Journal of Insect Physiology 44 (1998) 1225–1231 Aspects of excretion of antlion larvae (Neuroptera: myrmeleontidae) during feeding and non-feeding periods Amanda Van Zyl *1, M.C. Van Der Westhuizen, T.C. De K. Van Der Linde Department of Zoology-Entomology, University of the Free State, P.O. Box 339, Bloemfontein, 9300, South Africa Received 20 March 1998; received in revised form 15 June 1998 Abstract The main nitrogenous excretory products were determined for third instar Cueta sp. and Furgella intermedia larvae during periods of food abundance and for F. intermedia during starvation periods. Biochemical analysis indicated that allantoin was the main nitrogenous excretory product, with smaller quantities of ammonia, urea and uric acid. Respectively 9 and 13 amino acids of low concentrations (0.005–0.329 g/l) were detected by high pressure liquid chromatography in the excreta of Cueta sp. and F. intermedia larvae. The volume of urine produced and concentrations of the nitrogenous excretory products of fed Cueta sp. and fed F. intermedia larvae did not differ. F. intermedia excreted smaller volumes of urine and smaller quantities of nitrogenous excretory products during starvation than during periods of food abundance. Feeding conditions rather than the pitbuilding or non-pitbuilding lifestyles seem to be the major influence on the excretory products of these antlion larvae. 1998 Elsevier Science Ltd. All rights reserved. Keywords: Neuroptera; Myrmeleontidae; Excretion; Allantoin; Water 1. Introduction therefore be able to conserve water e.g. by excreting uric acid, during prolonged periods of food shortage. A strong correlation usually exists between the major On the other hand, antlion larvae are extra-intestinal nitrogenous excretory products and the nature of an digesters, i.e. they inject enzymes and probably ‘poison’ insect’s environment, where aquatic forms often excrete (Gaumont, 1976) into their prey, which dissolves the soft ammonia and terrestrial forms uric acid (Cochran, 1985). internal tissue of the prey. The fluid is then drawn out The antlion larvae, Cueta sp. and Furgella intermedia of the prey and into the alimentary canal of the antlion (Markl), live in the semi-arid to arid Kalahari desert larva. Van Zyl et al. (1997) demonstrated that third instar (28°21ЉE, 21°16ЉS), where food resources are unpredict- Cueta sp. larvae (body weight of 31 mg) extracted 73% able for these larvae (Van Zyl et al., 1996). As surface of the total wet weight of sixth instar Hodotermes mos- water is often absent in the natural environment of these sambicus (Hagen) larvae (body weight of 20 mg). antlion larvae, it has been suggested that food is their Antlion larvae ingest therefore large quantities of fluid primary water resource during the dry season (Van Zyl, during extra-intestinal digestion and a need exists for the 1995). Antlion larvae are also unable to take up atmos- storage or elimination of large quantities of excess fluid pheric water vapour as demonstrated for Myrmeleon after feeding. This was demonstrated in other fluid feed- medialis Banks, Cueta lanceolatus Navas and Syngenes ers, e.g. bloodsucking and plant-sucking insects where longicornis (Rambur)(Youthed, 1973). The pitbuilder an abundance of water is present after a meal and the Cueta sp. and non-pitbuilder F. intermedia larvae should urine voided is a crystal clear fluid (Wigglesworth, 1965). The study on the excretory products of antlion larvae is to a large extent facilitated in that the alimentary canal * Corresponding author. Fax: 00 27 12 3625242; e-mail: avanzyl@- is discontinuous (Lozı´nski, 1911; Van Zyl, 1995). No scientia.up.ac.za contamination by faeces occurs in the urine and the 1 Present address: 244 Carinus Street, Meyerspark, 0184, South excretory products are primarily of metabolic origin. Africa. Fax: (27 12) 3625242; E-mail: [email protected]. Unfortunately, only a limited number of studies have 0022–1910/98/$19.00 1998 Elsevier Science Ltd. All rights reserved. PII: S0022-1910(98)00100-0 1226 A. Van Zyl et al./Journal of Insect Physiology 44 (1998) 1225–1231 been conducted on the excretion of neuropteran species. were situated, it is unlikely that the prey carcass could Shaw (1955) and Staddon (1955) conducted an in depth have contaminated the excreta in any way. study on the ionic regulation of the larvae of the aquatic In the second treatment in total 16 F. intermedia lar- neuropteran, Sialis lutaria L. Ammonia is the main vae were each fed one termite of approximately 19 mg nitrogenous excretory product in these larvae. Razet once. These larvae are referred to as ‘starved larvae’. (1961, quoted by Bursell, 1967) demonstrated that uric The excreta was collected 10 times (on day 4, 5, 6, 8, acid