Measurement of cuticular water loss This has never been a simple matter (Loveridge 1980; Noble-Nesbitt 1991; Hadley 1994a). Essentially, there are three ways of measuring cuticular water loss—gravimetric, isotopic, and electronic— which have been applied to whole insects, portions of their cuticles, or in vitro cuticle preparations. Early gravimetric measurements on living insects inevitably included respiratory water loss. However, spiracular closure during continuous and sensitive recording of body mass enables periodic measurement of minimum or cuticular water loss; provided the insect in question exhibits discontinuous gas exchange (Kestler 1985; Machin et al. 1991). Isotopic methods allow unidirectional flux to be measured (while gravimetric methods measure net flux). Other advantages of isotopic methods are sensitivity, suitability for use at high av (gravimetric measurements are usually made in dry air), and direct comparison with aquatic animals (Croghan et al. 1995). Nicolson et al. (1984) developed a technique using ventilated capsules and tritiated water to measure cuticular transpiration of desert tenebrionid beetles, and Croghan et al. (1995) modified this technique to measure simultaneous water and CO2 loss from cockroaches. Electronic moisture sensing in flow-through systems, first developed by Hadley et al. (1982) and Nicolson and Louw (1982), is now widely used in concurrent measurement of water loss and metabolic rate by means of CO2/H2O analysers, but the emphasis is usually on respiratory water loss (see below). For cuticular water loss only, the ventilated capsule technique of Nicolson et al. (1984) and later variations thereof (for diagrams see Hadley 1994a: 72) allow measurement of transpiration across small areas of cuticle in vivo, and the same can be achieved with experiments using excised discs of cuticle in vitro. Both techniques can be used to investigate regional differences in permeability, which are averaged in whole-insect studies (for review see Hadley 1994a). Avoidance of damage during handling is critical with isolated cuticle preparations. The units used to express cuticular water loss data have also led to considerable confusion in the literature, discussed by Noble-Nesbitt (1991): commonly, the concentration gradient is expressed in terms of saturation deficit, so that transpiration rates in mgcm—2h—1 can be converted to permeability in mgcm—2h—1 Torr—1 (Loveridge 1980). Potential errors in estimating surface area are discussed by Loveridge (1980).

The possibility of hormonal control of cuticle permeability was suggested on the basis of increased mass loss after decapitation in Periplaneta americana and its reversal by injection of brain or corpora cardiaca homogenates (Treherne and Willmer 1975). There is still insufficient experimental evidence to confirm this (for different opinions see Noble-Nesbitt 1991; Hadley 1994a). Moreover, the adaptive value of such increased cuticle permeability has been questioned when other avenues of water loss in cockroaches vary greatly (Machin et al. 1991). Periplaneta americana has been the traditional subject of research on cuticular water loss, yet methodological differences have led to enormous variation in the data obtained for this one species. Machin and Lampert (1987) demonstrated that all previous permeability estimates for cockroaches were too high because of damage to the cuticle of individuals in crowded cultures (although this damage is continually repaired), and further damage incurred during experimental manipulation. When cuticular and respiratory water losses of quiescent P. americana were measured by continuous weighing, water loss rates were up to an order of magnitude less than those obtained during intermittent weighing. This was attributed to ventilatory water loss during the periodic disturbances required by intermittent weighing (Machin et al. 1991). Regardless of the difficulties involved in accurate measurement of cuticle permeability, the lipid component of the cuticle is accepted as the major barrier to water movement.

The lipid barrier

Lipids, by definition, do not interact with water: thus, it is not surprising that they have major waterproofing functions in plants as well as in arthropods (Hadley 1989). Insect epicuticular lipids include a diverse array of nonpolar, hydrophobic molecules, mainly long-chain saturated hydrocarbons, which differ in their physical properties. The composition of these lipids has been extensively studied (for a recent review see Gibbs 1998) and found to differ widely at various levels of organization. Individuals of a species vary in hydrocarbon composition and rates of cuticular water loss (Toolson 1984), while variation between species has been used as as a taxonomic character (Lockey 1991). Epicuticular lipids in the grasshopper Melanoplus sanguinipes vary in composition and biophysical properties between individuals, families, and populations, and with thermal acclimation (Gibbs et al. 1991; Gibbs and Mousseau 1994; Rourke 2000). There has long been broad consensus among insect physiologists that cuticular water loss is related to habitat water availability, and that there is a strong correlation between cuticular water loss and the quantity of epicuticular lipids (Edney 1977; Hadley 1989, 1994b). Waterproofing properties are also affected by structural differences in cuticle lipids; waterproofing increases with hydrocarbon chain length, but decreases with methyl-branching and unsaturation, both of which prevent close packing of molecules (Gibbs 1998).

Extreme development of surface lipids occurs in certain insects. Examples are the filamentous wax blooms which cover Namib Desert tenebrionid beetles in response to desiccating conditions (McClain et al. 1985), and the particulate wax deposits of whiteflies (Homoptera, Aleyrodidae) (Byrne and Hadley 1988).

Rates of cuticular water loss increase with temperature, gradually at first and then rapidly above a critical or transition temperature. This discontinuity is thought to signify a change in molecular organization of the lipid barrier, when surface lipids begin to melt and their resistance to water movement is impaired. The transition temperature is species-specific and may be ecologically relevant or may exceed the lethal temperature of the species concerned. There has long been controversy about the relationship between lipid melting and increased cuticular permeability, and indeed about the thermodynamics of water movement through cuticle, specifically whether vapour pressure deficit is the appropriate driving force (for review see Noble-Nesbitt 1991). Gilby (1980), for example, emphasized the complexity of the system and the need to measure cuticle as well as air temperatures during transpiration experiments: he concluded that transition temperatures were artefacts. A kinetic approach based on Arrhenius plots has been advocated by some authors (Wharton 1985; Noble-Nesbitt 1991). Arrhenius plots of ln [permeability] against the reciprocal of the absolute temperature give the same estimate for transition temperature whether or not the permeability data are corrected for vapour pressure deficit (Gibbs 1998), with phase transition indicated by deviation from a straight line relationship (Fig. 4.1).

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