The concept that insect respiration depends only on diffusion supplemented in larger species by ventilation is in need of an overhaul: the situation is much more complex.
Insects, like all living organisms, are far-from-equilibrium, dissipative structures. That is, they actively take up energy (and nutrients) and in doing so alter both themselves and their surrounding environment. Initially, the changes in both directions might appear insignificant, but on a longer time scale their impact can be profound (Brooks and Wiley 1988) (insect outbreaks are, perhaps, the best testimony to this). The dissipation of energy to the environment enables insects to undertake the enormous behavioural repertoire that has not only contributed to their success as a group, but which has also captivated human curiosity. This behaviour is usually the first response to a changing environment, and has been labelled the better part of regulatory valour (Bennett 1987). In response to unsuitable environmental conditions, insects might either leave by migrating or seek more suitable microclimates. If such behavioural regulation is insufficient to modulate the effects of change, insects can also avoid adverse conditions through physiological modification in the form of diapause, aestivation, or some form of dormancy (Danks 2000, 2002; Denlinger 2002). Active stages are also capable of considerable physiological responses to changing conditions.
This suite of responses to the environment, ranging from migratory behaviour to physiological regulation, is dependent on energy produced by the metabolism of substrates. Moreover, metabolism enables insects to acquire additional resources, to grow and reproduce, and to participate in the manifold interactions that characterize any given community. Although metabolism has both anabolic and catabolic components, in this chapter we will be concerned mostly, but not exclusively (see Wieser 1994), with catabolism. That is, we are concerned with the largely oxidative metabolism of substrates for energy provision, and the ways in which oxygen required for this process is transported to the tissues and carbon dioxide removed from them (water balance is dealt with mostly in Chapter 4). Although the rate of the entire process is often termed metabolic rate, especially where oxygen uptake and CO2 production rates are concerned, it is important to make a distinction between oxidative catabolism as a cellular level process (respiration), and gas exchange as the physical transfer of gases between the atmosphere and the tissues/haemolymph (Buck 1962; Lighton 1994).
In insects, metabolism during both rest and activity (especially flight) is generally aerobic. However, occasionally ATP provision can be via anaerobic metabolism (Gade 1985), although this is most common in special situations (Conradi-Larson and Somme 1973). During more normal pedestrian activity, anaerobic metabolism only accounts for a small proportion of ATP production, and it is virtually non-existent during flight. The demand for ATP is not consistent, but varies widely both over the short and longer terms. The most pronounced, virtually instantaneous rates of change are those associated with the transition from the alert quiescent state to flight, although endothermic heat generation is also responsible for substantial increases in metabolic rate. Metabolic rate is likewise dependent on body size, environmental temperature, and physiological status of the insect (such as dehydration and feeding), and it is responsive to both short- and longer-term changes in the partial pressures of oxygen and carbon dioxide. These physiological changes are not simply passive responses to the changing environment, but include active modulation of metabolic rate, sometimes via phenotypic plasticity, and often via evolutionary change.
Over the last several decades it has become increasingly clear that the circumstances and methods used to assess a given physiological variable have a considerable influence on the outcome of the experiment or trial in which they are used (Baust and Rojas 1985; Harrison 2001). This is certainly the case when it comes to the measurement of gas exchange rates. Although closed system (constant pressure and constant volume) methods are intrinsically accurate for the measurement of gas exchange (Slama 1984), in all but the most quiescent of stages (such as diapausing pupae) it is difficult to distinguish activity metabolism from standard metabolic rates (SMR) owing mostly to the poor temporal resolution and the integrative nature of the technique (Lighton and Fielden 1995). This means that comparisons of SMR, which are essential for determining whether insects modulate metabolic rates in response to different environmental circumstances (Chown and Gaston 1999), are unlikely to be possible. Comparisons between species and populations will be confounded by an experimental technique that fails to consider activity, especially because changes in levels of activity appear to be one of the responses of insects to a changing environment. For example, in laboratory-selected Drosophila melanogaster, desiccation-selected flies are far less active than control flies. Desert-dwelling flies appear to show a similar response compared to their more mesic congeners (Gibbs 2002a).
