber. The experimental chambers
consisted of double-necked Warburg chambers with
volumes from 1.5 to 2.1 mL, and the experimental
chambers were places in a temperature regulated water
bath at 25 1 C during measurements.
Oxygen concentrations were measured using fiber-optic
microsensors connected to an oxygen meter (PreSens
Microx TX3, Regensburg Germany). The sensor was built
into a syringe with a needle and it could be injected through
one of the rubber stoppers, such that the sensitive tip could
be placed in the middle of the chamber. The frequency of
measurements ranged from 1 to 30 s depending on the type
of experiments.
An injection needle was placed through each of the two
stoppers and attached to 3-way valves in the flow system of
a CO2 analyser (model LI-6251) connected to a data
acquisition and analyser system (Sable system international; Nevada, U.S.A., using Datcan V software) (Nielsen
et al., 1999). The flow of CO2 free air was kept constant at
150 mL per minute in the system.
Before the experiments, the chambers were switched to
the airflow system and flushed with CO2 free air through
the injection needles. At the start of the experiments the 3way valves were closed, thus sealing the chamber. The CO2
concentration in the chamber was measured after periods
ranging from 15 min to 4 h by switching the 3-way valves to
the flow system, so the amount of CO2 in the chamber
could be measured. The O2 concentration was measured
continuously during the same period.
In experiment A, 41 sets of measurements were made,
and the RQ was calculated from the O2 and CO2
concentrations.
In experiments B, the respiration chambers also contained 20 mg of cotton wool, and in eight sets of
measurements 50 mL of 0.1 N Na2 OH was added to absorb
the CO2 produced, and eight sets of measurements were
made with 50 mL of water placed in the cotton wool as a
control. The experiments ran for 100 min, or until the O2
concentration dropped below 4%. The mean respiratory
rates ðmL O2 =mg h) were calculated for both series. The
CO2 concentrations were measured directly for the
chambers without absorbent and for the chambers with
absorbent, an injection needle was forced through the
stopper to the cotton wool, and 100 mL of 0.1 N HCl was
added to release the absorbed CO2 , so it could be
measured.
The ants from all experiment were weighed just after the
measurements, and the respiratory rates are expressed as
mg live mass.
3. Results
The oxygen concentrations in the respiration chambers
in experiment A all followed the same pattern (see Fig. 1).
The first period is characterised by a linear decrease in O2
until it reached 18% O2 (mean r2 ¼ 0:95 for all experiments). The mean oxygen consumption at 25 1C for this
period was 2:95 1:45 mL O2 =mg h. The second period is
characterised by an exponential decrease in O2 concentration between 18% and 4% (ln y ¼ a þ bt, where y is O2
Fig. 1. Oxygen concentrations in a closed respiratory chamber containing
workers of Camponotus anderseni.
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concentration and t is time; mean r2 ¼ 0:97 for all
experiments). The mean oxygen consumption for this
period was strongly dependent on the O2 concentration,
so no mean values can be given. Finally, the decrease in
oxygen concentration below 4% was also exponential
(mean r2 ¼ 0:97) but with a much smaller slope.
The respiratory experiments B, with CO2 absorbent in
the chambers, showed a mean oxygen consumption of
1:75 0:26 mL O2 =mg h and the mean of similar measurements without absorbent was ð1:10 0:30 mL O2 =mg hÞ,
and a t-test of the means showed that it was significantly
lower ðPo0:0001Þ. The mean final O2 concentrations in the
chambers with and without absorbent were 2.9% and
3.2%, respectively, and the final CO2 concentration in the
chambers without absorbent was 20.3%. The O2 concentrations in all the experiments with CO2 absorbent
decreased linearly (mean r2 ¼ 0:99), and the experiments
without absorbent followed the pattern in experiment A.
Fig. 2 shows the RQ as a function of the final CO2
concentration in the chambers. The points fall into two
groups, described by linear functions:
CO2 concentration interval 0% to 15%:
Y ¼ 0:925 þ 0:000134X
ðr2 o0:001; N ¼ 15; P40:94Þ.
(1)
CO2 concentration 415%:
Y ¼ 0:147 þ 0:044X
ðr2 ¼ 0:80; N ¼ 26; Po0:0001Þ,
(2)
where y is the RQ and X is % CO2 .
The intersection of the equations is 17.8% CO2 .
Fig. 2. Respiratory quotient (RQ) for workers of Camponotus anderseni in
closed respiration chambers at different CO2 concentrations at the end of
the experiment. Line (1) represents the CO2 concentrations with constant
RQ, and Line (2) shows the CO2 concentrations associated with increasing
RQ, representing increasing anaerobic respiration.
