Respiratory concerts revealed by scanning microrespirography in a termite Prorhinotermes simplex (Isoptera: Rhinotermitidae)

Abstract

Respiratory metabolism of different developmental stages (larvae, pseudergates, nymphs, soldiers, neotenic reproductives; 0.6–4.5 mg body mass) of Prorhinotermes simplex was individually monitored by scanning respirographic method sensitive to subnanoliter amounts of O₂ consumption or CO₂ output per minute. Specimens exposed to dry air after removal from the colony performed enormously large, discontinuous bursts of CO₂ lasting usually 2 min. The volume of CO₂ produced during the burst often surpassed the volume of the whole body and it was 10- to 20-fold in excess of the air-filled endogenous tracheal volume. The initial velocity of CO₂ production during the burst was more than 90-fold faster in comparison to O₂ consumption. In the presence of enough moisture within the respiratory vessel, the termites breathed continuously without any larger outburst of CO₂. This fact fully corroborates validity of the so-called water retention theory in discontinuous CO₂ release. The highest rates of O₂ consumption were found in the second instar larvae (0.9 mg, 1000–2000 μl O₂/g/h), the soldier caste was intermediate (700 μl O₂/g/h) while pseudergates and neotenic reproductives consumed between 300 and 600 μl O₂/g/h, at 25 °C. All developmental stages feeding on a cellulose diet had CO₂/O₂ values (RQ) over 1 (1.2–1.4, i.e. carbohydrate metabolism), pigmented soldiers fed by the workers had RQ around 0.75 (predominating lipid or protein metabolism). The unusually large, sudden eruptions of CO₂ in specimens exposed to dry air allow us to make the following conclusions: (1) the bursts were due to special chemical processes, such as by enzymatic hydration of carbonic acid by carbonic anhydrase and; (2) the bulk of chemically evolved gaseous CO₂ escaped from the body by a mass flow supported by active ventilation, not by a passive diffusion. These results demonstrated that the periodic emissions of CO₂ and the associated homeostatic regulation of the respiratory acidaemia were under perfect physiological control. The termites could thus actively select the type of CO₂ release best suited to the extant environmental or internal physiological conditions, i.e. from a completely continuous respiration to occasionally cyclic or completely discontinuous CO₂ release.

Introduction

The field of insect respiration is a subject of prolonged scientific discussions. The problem started 85-years ago by a mechanistic theory of insect respiration created by Krogh (1920). According to his “diffusion theory of insect respiration”, insects could satisfy their demands for oxygen by a simple, passive diffusion of CO₂ and O₂ through open spiracles, without the need of special respiratory movements. The theory was later applied to explain discontinuous cycles in CO₂ release (Buck, 1956; Schneiderman, 1960; see Kestler, 1985; Lighton, 1996 for a review). According to the theory, CO₂ diffuses out and O₂ into the tracheal system when the spiracles open widely for several minutes. The resulting outburst of CO₂ is terminated when the spiracles are again closed. The metabolic CO₂ slowly accumulates within the tracheal system until the next burst (Schneiderman, 1960).

The periodically repeated bursts of CO₂ were found and most investigated in diapausing lepidopteran pupae of large body size. It was generally assumed that the opening, closure and “fluttering” of the spiracular valves depended primarily on the concentration of respiratory gases within the tracheae. This resulted in a purely mechanistic doctrine of the triphasic, discontinuous gas cycles (DGC), sometimes referred to as OCF cycles (for open, closed and “fluttering” spiracle), which has survived conservatively until this time (Buck, 1956; Schneiderman, 1960; Kestler, 1985; Lighton, 1996; Chown et al., 2006). The most recent review of the DGC (Chown et al., 2006) offers 5 hypotheses for the evolution and occurrence of these respiratory cycles, all of them being derived from Krogh's diffusional theory of insect respiration. The possibility of the physiological regulation of respiratory cycles by a special neuroendocrine system (Sláma, 1991), which can actively restrain the respiratory acidaemia by means of chemically produced mass outflow of CO₂ (Sláma, 1988, Sláma, 1994, Sláma, 1999, Sláma, 2000a, Sláma, 2000b), has been ignored.

Due to their relatively small size, previous respirometric measurements on termites were mostly made on groups of up to 50 specimens, using the conventional Warburg manometric techniques. The results revealed that termites released CO₂ by rates equal or higher than was O₂ consumption (RQ values equal or higher than 1), indicating catabolism of a carbohydrate (Cook, 1932; Ghidini, 1939; Hébrant, 1964). These results were true for most termite species (Lűscher, 1955; La Fage and Nutting, 1979) as well as for the termite colonies measured as a whole (Hébrant, 1970). The colony schowed diurnal cyclicity in the gas exchange (reviews by Grassé, 1982; Nunes et al., 1997).

The progress of gas chromatography methods during the 1990s, especially the improved manometric-GC techniques, opened new insights into termite respiration. This is mainly related to the production of CH₄ and H₂ by the endosymbiotic microorganisms helping termites to digest cellulose. In addition, certain species of termites contain nitrification bacteria which cause fixation of aerial N₂. To the present day, a number of termite species have been investigated with respect to the rather cumbersome proportions in CO₂, CH₄ and H₂ release and reciprocal consumption of O₂ and N₂ (Wheeler et al., 1996; Nunes et al., 1997; Jeeva et al., 1999). This situation is further complicated by relatively large proportions of anaerobic metabolism (Slaytor et al., 1997).