Fortunately, modern flow-through gas analysis (Lighton 1991a), continuously recording calorimet-ric (Acar et al. 2001), and activity detection (Lighton 1988a) methods allow periods of activity to be separated from those where the animal is at rest, so facilitating accurate estimation of SMR (because insects do not have a thermoneutral zone these rates cannot be referred to as basal). The difference between these open system methods and the closed system techniques previously used is usually substantial (Lighton 1991b). Lighton and Wehner (1993) provided the first quantitative data on beetles and ants highlighting this problem, pointing out that VO2 is not necessarily reduced in xeric species. In a later, more detailed analysis, Lighton and Fielden (1995) showed that previous estimates for insects based on closed system methods may have errors of 134 per cent of the true value for an insect weighing 0.001 g, and 18 per cent for a 10 g individual. These errors are a consequence of different estimates of both the coefficient and exponent in the scaling relationship between metabolic rate and mass. The general overestimation of SMR was borne out in a later comparative analysis (Addo-Bediako et al. 2002), but the exponent of the relationship between mass and metabolic rate remains contentious (Section 3.4.5).
The influence of technique on the outcome of experimental work has also been highlighted recently in three other areas of investigation. These are the measurement of oxygen partial pressures in tissues, the importance of the coelopulse system for gas exchange and its regulation, and the 'postural effect' that might be responsible for the lack of congruence between resting metabolic rate estimates for insects running on a treadmill and those measured during quiescence. In the first instance, oxygen partial pressures in tissues are often measured using oxygen microelectrodes inserted into the tissue (Komai 1998). Although care is usually taken to assess the effects of this invasive technique, it has recently been demonstrated that tissue damage might be responsible for underestimates of pO2. The use of implanted paramagnetic crystals of lithium phthalocyanine and in vivo electron paramagnetic resonance oximetry has shown that pO2 might be considerably higher than estimated by oxygen micro-electrodes, owing to acute mechanical damage caused by the latter, which probably stimulates oxygen consumption in the fluid surrounding the electrode (Timmins et al. 1999). Implantation of paramagnetic crystals and a subsequent period allowed for tissue repair mean that the effects of tissue damage could be substantially reduced.
The second example concerns the debate on the importance of extracardiac pulsations and the coelo-pulse system for gas exchange and its regulation, respectively (Slama 1999) (Section 3.4.1). These extracardiac pulsations are visually imperceptible abdominal segmental movements (caused by contraction of abdominal intersegmental muscles), but with an apparently significant effect on haemo-coelic pressure, and therefore on gas exchange (Slama 1988, 1999). Later investigations by Tartes and his colleagues (review in Tartes et al. 2002) of several species and stages identical to those used by Slama (1999) have failed to confirm the importance of these pulsations for the regulation of gas exchange. Indeed, both Tartes et al. (2002) and Wasserthal (1996) ascribe many of the abdominal movements recorded in earlier reasonably invasive experiments (e.g. connection of the abdominal tip to a position sensor) to artefacts of the experimental design. Furthermore, Tartes et al. (2002) concluded that these miniscule abdominal movements are functionally similar to large abdominal movements, though reduced in magnitude, with only a small influence on ventilation, and then only in a few species.
The effects of measurement technique are also obvious in the calculation of the costs of transport. It has long been known that in both vertebrates and invertebrates the y-intercept of the relationship between running speed and metabolic rate, usually obtained from data gathered using animals on a treadmill, provides an estimate of resting metabolic rate that is higher than empirical measurements of resting metabolic rate. This elevated metabolic rate was ascribed to a 'postural effect' by SchmidtNielsen (1972). In subsequent investigations of insects, this explanation, and several others that have been mooted to explain the elevated y-intercept, were questioned (Herreid et al. 1981). It was subsequently demonstrated that the elevated y-intercept in insects is a consequence of the use of treadmills. In ants running voluntarily in a respirometer tube, there is no y-intercept elevation (Lighton and Feener 1989), and this has also recently been confirmed for ants in the field (Lighton and Duncan 2002).
