Use of a Ventilated Capsule and Tritiated Water to Measure Evaporative Water Losses in a Tenebrionid Beetle

Evaporative water losses (EWL) in arthropods have traditionally been measured by gravimetric methods (Loveridge, 1980), although electronic sensing of water has recently been employed (Hadley, Stuart & Quinlan, 1982; Hadley & Quinlan, 1982). Difficulties are encountered in separating the cuticular and respiratory components of total transpiration. Cuticular water loss has frequently been measured in freshly killed specimens with blocked spiracles, and the results subtracted from the total EWL of living animals to obtain values for respiratory transpiration. Unfortunately, however, cuticular transpiration is higher in dead than in living arthropods (e.g. Hadley, 1970) and is further augmented by the appreciable damage to the cuticular waterproofing caused by blocking the spiracles (Beament, 1959). An additional problem arises when surface areas are estimated from standard equations, in order to express cuticular EWL in units independent of body weight. Loveridge (1980) has suggested that such estimates of surface area may be as much as an order of magnitude too low. We describe here an alternative technique using tritiated water (THO) and ventilated capsules to determine transpiration rates across small, known areas of cuticle in living insects. Tenebrionid beetles were selected as experimental animals and, because of the anatomy of their respiratory system, our method has also proved suitable for direct measurement of water losses from the subelytral cavity. The beetles (Onymacris plana P6ringuey, mean weight 0-852g) were collected from sand dunes near Gobabeb, Namibia, and maintained on a diet of oats and lettuce. Before each experiment a weighed beetle was completely immobilized by covering the legs with adhesive tape, and injected with 20/ilTHO (specific activity 630 000 c.p.m. /il"), using a Hamilton syringe and 26 gauge needle. The injection site between the abdominal sternites was sealed immediately with a wax-resin mixture. An initial experiment established the time needed for equilibration of injected THO with total body water. Seven O. plana were injected with THO and 1-fA haemolymph samples were taken after 15min and then every hour for 7h. Fig. 1 shows that equilibration was complete 3 h after injection. Water loss experiments were therefore started after this equilibration period, by connecting the beetle to the apparatus shown in Fig. 2. Room air was pumped (Beckman Gas Sampling System Model Y102) at a constant rate of ISOmlmin" through the ventilated capsule attached to the cuticle of the insect. Flow rates were measured