J. Exp. Biol. (1963), 40, 563-571 563 Jtjth 4 text-figures ^^mted m Great Britain WATER BALANCE IN THE AQUATIC BUGS NOTONECTA GLAUCA L. AND NOTONECTA MARMOREA FABR. (HEMIPTERA; HETEROPTERA) BY B. W. STADDON Department of Zoology, University College of South Wales and Monmouthshire, Cardiff (Received 29 April 1963) INTRODUCTION Several workers have investigated mechanisms of water balance in aquatic insect larvae. An account of the mechanism of water balance in the mosquito larva Aedes aegypti L. will be found in papers by Wigglesworth (1933) and Ramsay (1950, 1951, 1953). Beadle (1939) and Ramsay (1950) give an account of the situation in the larva of Aedes detritus Edw. The alderfly larva Sialis lutaria L. has been studied by Shaw (1955a, b); Limnephilus affinis Curt, and other trichopterous larvae by Sutcliffe (1961a, b, 1962). Aquatic imagines have attracted little attention and the work to be described in this paper goes some way towards remedying this situation. MATERIAL AND METHODS The investigation was carried out on two closely related species of aquatic bug, Notonecta glauca L. and N. marmorea Fabr. The bugs were collected from ponds and ditches in Monmouthshire and Glamorgan. Both species occurred together in ponds lying very close to the sea; elsewhere N. marmorea appeared to be absent. N. marmorea is reported to occur mainly in brackish waters in coastal areas (Southwood & Leston, 1959), unlike N. glauca which appears to be confined to fresh waters, and it was hoped that a comparison of the two species would throw some light on these differences in distribution. Adult specimens varied a great deal in weight, but no attempt was made to obtain uniformity in this respect. Adults of N. glauca ranged in weight from about no to 150 mg. and adults of the somewhat smaller N. marmorea ranged from about 100 to 120 mg. To facilitate comparison uptake and output measurements have been calculated in terms of a standard body weight of 100 mg. In the laboratory the bugs were kept without food in separate containers until required, and the experiments were carried out at a temperature of about 180 C. Collection and analysis of rectal fluid. Measurements were made on the clear fluid voided from the anus of the intact bug. In order to obtain this fluid a bug was carefully dried on filter-paper and placed in a wax-lined dish. The specimen was gently secured in order to induce the ejection of fluid which was collected in a capillary pipette and analysed without delay. For the purpose of routine ammonia measurements bugs were narcotized with CO2 in order to induce the ejection of fluid from the rectum and this had the advantage that handling was reduced to a minimum. 36 Exp. Biol. 40, 3 564 B. W. STADDON Ammonium, bicarbonate and conductivity measurements were made in dupli^B or triplicate and 0-2 /xl. of fluid was used for each determination. Ammonium was measured by the diffusion method described by Shaw & Beadle (1949) and o-oi N-HC1 was used for the titration. In order to facilitate the mixing of the sample and alkali in the diffusion chamber a clear lid was employed and the absorbing acid was placed on the floor of the chamber. Bicarbonate was measured by direct titration of the sample with o-oi N-HC1 which contained 5 ml. of B.D.H. '4-5' indicator per 100 ml. A measure of the total ionic concentration was obtained by comparing the conductivity of samples diluted in about 44 (A. of de-ionized water in a Perepex conductivity cell (Shaw & Staddon, 1958) with the conductivity of standard solutions of ammonium bicarbonate prepared in the same way. Ammonium was measured with an average error of ± 2 mM./l., bicarbonate with an error of ± 4 mM./l. and conductivity measure- ments with an average error of ± 2 mM./l. Output of water in the rectal fluid. This was deduced from measurements of the rate of ammonia output and of the concentration of ammonia in the rectal fluid. Given the rate of ammonia output (in /*M./day) and the concentration of ammonia in the rectal fluid (in pM.