The fat body is the principal site of synthesis and storage of carbohydrates, lipids, amino acids, and proteins. It is the major location of trehalose and glycogen synthesis, respectively the main haemolymph and storage carbohydrates in insects, the principal site of proline synthesis from alanine and acetyl CoA, and the principal site of lipid synthesis and storage (mostly as di- or triacylgly-cerol) (Friedman 1985). When either flight muscles, or other muscle groups, place a demand on the system for fuel, initially this fuel is provided from stores in the muscles themselves, but shortly thereafter energy-providing macromolecules from the haemolymph and eventually from the fat body must be used. Small peptide hormones produced by the corpora cardiaca are responsible for the control of fuel mobilization, and many members of this adipokinetic (AKH) family of hormones, which are responsible for lipid, carbohydrate and proline mobilization, are now known from insects (Gade 1991; Gade et al. 1997; Gade and Auerswald 2002).
The bulk of the energy provided by carbohydrates, lipids, and proline is made available by aerobic metabolism. Indeed, insect flight, one of the most energetically demanding activities known in animals, is wholly aerobic. In contrast, pedestrian locomotion can be fuelled partly by anaerobic metabolism, and anaerobic metabolism serves to provide energy during periods of environmentally induced hypoxia.
Because flight is such an energetically demanding activity, and because so many insect species use flight as a major method of locomotion, much of the work done on substrate catabolism has been concerned with the aerobic provision of ATP for flight. Nonetheless, it is worth noting that only adult insects are capable of flight, that in ants and termites only the alates fly and then usually for just a brief period, and that in many species the adults are flightless (Roff 1990), although, amazingly, some stick insects have secondarily regained their powers of flight (Whiting et al. 2003). Even among those adult insects that can fly, some, such as dung beetles, parasitic wasps, flies, leafhoppers, and grasshoppers, spend much of their time walking, running, or hopping (Chown et al. 1995; Gilchrist 1996; Krolikowski and Harrison 1996).
Carbohydrates are among the most widely known flight fuels in insects. Small amounts of trehalose and glycogen (the most common carbohydrate stores) can be found in the muscles, but the former is usually found in large quantities in the haemolymph, and the latter is stored in the fat body. Carbohydrates are utilized by most flies and hymenopterans, although they are also used in species, such as locusts, that shift from one kind of fuel to another or that alter their reliance on different fuels depending on food availability (Joos 1987; O'Brien 1999; Dudley 2000). The biochemical pathways involved in the use of glycogen and trehalose have recently been reviewed in detail (Nation 2002).
Lipids (fatty acids) are also widely known as fuels for insect flight. In locusts they provide a major source of energy in flights that last for longer than approximately 30 min, as they do for non-feeding or starved hawk moths (Ziegler 1985; O'Brien 1999; Dudley 2000). Adipokinetic hormone mobilizes triacylglycerol stored in fat body cells as diacylglycerol (via triacylglycerol lipase), and this is subsequently transported as a lipoprotein complex (the apolipoprotein is apoLp-III, and the complex known as lipophorin) through the haemo-lymph to the flight muscle cells, where it is utilized via b-oxidation (Ryan and van der Horst 2000).
Although proline was first thought to be important only in tsetse flies and Leptinotarsa decemlineata (Coleoptera, Chrysomelidae), or as a primer of the Krebs cycle in some species (such as the blowfly, Phormia regina) it has now been shown to be an important flight fuel especially in beetles (Gade and Auerswald 2002). In species that make use of both proline and carbohydrates for energy provision, proline varies in its contribution to flight metabolism from approximately 14 per cent in the meloid beetle Decapotoma lunata, to about
50 per cent in the cetoniine scarab beetle, Pachnoda sinuata. As much as half of this energy may come from haemolymph stores in beetles with large haemolymph volumes, and although measurable levels of lipids are present in the haemolymph, they are not used for flight metabolism (Gade and Auerswald 2002). In P. sinuata, warm-up prior to flight is powered entirely by proline (Auerswald et al. 1998), and in those true dung beetles (Scarabaeinae) that have been examined, it appears that flight (or walking in flightless species) is powered entirely by proline (Gade and Auerswald 2002). The pathways involved in proline utilization are discussed in Nation (2002), and regulation of its use is reviewed by Gade and Auerswald (2002).
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