Cuticular water loss in the smokybrown cockroach, Periplaneta fuliginosa

J. Insect Physiol,

Vol. 32, No. 7, pp. 623-628, 1986 Printed in Great Britain.

0022-1910/86$3.00+ 0.00 Pergamon Journals Ltd

CUTICULAR WATER LOSS IN THE SMOKYBROWN COCKROACH, PERIPLANETA FULIGINOSA A. G. APPEL*t, D. A. REIERSON and M. K. RUST Department of Entomology, University of California, Riverside, CA 92521-0314, U.S.A. (Received 20 May 1985; reuised 31 Ocrober 1985)

Abstract-The effects of temperature and humidity and body water and lipid content on the cuticular water loss and permeability of the smokybrown cockroach, Periplaneta fuliginosu, were examined. Cuticular water loss (pg crnm2h-‘) was directly correlated to body water content and inversely related to body lipid content. Density of epicuticular lipids was not associated with water loss. Cuticular water loss and cuticular permeability @gcrne2 h-’ mmHg-‘) increased exponentially with temperature. There was no defined transition temperature. Cuticular water loss decreased arithmetically with increasing ambient relative humidity although calculated cuticular permeability increased exponentially. Watervapour flux experiments with tritiated water revealed that vapour eflux was independent of relative humidity. Cutkular permeability calculations that indicate relative humidity dependency under constant temperature and variable relative humidities are therefore mathematical artifacts. Water-vapor influx, however, was dependent of relative humidity as was net flux, the vector sum of efflux and influx. Key Word Index: Cuticular permeability, cuticular water loss, water flux, tritiated water, smokybrown

cockroach

INTRODUCTION Water loss in insects occurs through several parallel pathways: the cuticle, respiratory surfaces and oral and anal openings (Cooper, 1983). The cuticle in insects is the primaq water-efflux pathway because of the large surface area to body volume ratio. The epicuticular lipid layer is considered the major barrier to cuticular water loss (Beament, 1958; Edney, 1977); however, abiotic factors such as temperature and humidity reportedly mediate not only rates and amounts of water loss but the permeability of the cuticle itself (Edney, 1957; Beament, 1958). High temperatures, at a given relative humidity, tend to increase water loss by increasing the saturation deficiency of the surrounding environment (Edney, 1957) and perhaps by also altering the physicochemical state of the epicuticular lipids (Locke, 1965). also elevate saturation Decreasing humidities deficiency, increasing water loss (Edney, 1957). Cuticular resistance to water loss may also be affected by humidity acclimation (Appel and Rust, 1985), seasonal acclimatization (Hadley, 1977, 1978; Toolson, 1982; Toolson and Hadley, 1977, 1979) and changes in cuticular water content (Yokota, 1979). Respiratory water ioss may be limited by spiracular closure triggered by rapid temperature or humidity changes (Bursell, 1957; Loveridge, 1968b; Ahearn. 1970; Cooper, 1983.). Similarly, oral and anal water

loss may be affected by temperature, humidity and availability of water rich food (Loveridge, 1974; Appel and Rust, 1985). The smokybrown cockroach, Periplaneta fuliginosa (Serville), is most abundant in temperate humid environments (Appel, 1985). Edney and McFarlane (1974), Cohen and Cohen (1981), and Appel er al. (1983) related distribution patterns of cockroaches to habitat humidity and to the degree of cuticular waterproofing, some species having distinctly different cuticular permeabilities directly related to habitat moisture. High cuticular permeability at high relative humidity is thought to facilitate water loss and prevent water poisoning while low cuticular permeability at low humidity promotes water retention and conservation (Edney, 1977). Several previous studies (Loveridge, 1968a; Yokota, 1977) have reported that rates of water loss through the cuticle at constant environmental temperature may be altered by ambient humidity. The present study examines the effects of temperature and humidity and body water and lipid composition on cuticular water loss and permeability in P. fuliginosa. MATERIALS AND METHODS Insects

Adult male P. fuliginosa, obtained from laboratory cultures, were used because of their relatively homogenous physiological milieu and more similar size and weight compared to adult females or nymphs. Several thousand cockroaches were cultured in 120-l rubbish bins maintained at 25.5 &-2”C, 50 + 10% r.h., exposed to an irregular photoperiod and supplied water and dry dog chow ad fib. To ensure hydration, groups of 50 individuals were confined for 24 h in 3.8-l glass jars with cardboard harbourage,

