Nitrogen Excretion in Nymphs of Aeshna Cyanea (Müll.) (Odonata, Anisoptera)

The present paper reports the results of a brief study on the excretion of ammonia and uric acid in nymphs of Aeshna cyanea. It is well known that terrestrial insects excrete waste protein nitrogen mainly in the form of uric acid. Among aquatic insects, however, a reversal to ammonia as the main end-product of protein catabolism appears to be widespread (Staddon, 1955), although some variation in nitrogen excretion is to be expected since the freshwater habitat has been colonized independently at the ordinal, and in some cases family, level. In some species it would not be surprising to find that waste protein nitrogen (a-amino-N) is in part excreted as ammonia and in part transformed into uric acid before expulsion. It was the primary object of the present work to clarify the position in nymphs of Aeshna.

This paper also includes some preliminary observations on nitrogen gain after feeding.

Nymphs of A. cyanea (Müll.) were collected from a small pond in Northumberland, near Newcastle upon Tyne. A few adults were bred from nymphs to ensure proper identification. I am greatly indebted to Dr E. T. Burtt of the Zoology Department, King’s College, Newcastle upon Tyne, for kindly arranging the collection and dispatch of living material when required.

Animals were starved in clean water at 20° C. for a period of 7-14 days before experiment. During this period unabsorbed food materials were eliminated from the gut.

(a) Collection of excreta. The method consisted in partly immersing a nymph in 2-5 ml. of an acetate buffer solution at pH 3·7 for a period of 24 hr. at 20° C. The stock solution contained 13·6 g. sodium acetate 3H₂O and 44 ml. acetic acid/100 ml. This solution was diluted 1/150 with distilled water before use.

The choice of an acetate buffer solution was determined by the need to prevent decomposition of the excreta during the period of collection. Using distilled water it is estimated that as much as 20 and 80%, respectively, of the total quantities of ammonia and uric acid excreted may be lost during a 24 hr. collecting period.

Decomposition of the excreta in distilled water is attributed to the presence of contaminating micro-organisms derived from the gut or body surface of the animal, although a small quantity of ammonia may be lost by diffusion into the air. The low pH of the acetate buffer solution both prevents loss of ammonia by diffusion and makes conditions unfavourable for micro-organisms; acetic acid has a disinfectant action independent of pH, a property of the whole molecule or anion (Wilson & Miles, 1946).

The efficacy of the acetate buffer solution was tested. Samples containing excreta, collected during a period of 24 hr., were cultured using a routine bacteriological technique. The samples appeared to be sterile (in contrast, washings taken from nymphs with sterile distilled water yielded heavy growths of bacteria). There was no detectable loss of ammonia in samples left for a further period of 24 hr. at 20° C. after the nymphs had been removed. Uric acid recoveries of over 90% were obtained in tests in which uric acid was added to the medium before insertion of the nymphs. Since the acetate buffer solution preserves the excreta and in no way appears to affect the vitality of nymphs which were at times immersed continuously for over a week, it was consequently considered entirely satisfactory for the purpose of the present investigation.

The elimination of urine from the gut appears to occur frequently. Measurements of the ammonia content of the medium, made at 90 min. intervals, rarely failed to reveal an increase in ammonia level. This emphasizes that daily measurements must approximate closely to the actual daily output of excreta, negligible error being incurred through retention in the gut.

The apparatus employed for the purpose of collecting excreta is shown in Fig. 1. The inner tube, of internal dimensions 11·4 × 1·4 cm., is made of ‘Pyrex’ and bears a 5·0 ml. graduation mark. In this tube the excreta are collected and later incinerated if a total nitrogen determination is required. After the introduction of 2·5 ml. of the buffer solution the nymph is secured in position with nichrome wire. One end of the wire is tied round the ‘waist’ of the animal, the other is folded over the mouth of the tube and gripped by an elastic band. The head of the nymph is directed towards the mouth of the tube. This prevents loss of fluid which may be voided forcibly from the rectum. The outer tube, approx. 20 × 2·5 cm., serves to keep the inner tube clean when immersed in a water-bath and also increases the volume of air available. The mouth of the outer tube is covered, first with a piece of sheet Polythene, secured with an elastic band, and then with the cut end of a rubber balloon to make it completely water-tight. The apparatus is inclined at an angle of 5-10⁰ from horizontal to make surface breathing possible.

