Respiratory concerts revealed by scanning microrespirography in a termite Prorhinotermes simplex (Isoptera: Rhinotermitidae)
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.
Section snippets
Material and methods
The material of P. simplex (Hagen, 1858) originated from a colony of termites collected in Soroa (Pińar del Rio, Cuba) in 1964. The colony was maintained and reproduced in our laboratories until this time. It was kept constantly on spruce and pine wood blocks under conditions of high moisture, permanent darkness, at 26±1 °C. Here we use the term pseudergates for all older larvae irrespective of their origin, nymph for unpigmented fully grown larvae with wing buds and neotenic reproductive for
The scanning microrespirography of termites
Termites live in underground colonies or in special wood tunnels with more or less constant conditions of humidity and gas composition. A termite removed from the colony into free air undergoes a sudden stress. It starts running desperately around the respiratory vessel in order to find way to escape back to the colony. After several minutes of this isolation, it usually slows down locomotory activity and starts to economise energy expenditure. Due to this, the actual respiratory patterns
General principles of CO2 release
The retention and discontinuous release of CO2 is a common habit of most terrestrial arthropods. Undoubtedly, this homeostatic mechanism is used to moderate the respiratory acidaemia and prevent water loss. Theoretically, there are several physiological models for the regulation of respiratory acidaemia in insects: (1) CO2 is continuously liberated from tissue or hemolymph buffers into the tracheal system from which it diffuses out through the widely opened spiracles; (2) CO2 is continuously
Acknowledgements
This research was supported by the Czech Science Foundation (206/06/1643). We wish to thank the Z4 055 0506 project realized in IOCB AS CR, Prague.
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