*Present address: Department of Zoology-Entomology, Auburn University, AL 36849-4201, tTo

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food and 2 watering devices. This procedure resulted in a stable level of initial hydration (about 70% of the wet weight). Water and lipid content Total body water content was determined gravimetrically. In all experiments it was assumed that weight lost by specimens was due to loss of water. Data from specimens that defecated or salivated were not included. Dry weight was determined by weighing specimens kept at 5OC until two successive daily weighings differed ~0.1 mg. Percentage total body water and mg of water per g wet weight were calculated as the proportional difference between the weight of freshly HCN-killed specimens and their dry weights. Cockroach whole-body lipid content was determined by chloroform extraction. Individual cockroaches cut into 8-10 pieces were soaked in 10ml chloroform for 24 h. the solution was passed through Whatman No. 1 filter paper, the chloroform evaporated, and the amount of lipid determined. The amount of cuticular lipids was determined by immersing pre-weighed, freshly killed specimens in distilled hexane for 10 min. The solution was dried in a nitrogen atmosphere and the lipids remaining weighed (Blomquist and Kearney, 1976). Cuticular water loss and permeability Cuticular water loss and permeability of live and dead cockroaches were determined by the rate of weight loss (Appel et al., 1983). Live cockroaches were confined in screen cages and freshly HCN-killed cockroaches were arranged on their tegmina in numbered weighing pans. Groups of live and dead cockroaches were placed in 1 l-l desiccator jars that contained 0, 32, 50, 75 or 100% r.h. (Winston and Bates, 1960) at 30°C. Similarly, groups were incubated at 040°C and 0% r.h. The air in the desiccators was not circulated since relative humidity equilibrium occurred less than 15 min after the desiccators were sealed. The weight (mg) lost at 4 h was used to calculate cuticular water loss and permeability. Cuticular water loss was calculated as the change in weight of the specimen per cm’cuticle/h. Permeability was calculated as cuticular water loss per unit saturation deficiency (mmHg). Surface area @A), in cm*, was estimated by Meeh’s formula, SA = k(wt)0,667where k represents a species or groupspecific constant (Edney, 1977); 12 is often used for cockroaches (Mead-Briggs, 1956; Edney and McFarlane, 1974; Appel et al., 1983); and wt is the gram weight of the insect. To examine the rate of water loss over time, groups of live and dead cockroaches were desiccated at 30°C and 0% r.h. and weighed every other hour for 8 h and at 24 h. Water +apour Jux Water-vapour flux was determined by changes in concentration of tritiated water in live cockroaches. Fully hydrated male cockroaches were lightly anesthetized with CO,, injected between the 4th and 5th abdominal sternites with 1 ~1 of tritiated water, containing 4nCi (ICN Biomedicals Inc.), with a 225-gauge needle and weighed to the nearest 0.1 mg. Following injection, the cockroaches were confined

for 6 h in a 3.8-l jar with food, water, and cardboard harborage. Since haemolymph sampling results in high cockroach mortality and since the body water pool is uniformly labelled within 6 h (Appel, unpublished), an initial tritium concentration was determined from a 5 ~1 saliva sample obtained by gently palpating the thorax and abdomen of each injected individual. The cockroaches were again anaesthetized, weighed, their mouthparts and anal openings sealed with melted Tissueprep wax (SO’C), and reweighed. The cockroaches (N = 10) were confined in individual screened cages and exposed at each humidity for 24 h at 3OC. They were then reweighed and a 5-~1 haemolymph sample taken by piercing the membrane between the metathoracic coxae and the first abdominal sternite. In those cockroaches that could not be bled. the entire abdominal contents were removed and micro-distilled to obtain pure water (Wood et al.. 1975). There was no significant difference in activity or quenching between body fluids and their distillates so samples were counted for tritium activity as obtained. Fluid samples (5 ~1) were expelled into 10 ml of Beckman HP/b scintillation medium in plastic counting vials and counted for 10 min, or 1% error, using a Beckman LS-100 liquid scintillation counter. Unidirectional fluxes were calculated using the linear change equations of Nagy (1975). Data were fitted by least squares regression and means compared with Student’s t-test.