At the end of the 24 hr. collecting period the inner tube is removed and clamped in a vertical position. The nymph is drawn half-way up the tube and then washed with water or with a solution of sodium carbonate anticipating uric acid estimations. The nymph is provoked to expel fluid retained in the rectum. The washing is then repeated. The nymph is now removed, the tube centrifuged to collect any fluid adhering to the sides, and the volume made up to 5·0 ml. with distilled water. Transfer of the nymph from one tube to another is effected with speed and without loss of fluid adhering to the body surface.

Faeces nitrogen was estimated separately. Since faeces are constrained within a peritrophic membrane they can be transferred intact to a clean tube by means of a hooked glass rod.

Parallel experiments were periodically carried out in which nymphs were absent.

(b) Analytical methods. Ammonia nitrogen was at first estimated by the diffusion/titration method of Shaw & Beadle (1949), and later by the diffusion/conductimetric method of Shaw & Staddon (1958). A comparison of results obtained by the diffusion/titration method with results obtained by the Nessler colorimetric method showed that ammonia was the only volatile base separated in the diffusion process.

Total nitrogen was estimated by a micro-Kjeldahl method. After removal of samples for ammonia analysis the fluid remaining was acidified, evaporated overnight, digested vigorously for a period of 1 hr. and then made up to 5·0 ml. with distilled water. The ammonia content was determined by one of the methods noted previously. For the ratio potassium sulphate/sulphuric acid in the digest mixture see McKenzie & Wallace (1954). Copper sulphate was used as catalyst. A mean recovery of 99·3% (min. 98; max. 101) was obtained in three recovery tests performed on a standard solution of ammonium sulphate. A mean recovery of 98·8% (min. 97; max. 99·4) was obtained in four tests on tryptophane. In estimating the total nitrogen content of the whole sample no correction was considered necessary for the very small quantity of fluid removed for ammonia analysis.

Uric acid was estimated by the method of Brown (1945) as modified by Dresel & Moyle (1950). Estimations were made in duplicate using 2 ml. samples. The procedure of Dresel & Moyle (1950) was used for extracting uric acid from whole nymphs. The method was tested in the manner described by these authors and was similarly found very satisfactory.

(c) Method of feeding. Nymphs were quantitatively fed on heat-coagulated eggwhite. The dry weight of egg-white is about 12% of the wet weight and the bulk of the dry material is in the form of protein. A 100 mg. (wet weight) contains approximately 1·7 mg. nitrogen. Samples were obtained from the middle thick white (Brooks & Taylor, 1955) and weighed to the nearest 0·5 mg. on a 500 mg. torsion balance. The Kjeldahl method was used to determine the nitrogen content.

Feeding was accomplished by hand under the low power of a binocular micro scope. The nymph is held in one hand, ventral side uppermost. With forceps held in the other hand a portion of the weighed sample of egg-white is inserted between the distal extremities of the labrum and labium. By stroking the edge of the labrum the animal is induced to feed. The egg-white is seized by the maxillae from whence it is transferred to the mandibles, briefly masticated then swallowed. Further pieces of egg-white are given to the animal until the whole sample has been ingested, care being taken to see that no small particles remain trapped behind the mouth under the labium.

(d) Collection of mid-gut contents. Unabsorbed food in the midgut is collected in the following way. The animal is killed by crushing the head and then dissected to expose the gut. The body cavity is washed with saline. Surplus fluid is then removed from the surface of the gut with filter-paper. The contents are exposed by making a small incision in the midgut wall, care being necessary to prevent damage to the peritrophic membrane. Gut contractions cause the contained mass to protrude through the incision. The midgut contents are now removed entire within the peritrophic membrane by gently seizing and pulling the protruding portion with blunt forceps.

Daily measurements were made to determine the ammonia nitrogen and total nitrogen output of nymphs for a period of 2 days during fasting and then for a further period of 3-5 days after feeding on egg-white (Table 1; Fig. 2).

The total nitrogen output prior to feeding ranged from 2–6 to 16–6 μg. (av. 7 μg. N)/100 mg. wet weight/24 hr. at 20° C. Of the total nitrogen excreted 41-91 % (av. 74%) was in the form of ammonia.

Feeding was followed by a large increase in nitrogen output which lasted for 1 or 2 days. The total nitrogen output measured at the end of the first day after feeding ranged from 17·7 to 46 /μg. (av. 32 /zg. N)/100 mg. wet weight. Of the total nitrogen excreted 78-96 % (av. 87 %) was in the form of ammonia.

