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: [email protected] (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. ARTICLE IN PRESS 506 M.G. Nielsen, K.A. Christian / Journal of Insect Physiology 53 (2007) 505–508 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. ARTICLE IN PRESS M.G. Nielsen, K.A. Christian / Journal of Insect Physiology 53 (2007) 505–508 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 ARTICLE IN PRESS 508 M.G. Nielsen, K.A. Christian / Journal of Insect Physiology 53 (2007) 505–508 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. 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