507
4. Discussion
The oxygen concentration in the respiration chambers in
experiment A depended on the mass of ants, volume of
chambers, temperature, and time; and the pattern of
decreasing O2 concentrations can be described with a three
component curve. The first part was a linear decrease with
O2 concentrations between 21% and 18%, and these are
the concentrations the ants experience in normal open nests
in the mangrove (Nielsen and Christian, in Prep.). The
second component of the curve is the decrease in O2
concentration between 18% and 4% O2 , and it can best be
described by an exponential function. Within this interval,
both O2 uptake and CO2 production decrease with the
same ratio, resulting in a constant RQ. The third part of
the curve can also best be described by an exponential
function. At these low concentrations of O2 , there is an
increase in anaerobic respiration (see below) in which the
O2 uptake gradually stops, but the CO2 production
continues.
The CO2 production in C. anderseni nests was previously
described by a single logarithmic function (Nielsen et al.,
2006). The exact pattern of change in CO2 concentration
was not determined in the previous study, and it is therefore
possible that the CO2 concentration increases linearly in the
first short component, followed by a logarithmic increase.
The respiratory rates in the first component were 2:95 1:45 L O2 =mg h and 2:72 1:33 mL CO2 =mg h, respectively,
which was significantly higher than the CO2 values of
1:93 mL CO2 =mg h found by Nielsen et al. (2006). The very
high SD on the mean respiratory rate in this experiment
indicates variation in the measurements, which is due to
very high activity by some ants in the chambers. The
respiratory measurements lasted only a few minutes,
without time for the ants to acclimatise after the airflow
had been switched off, and therefore we would expect the
values to be higher than values obtained from longer
experiments on acclimatised ants.
The O2 uptake rate with absorbent in experiment B is the
same as the O2 uptake rate measured by Nielsen et al.
(2006), which was expected due to the much longer
measuring period in experiment B. The small SD also
indicates a much less ant activity. The O2 uptake for
C. anderseni is generally higher than other ant species of the
same size (Nielsen, 1986).
The linearly decreasing pattern indicates a constant
respiratory rate independent of the O2 concentration between 21% and about 4%, which is the lower
critical limit for the survival of colonies of C. anderseni in
CO2 free atmosphere (Nielsen, unpublished data). Therefore, the significantly lower O2 uptake in the experiments
without CO2 absorbent verified that it is the CO2
concentration that depresses the respiratory rates and not
the decreased O2 concentrations as long as the O2
concentrations are 44%. This tolerance to low O2
concentrations was also shown by Holter and Spangenberg
(1997) who demonstrated that dung beetles maintain
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normal respiration and movements in environments with
1–2% O2 .
In many microhabitats of insects, hypoxic and hypercarbic conditions are strongly negatively correlated
(Anderson and Ultsch, 1987), so it is often impossible to
separate the effect of high CO2 and low O2 concentrations.
During normal aerobic respiration, the RQ will always
be in the interval 0.7–1.0. A RQ above 1.0 can only be
achieved during anaerobic respiration or if the organism
transforms carbohydrate to fat (Wigglesworth, 1965;
Keister and Buck, 1964; Nielsen et al., 2006). It seems
unlikely that the ants would have accumulated fat during
this experiment, therefore, an increase in RQ to 41:0 is
likely to be an indicator for anaerobic respiration. Line (1)
in Fig. 2 shows that C. anderseni have a constant RQ of
about 0.92 in CO2 concentrations up to about 15–18%,
and Nielsen et al. (2006) found a RQ of 0.94 under natural
above-water conditions in the mangroves. This indicates
that there was no anaerobic respiration in this range of
CO2 concentrations. The second line (2) shows an
increasing RQ with increasing CO2 concentration in the
respiration chamber. This line indicates that the anaerobic
respiration is initiated at concentrations around the
intersection of lines 1 and 2, which is at 17.8% CO2 .
When insects experience hypoxic and hypercarbic conditions, it is often due to conditions in the environment or
microhabitat, and their exchange of respiratory gases has
marginal influence on the O2 and CO2 concentrations
(Anderson and Ultsch, 1987; Nielsen et al., 2003). In
contrast to this, the hypoxic and hypercarbic conditions in
the nests of C. anderseni are entirely due to their own
respiration after they have sealed the nest chamber to avoid
drowning. These experiments were carried out at 25 1C,
which is close to the mean daily air temperature for
Darwin in July (mean minimum ¼ 19:8 C and mean
maximum ¼ 30:6 C) and August (mean minimum ¼
20:9 C and mean maximum ¼ 31:7 C) (Australian Bureau
of Meteorology: www.bom.gov.au). During other times of
the year the temperatures are markedly higher, and the
conditions for the ants are even more unfavourable
(November mean minimum ¼ 25:5 C and mean
maximum ¼ 34:2 C). The very extreme conditions in the
intertidal mangrove habitat require remarkable adaptations for the survival of insects, and it is therefore not
surprising that anaerobic respiration in social insects has
been demonstrated for the first time in such an environment.
Acknowledgements
This work was supported by grants from the Danish
Agency for Science, Technology and Innovation. Further,
we want to thank Dorthe Birkmose for valuable assistance
during the field and laboratory work and Charles Darwin
University for providing the necessary laboratory facilities.
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