The non-ammonia nitrogen output prior to feeding ranged from 0·6 to 5·2 /μg. (av. 1·8 μg. N)/100 mg. wet weight/24 hr-Measurements obtained at the end of the first day after feeding ranged from 0·7 to 9·8 (av. 3·9 μg. N)/ioo mg. wet weight. This increase is small when compared with the increased output of ammonia after feeding.

The results clearly show that ammonia is quantitatively the most important nitrogenous excretory substance in the excreta of nymphs, both during starvation and after feeding on egg-white.

It is evident from results shown in Tables 2 and 3 that uric acid is quantitatively a minor excretory product in nymphs of A. cyanea. The uric acid output of fasting nymphs ranged from 6·2 to 13·1% (av. 8%) of the total ammonia nitrogen and uric acid nitrogen output, and no increase in output was apparent after feeding on egg-white (Table 2). The uric acid content of whole nymphs (Table 3) ranged from 5·3 to 9·3 μg. N/100 mg. wet weight. These quantities are approximately equivalent to a 24 hr. output of ammonia nitrogen during starvation.

It is of interest here to note that Florkin & Duchâteau (1943) failed to find uricase in extracts of Aeshna nymphs thereby indicating that uric acid is not degraded before excretion. The results obtained in the present work are taken to confirm this.

Estimates have been obtained of the nitrogen gained by nymphs after feeding on egg-white.

That the nitrogen ingested is ultimately wholly absorbed is indicated by the fact that faeces expelled after feeding contain very little nitrogen (Table 1). It became apparent, however, that a portion of the egg-white ingested may remain unabsorbed in the midgut for some days after feeding, and that the elimination of faeces (and fall in nitrogen output after the post-feeding peak) may not necessarily be associated with complete absorption of the egg-white ingested. Consequently, the estimation of nitrogen gain over a short period necessitates an estimation of the nitrogen remaining unabsorbed in the gut at the end of the selected experimental period.

In Exp. 6 (Table 1) the nymph moulted on the sixth day after feeding and died, presumably due to asphyxiation since withdrawal of the abdomen from the old cuticle was precluded by the presence of the securing wire. The ingested protein appeared to have been completely absorbed since the gut was empty but for the presence in the midgut of a number of peritrophic sacs concentrically disposed one within the other. It can be calculated, therefore, that during the 6 days after feeding the nymph gained 270 μg. nitrogen, an amount equivalent to 40% of the nitrogen ingested and absorbed.

In Exp. 7 (Table 1) the nymph was killed and dissected 4 days after feeding. The foregut appeared empty, but the midgut was partially distended with semi liquid, homogeneous, pale yellow-brown contents, presumed to be partially digested egg-white (it is to be noted that faeces had already been expelled on the second and fourth days after feeding). The mass within the midgut was removed entire within the peritrophic membrane and estimated to contain 230 μg. nitrogen. From the figures included in Table 1 it can be calculated that, of the nitrogen ingested, 22% remained unabsorbed 4 days after feeding. Further, it can be calculated that the quantity of nitrogen gained during the 4-day period after feeding amounted to 239 μg., an amount equivalent to 26% of the total nitrogen absorbed.

Table 4 includes further data on nitrogen absorption, nitrogen gain and nitrogen excretion after feeding. The foregut in all cases appeared empty at the end of the experimental period but unabsorbed protein still remained in the midgut. In one case there was a small nitrogen deficit, in the remaining three cases the quantity of nitrogen gained ranged from 10 to 23 % of the total nitrogen absorbed.

The results presented in this paper affirm that ammonia is the main end-product of protein catabolism in nymphs of A. cyanea. Uric acid, a minor excretory product, is taken to originate solely from the breakdown of purines. The alternative possibility, that uric acid is in part derived synthetically from the a-amino-N of protein, is discounted on the grounds that output does not increase after feeding on a protein-rich diet. It is understood that measurements on the excreta give a true indication of output since measurements on extracts of whole nymphs revealed that very little retention is occurring.

Feeding is followed by a large, temporary increase in the amount of ammonia excreted. It has been estimated that during the 24-48 hr. period after feeding a quantity of nitrogen is excreted equivalent in amount to the greater part of the food nitrogen absorbed during that period. It would be of interest to take this aspect further since little is known about the factors influencing nitrogen excretion and retention in insects. For the purpose of investigating these problems Aeshna nymphs would appear to offer admirable material.