dominated in the excreta of the antlion larva, Uro- 10, 12, 14, 18, 25 and 36) over a 36 d period and pooled leon nostras (Fourcroy), with smaller quantities of allan- for each individual. Presence of excreta was noted on toin present. Spiegler (1962b) observed urate storage in the collecting days. the larvae of the green lacewing, Chrysopa carnea The uric acid, allantoin, ammonia and urea concen- Steph. Of all these examples only U. nostras occurred trations were determined for the excreta of the fed and in a terrestrial environment comparable to that of Cueta starved antlion larvae, respectively. The volume of urine sp. and F. intermedia larvae. excreted by the antlion larvae was estimated by In the present study the main nitrogenous excretory determining the quantity of water that saturated a known products (uric acid, allantoin, urea, ammonia and amino weight of dry sterilised Kalahari sand viz. 0.1018 Ϯ acids) of Cueta sp. and F. intermedia larvae were ident- 0.017 ml of water/g dry sand (n ϭ 9). The dry weight ified. Excretion during periods of food abundance and of the collected pellets (urine mixed with sterile sand) food shortage was then related to survival in the semi- was then used to estimate the volume of urine excreted arid to arid Kalahari desert. by the antlion larvae. For analysis the collected dry excreta of fed larvae and of starved larvae were homogenised in 1.0 ml and 2. Materials and methods 0.5 ml of 0.6% Li2CO3, (pH 11.53) respectively. All nitrogenous products tested, are soluble in this subst- ance. The antlion excretory extract was heated for Third instar Cueta sp. and F. intermedia larvae were 10 min at 100°C, centrifuged for 10 min at 4 000 g and collected in the Kalahari desert and transferred to glass ° ϫ kept at 4 C. A sample volume of 0.1 ml of the super- vials (120 mm in depth 20 mm in diameter) filled to natant was used in all the determinations. a depth of 60 mm with sterilised sand from their natural Uric acid was determined by the method of Liddle et environment. Antlion larvae were acclimated to 28 Ϯ ° al. (1959) as summarised by Potrikus and Breznak 2 C with a diel cycle of 12:12 (L:D). Termites, sixth (1980). An assay cuvette contained 2 ml of 0.1 M gly- instar H. mossambicus larvae, were used as their food cine buffer (pH 9.4), 0.1 ml antlion excretory extract and supply. approximately 0.03 enzyme units (U) of purified uricase (hog liver, type V, Sigma). Uric acid (free acid, Sigma) 2.1. Uric acid, allantoin, ammonia and urea was used as standard. determinations The allantoin content was determined by the method of Borchers (1977). Allantoin was converted to allantoic Antlion larvae were starved for 42 days prior to the acid by dilute alkaline hydrolysis. This was done by experiments. Excretory products were collected from fed heating 0.1 ml antlion excretory extract with 0.25 ml of and starved larvae in two treatments. In the first treat- 0.6 M NaOH at 100°C for 12 min. The allantoic acid ment nine Cueta sp. (body weight 29.8 Ϯ 7.0 mg) and was hydrolysed by the addition of 0.5 ml of 0.1% 2,4- 14 F. intermedia larvae (body weight 52.9 Ϯ 10.0 mg) dinitrophenylhydrazine (crystalline, Sigma) dissolved in were each fed one termite (approx. 12 mg) every second 2 M HCL. Heating was continued for 4 min and the day over a 14 d period. These are referred to as ‘fed hydrazone of the resulting glyoxylic acid was formed. larvae’. The excreta of the individual antlion larvae and The tubes were cooled and alkalified with 2.5 ml 2.5 M the termite carcasses were collected at the next feeding NaOH. Absorbance was read at 520 nm after 11 min. and frozen at Ϫ 12°C. Presence of excreta and wet Standard solutions of 50 ␮M allantoin, 0.83 ␮M urea weight of termites before feeding were recorded every and 1.2 ␮M uric acid were used. The small interference second day. Excreta was thus collected on seven of the due to uric acid or urea could then be corrected for. 14 experimental days and pooled for each individual. Urea and ammonia were determined with the ultra- The liquid excreta forms pellets in the sterile sand. These violet method described by Anonymous (1980). An could be removed by sieving the sand through a 2 mm assay cuvette consisted of 1 ml of 0.5 M triethanolamine mesh screen. Prey carcasses were removed before siev- buffer (pH 8.6), 0.1 ml of 6 mM NADH, 1.9 ml distilled ing. As antlion larvae eject the prey carcass above the water and 0.1 ml antlion excretory extract. The enzymes soil surface within an hour after feeding and excreted L-glutamate dehydrogenase (0.2 mg, bovine liver type the urine several hours later in the sand, at a depth of III, Sigma) and urease (0.05 mg powder, Sigma) were 20 mm below the soil surface where the antlion larvae added.