The results of the above studies can be basically summarized as follows: (1) the rate of O₂ consumption ranged in most termite species from 80 to 500 μl O₂/g/h (at 25 °C); (2) the rate of CO₂ output was usually equivalent or slightly higher than the respective rates of O₂ consumption; (3) the release of CO₂ usually predominated over O₂ consumption in well nourished specimens (RQ=1.2–1.6), while O₂ consumption predominated in starved specimens or in soldiers nourished by the workers (RQ=0.73–0.83); (4) the rates of CH₄ and H₂ emissions varied considerably in different species, but in most cases the production of CH₄ and H₂ did not surpass 10% of O₂ consumption (Ebert and Brune, 1997); and (5) aerial fixation of N₂ took place only in certain particular species of the termites and the rates of N₂ fixation represented only a small part of the total gas exchange (for review see Nunes et al., 1997).

The production of H₂ and CH₄, combined with N₂ fixation, make the study of termite respiration the most difficult field of insect physiology. Shelton and Appel (2000) measured the output of CO₂ in a single termite by means of sensitive, flow-through IR analysis methods. In Zootermopsis and other termite species (Cryptotermes, Incisitermes; see Shelton and Appel, 2001a) they found discontinuous bursts of CO₂ release, similar to the DGC cycles described in ants (see Lighton, 1998 for a review). In other species (Coptotermes and Reticulitermes), however, they were unable to see the regular DGC (Shelton and Appel, 2001b), which led them to propose several hypotheses for the possible roles of DGC in insect respiration (review by Shelton and Appel, 2001c).

Insect specimens measured by flow-through methods of CO₂ analysis (Lighton, 1996, Lighton, 1998; Lighton et al., 2004; Shelton and Appel, 2001c; Chown and Holter, 2000) were exposed to a flow of absolutely dehydrated air. This treatment is harmful to most insects, especially to ants or termites which are accustomed to living in underground shelters with relatively constant conditions of humidity. An unfortunate termite or ant that is exposed suddenly to a dehydrating air stream must keep the spiracles tightly closed in order to survive. The ants dehydrated in this way appeared to be the favorite examples of the DGC cycles (Lighton, 1996 for a review). They served as a basis for the so-called “chthonic theory” for the origin of DGC in insects living underground (Lighton, 1998). However, the theory has been recently challenged by Lighton and Ottesen (2005) who found that termites showing beautiful DGC in dehydrated air (Zootermopsis, Shelton and Appel, 2000) released CO₂ continuously in humid air streams. It is thus possible that the stereotypic DGC cycles described by Lighton (1996) could be artifacts resulting from exposure of ants to the absolutely dehydrated air stream.

Originally, the discontinuous type of respiration was assumed to occur only in diapausing pupae of large body size (Schneiderman, 1960). At the same time, however, discontinuous CO₂ output was also found in miniature pupae of a geometrid Bupalus (100-fold smaller than cecropia), while diapausing soft bodied prepupal stages of sawflies (Cephalcia) exhibited only continuous respiration without cyclicity (Sláma, 1960). After the invention of the scanning microrespirographic method (Sláma, 1984b), the presence of very short, actively regulated emissions of CO₂ were discovered in a miniature adult beetle (Bruchus, 5 mg body mass; Coquillaud et al., 1990; Sláma and Coquillaud, 1992). Similar discontinuous bursts of CO₂ were also found in arthropods other than insects, i.e. Arachnids (Dermacentor, 5 mg, Sláma, 1991; Ixodes, 1 mg; Sláma, 1994), pseudoscorpions and solpugids (Chelifer, 3 mg; Galeodes, 200 mg; Sláma, 1995 (see Table 1)). Later on, the scanning microrespirographic method revealed cyclic CO₂ emissions in worker ants as small as 0.1 mg (Monomorium, Sláma, 1999) and other insects of very small body size (reviews by Sláma, 1988, Sláma, 1994, Sláma, 1999, Sláma, 2000a).

The method of microrespirographic scanning demonstrated that the discontinuous emissions of CO₂ could not depend simply on a passive diffusion of CO₂ through the spiracles. Some insects and mites, for example, were able to exhale within a few seconds volumes of CO₂ surpassing the total body volume. Evidently, CO₂ was suddenly discharged from the body by a mass flow, because the amounts of CO₂ released during the short burst exceeded the volume of the whole tracheal system 10- to 100-times. These findings do not match with the mechanistic view of Krogh and the theories based on this view (Lighton, 1996; Hetz and Bradley, 2005; Chown et al., 2006). The gaseous CO₂ is 26-fold more soluble in tissues than O₂ or N₂. In a dry environment, a passive diffusion of CO₂ through the narrow slits of spiracular valves would be inevitably associated with pernicious water loss. Insects can survive, however, due to the evolution of actively controlled breathing which is regulated by an autonomic neuroendocrine system, known as the coelopulse system (Sláma, 1991). According to this concept, gaseous CO₂ is released from endogenous carbonate buffers by a special chemical process and the bulk of CO₂ leaves the body by a mass flow or ventilation through one or just a few spiracles (Sláma, 1988, Sláma, 1991, Sláma, 1994, Sláma, 1999, Sláma, 2000a, Sláma, 2000b; Sláma and Coquillaud, 1992; Sláma and Neven, 2001). The chemically produced outbursts of CO₂ were originally named “the Prague respiratory cycles” (Sláma and Coquillaud, 1992), but later they were renamed to “the respiratory cycles of Babák” (Sláma, 1994, Sláma, 1995, Sláma, 1999) to honor their discovery and first description by Babák, 1912, Babák, 1921 in Dytiscus.

The scanning microrespirographic technique has been used for monitoring the respiratory physiology of various insect species, including termites. Here we report on some results obtained with various castes and developmental stages (larvae, pseudergates, soldiers, neotenic reproductives) of a termite Prorhinotermes simplex which appears to be a good experimental model (see Štys and Šobotník, 1999 for a review). The genus Prorhinotermes is a representative member of the advanced family Rhinotermitidae (Austin et al., 2004), which is considered as a more or less representative group of termites.