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Cuticular water relations RESULTS

Fully hydrated adult male P. filiginosa weighed 649.6 & 89.0 mg (r + SD) and % composition was 70.6 f 2.7% water and 8.4 k 3.8% lipid. There was no relationship between water or lipid content and initial body weight. Initial water content was inversely proportional to body lipid content (Fig. 1C). Rate of water loss was significantly

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proportionate amount of body water and body lipid (Fig. IA-B). Epicuticular lipids represented ~0.3% of initial wet weight and < 6.2% of total extractable lipids. The amount of epicuticular lipids per cockroach was proportional to estimated surface area (Fig. 2) but not to the rate of cuticular water loss or permeability. Extraction of surface lipids significantly increased the rate of cuticular water loss and permeability both increased significantly by 52.1% (Student’s t-test, P > 0.01, N = 20). Water loss (mass) of live and dead cockroaches was analyzed as the ratio of the water mass at time t (M,) to initial water mass (M,) plotted as a function of time. The proportion of water lost per unit time is the slope of the linear regression model. This rate of water loss from live and dead cockroaches was significantly higher for the first 2 h than afterwards (Fig. 3). In dry air, cuticular water loss increased exponentially with increasing temperature, from an average of 183.1 to 10,780.3 pgcm-2h-1 at 0 and 40°C respectively (Fig. 4). Cuticular permeability increased exponentially, ranging from also 34.6 pg cm-* h-’ mmHg-’ at 0°C to 116.5 at 40°C. There was no obvious transition temperature; cuticular permeability increased smoothly with temperature. At 3O”C, cuticular water loss decreased with increasing relative humidity. The rate of water loss declined linearly from 1559.9 pg cm-* h-’ at 0% to 380.7 at 98% r.h. Cuticular permeability, however, increased exponentially from 33.8 pg cme2 h-’ mm Hg-’ at 0% to 564.9 at 98% r.h. (Fig. 5). Even though cuticular permeability at a constant temperature is not completely independent of relative humidity because of the saturation-deficit factor in the calculation, water-vapour pressure is independent of relative amounts of water vapour (Nobel, 1974). Tritium-determined unidirectional fluxes of water vapour and net flux at 30°C and t&98% r.h. are shown in Fig. 6. Water-vapour efflux was not significantly correlated with humidity. Influx, however, increased with relative humidity. Net flux, the .

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vector sum of influx and efflux, decreased with humidity. The relationship of net flux and cuticular water loss to relative humidity is explained by the significant relationship of water-vapour influx to ambient relative humidity. Cuticular permeability calculations that indicate humidity dependency under constant temperature and variable humidity are, therefore, clearly mathematical artifacts. DISCUSSlON

Cuticular water loss from adult male P. fuliginosa is affected by both intrinsic and extrinsic factors. The amounts of body water and body lipids are inversely related (Fig. 1C) but body lipids account for more of the variability the rate of water loss than does water content. Loveridge (1973) and Tucker (1977) found a similar inverse relationship between body water and lipid contents of Locusta migratoria R. and F. and P. americana, respectively, and Tucker concluded that higher body lipid contents contributed to lower rates

of water loss. The quantity of epicuticular lipid, however, is apparently not related to the permeability of the cuticle as much as its composition. The importance of surface lipids in waterproofing was evidenced in this study by a 52% increase in cuticular permeability of P. fuliginosa after hexane extraction. Increased rates of water loss following removal of surface lipids have also been reported for scorpions (Toolson and Hadley, 1977, 1979), cicadas (Toolson, 1984) and other acarines and insects (see Edney, 1977, for references). Epicuticular hydrocarbon composition has been correlated with degree of waterproofing. Long-chain alkanes and alkenes are found on more xeric adapted species tending to limit cuticular water loss (Hadley, 1978). Differences in epicuticular hydrocarbon composition accounted for >64% of the interindividual variation in the rate of cuticular water loss of a desert cicada (Toolson, 1984). Appel et al. (1983) however, found that cockroaches with qualitatively identical and quantitatively similar cuticular hydrocarbons had over a