//tl.) then division of one term by the other gives directly the output of water in the rectal fluid (in /xl./day). In order to measure the rate of ammonia output a bug was placed in 25 ml. of de-ionized water in a covered beaker. At the end of 24 hr. the ammonia content of the water was measured colorimetrically and for this purpose Nessler's reagent was used. This method of collecting ammonia appeared to be reliable as shown by the fact that on removing a bug at the end of the 24 hr. period the concentration of ammonia in the water underwent little or no change during a further period of 24 hr. Now the concentration of ammonia in the rectal fluid was measured by diffusion analysis and the output of ammonia was measured colorimetrically. Dissimilar methods of analysis may yield dissimilar results, and it was clearly necessary to check this possibility. To make this check several samples of rectal fluid were pooled and ana- lysed by both methods. The results which were obtained by the two methods did not differ significantly. Ingestion of water. The uptake of water into the gut was measured with the aid of amaranth. A specimen was placed in an o-oi M solution of the dye for a period of 8 hr. It was then removed, washed with water and narcotized with COj. The dye was extracted from the gut and for this purpose the method devised by Treherne (1957) was closely followed. The gut was dissected out in isotonic saline and then transferred to 5 ml. of phosphate buffer at pH 10. It was finely ground up with the aid of glass powder. The extract was centrifuged and the concentration of amaranth in the clear supernatant was measured with a Unicam absorptiometer at an absorption maximum of 525 m/i. A slight correction had to be made to each measurement to allow for the effect of soluble gut material on absorption. Given the quantity of dye in the gut (in m/xM.) and the concentration in the external medium (in m^.M.//il.) then division of one term by the other gives the volume of water (in /J.) ingested in the 8 hr. period. The procedure was tested by feeding individual bugs on a measured quantity of dye. To do this each bug was narcotized by means of CO2 and the proboscis was sealed into a small wax-lined tube with a warm mixture of beeswax and rosin. A quantity of Water balance in aquatic bugs 565 ^Raranth, 10 /A. of an o-oi M solution, was introduced into the tube which was then covered in order to reduce evaporation. The bug was left in air to consume the fluid overnight. The small traces of amaranth which remained on the sides of the tube were washed out and measured in order to obtain an accurate measure of the quantity actually ingested. The quantity of amaranth recovered from the gut was always over 97 % of that ingested which shows that there can be little, if any, absorption from the gut lumen. The reason for selecting an experimental period of 8 hr. was to avoid the loss of amaranth from the gut by evacuation in the rectal fluid. In 8 hr. the dye may have reached but not travelled beyond the pyloric valve; in 12 hr. the rectal fluid is frequently coloured with the dye. A more direct method was also used to measure the ingestion of water. This entailed sealing the proboscis into a capillary tube of about 1 mm. external diameter with the beeswax-rosin mixture. The tube was filled with water to a mark and then secured vertically so as to suspend the bug in water in its normal resting position just beneath the surface. The decreased volume of water in the tube was recorded 24 hr. later. This method had the advantage that simultaneous measurements of water output could be made. RESULTS (a) Excretion of ammonia. Ammonia is quantitatively the most important nitro- genous excretory product of adult bugs kept without food. Measurements of total-N and ammonia-N showed that approximately 75 % of the total-N in the rectal fluid of N. glauca is in this form. The rectal fluid is not a dilute one for the mean conductivity (92 mM./l.) of the measurements which are recorded in Table 1 is as much as 56 % of that of the haemolymph (165 mM./l. as equiv. NaCl) and the greater part of the ionic material is in the form of ammonium bicarbonate. A fluid of similar composition is produced by the larval SiaUs hitaria L. (Shaw, 19556). Table 1. The composition of the rectal fluid of Notonecta glauca Sample 1 a 3 Mean Conductivity as equiv. 107 90 78 9a ammonium bicarbonate soln. (mM./l.) Ammonium (mM./l.) 97 75 54 75 Bicarbonate (mM./l.) 84 78 62 75 The output of ammonia fluctuates a little from day to day as shown by the results of measurements which are recorded in Table 2. It is not known what causes these fluctuations in ammonia output but small fluctuations in the output of rectal fluid may be partly responsible. (b) Relationship between water output and ammonia output. In view of the fact that ammonia is the major end product of protein catabolism in Notonecta adults it is of interest to know whether the output of water in the rectal fluid is closely related to the output of ammonia.