Cuticular water relations

3-fold difference in cuticular permeability. Both epicuticular hydrocarbons and internal lipids contribute to cuticular waterproofing. A change in the rate of water loss over time has been considered evidence for water compartmentalization (Edney, 1977). The significantly greater rate of water loss during the first 2 h of desiccation indicates a rapidly exchanging water compartment that may represent water weakly bound near the cuticle surface (Edney, 1977) or components of the cuticle interior (Toolson, 1984). The rapidly exchanging compartment also includes haemolymph water; after 2 h of desiccation, haemolymph volume in P. fuliginosa decreases 32% (Appel, unpublished). This compartment represents about 10% of the total body water of P. jkliginosa of which some 77% is haemolymph water. In this and several other studies with cockroaches (see Edney, 1977), there is no apparent transition temperature; cuticular permeability increases exponentially with te-mperature. Comparative studies with cockroaches indicate that a transition phenomenon is subtle or absent in species that have especially high cuticular permeabilities (Appel, unpublished). The more grease-hke epicuticular lipids of these species might not become disorganized at a specific temperature, as has been proposed for waxy lipids. Gilby (1981) reviewed the effects of temperature on cuticular water loss and permeability. Even though there is a paucity of theoretical models, one fact is clear viz. that the rate of water loss increases with temperature. Saturation deficiency has long been considered the driving force of water loss (Wigglesworth, 1945), but more recent approaches have discounted saturation deficit in favour of chemical potential gradients based on irreversible thermodynamics theory (Toolson, 1978, 1980). This model has been used to investigate the transition or critical temperature at which cuticular permeability dramatically increases. Toolson’s results indicate that transition temperatures are artifacts of improper permeability calculation. Yokota (1979), however, concluded that the chemical potential gradient and its necessary conductivity coefficient do not appear to offer any benefits for analysis and that transition temperatures may not be artifacts. A kinetic approach was proposed and an Arrhenius plot (in cuticular water loss plotted by the reciprocal of the absolute temperature) used to detect changes in activation energy. A Istraight line relationship should indicate a cuticle that is not physically altered by temperature. An Arrhenius plot of the cuticular water loss data in Fig. 4 results in a straight line with an r* value of 0.97. A transition temperature would be particularly relevant if the animal encountered that but these temtemperature in its, environment, peratures are in the range of 35-37°C and are often lethal. Thus, although the transition phenomenon is of theoretical interest, it is probably not ecologically relevant. At a constant temperature, water loss, determined gravimetrically, decreased with increasing relative humidity, but calculated cuticular permeability increased. This discn:pancy is due to the division of cuticular water loss by the value of saturation deficiency with increasing humidity. Increasing tem-

627

perature increased cuticular permeability but it is unlikely that increasing relative humidity could accomplish the same thing. Results from the tritiated water experiment clearly show that actual cuticular permeability does not change with humidity. Similar results have been obtained with ticks (Kniille and Devine, 1972) and beetles (Cooper, 1983). Net weight loss is the difference between water-vapour efflux and influx. Efflux is constant over the humidity range whereas influx is significantly correlated with humidity. These data support the conclusion that saturation deficiency alone cannot be used to describe

permeability, but is adequate to describe net water loss. The regression lines of water-vapour efflux and influx should theoretically intersect at 100% r.h., indicating no net weight change (Cooper, 1983). Although the confidence intervals of the 2 regression lines intersect, male P. fuliginosa had a net loss of about 75mgg-‘day-’ at 100% r.h. House (1974) proposed that water flux at high humidity may be associated with thermo-osmosis, i.e. heat transfer increasing the kinetic energy of water molecules within the animal. Galbreath (1975) measured a 0.22”C difference between the cuticle of a soildwelling beetle and ambient temperature and calculated a chemical potential difference of 340 J kg-‘. He concluded thermoosmosis was the mechanism accounting for water loss at high soil water potentials. Cuticle temperatures of P. fuliginosa at 30°C and 100% r.h. were 0.3% higher than ambient and represent about a 480 J kg-’ (Appel, unpublished) water potential difference that drives water efflux at 100% r.h. Acknowledgements-This research was supported in part by a grant from the UCR Chancellor’s Patent Fund and UCR Academic Senate Intercampus Opportunity Fund. We thank Raloh B. March and Edward G. Platzer for their critical reviews and useful discussions.

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