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Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta
Experimental Parasitology 123 (2009) 17–23
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Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta H.V. Simpson a,*, N. Muhamad b, L.R. Walker a, D.C. Simcock c, S. Brown d, K.C. Pedley c a
Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand Faculty of Medicine and Health Sciences, UNIMAS, University Malaysia Sarawak (UNIMAS), 93150 Kuching, Sarawak, Malaysia Institute of Food, Nutrition & Human Health, Massey University, Private Bag 11-222, Palmerston North, New Zealand d School of Life Sciences, University of Tasmania, Locked Bag 1320, Launceston, Tasmania 7250, Australia b c
a r t i c l e
i n f o
Article history: Received 7 February 2009 Received in revised form 30 March 2009 Accepted 7 May 2009 Available online 20 May 2009 Keywords: Nematode Teladorsagia (Ostertagia) circumcincta Nitrogen excretion Ammonia
a b s t r a c t Excretion of nitrogenous substances by Teladorsagia circumcincta was investigated during incubation of L3 in phosphate buffer for up to 30 h and adult worms for 4–6 h. Ammonia was the main excretory product, with about 20% urea. For the first 4–6 h, ammonia excretion by L3 was temperature dependent, directly proportional to the number of larvae, but independent of the pH or strength of the phosphate buffer. Later, ammonia excretion slowed markedly in L3 and adults and reversed to net uptake in L3 by 30 h. An initial external ammonia concentration of 600 lM did not alter the pattern or magnitude of excretion. Re-uptake of ammonia did not occur at extremes of pH or low buffer strength and was slightly reduced at the highest external concentrations. Ammonium transporters and enzymes of glutamate metabolism, including glutamate dehydrogenase, glutamine synthetase and possibly glutamate synthase, are worthy of further investigation as anthelmintic targets. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Novel control measures are being sought to supplement or extend the life of currently used anthelmintic drenches to which nematode parasites of livestock are becoming increasingly resistant. Understanding the biology of these parasites has therefore taken on renewed importance in the search for new anthelmintic targets in the worms. Rapid growth and the demands of egg-laying must require very active energy and nitrogen metabolism in nematodes, yet surprisingly little is known about the sources of nitrogen, nutrient uptake, excretion and the key metabolic enzymes in species parasitising the sheep abomasum. Sources of nutrients for Teladorsagia circumcincta have not been clearly identified either for the free-living larval or parasitic stages. It is generally accepted that the free-living stages feed on microbes or their components in the sheep faeces. Transition to parasitism, first during development in the gastric gland lumen and subsequently in mucus overlying the abomasal mucosa (Sommerville, 1954; Armour et al., 1966), may require changes in both nutrient uptake and metabolism. Alternative sources of nitrogen for parasitic stages are mucins, epithelial cells or cell debris, substances in leaked interstitial fluid or components of the abomasal digesta. Nitrogen could be available as ammonia, urea or protein, or as peptides or amino acids after breakdown by the proteolytic enzymes present on the intestinal brush border and/or secreted by T. circumcincta (Young et al., 1995). Both proteases (Cox et al., 1990; * Corresponding author. Fax: +64 6 350 5636. E-mail address: [email protected] (H.V. Simpson). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.05.003
Redmond et al., 1997; Sajid and McKerrow, 2002) and glycosidases (Gamble and Mansfield, 1996; Irwin et al., 2004) have been identified in abomasal nematodes and their secretions. The nitrogenous compounds excreted by abomasal parasites are assumed to be similar to those excreted by other nematodes, but the relationship of these end-products to worm metabolism or to the changing local environment are largely unknown. The chemicals released during in vitro incubations are known as excretory/ secretory (ES) products and may be excreted metabolic end-products, components of the gut cells or cuticle (Rhoads et al., 2001), secretions from glands or material passing out of the intestine of adult worms (Rothstein, 1963; Wright, 1975; Wright and Newall, 1976). Adult worm ES products contain numerous proteins, e.g. incubates of adult Haemonchus contortus in RPMI contained in excess of 100 proteins on 2D gel electrophoresis (Yatsuda et al., 2003). Some of these may be involved in causing host pathophysiology, modifying the host response or supplying essential nutrients and have potential as protective vaccine candidates. Both free-living and parasitic nematodes predominantly excrete ammonia, with urea as a minor component not usually greater than about 20% of the total nitrogen excretion (Rogers, 1952; Rothstein, 1963, 1970; Wright, 1975). Significant amounts of amino acids are occasionally excreted and may result from stress, rather than reflecting changes in metabolism. Wright (1975) reported this to be the case for the free-living nematode Panagrellus redivivus. The present study addressed the excretion of nitrogenous substances of adult and L3 T. circumcincta during in vitro incubation under various conditions, particularly changing external pH and concentration of the medium. As expected from studies of other
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nematodes, ammonia was the main excreted compound, but an unexpected finding was the apparent re-uptake of ammonia during several hours of incubation in phosphate buffer. 2. Materials and methods All chemicals were supplied by Sigma Chemical Co. (Mo, USA). 2.1. Parasites L3 T. circumcincta were cultured from the faeces of sheep infected with a pure strain of parasite and stored at 4 °C in RO water until used. Prior to each experiment, L3 were Baemannised in RO water to remove inactive worms, counted and suspended in incubation medium. L3 were used sheathed or after exsheathing with sodium hypochlorite or CO2. L3 were incubated at 37 °C in 0.2% sodium hypochlorite for 15 min, filtered, washed and resuspended in medium. CO2 exsheathing was carried out at 40 °C by bubbling CO2 through the suspension for 20 s, shaking for 2 min and exsheathing overnight. Adult worms were recovered from the abomasa of infected sheep using the technique of Simpson et al. (1999). Briefly, abomasal contents were mixed 2:1 with 3% agar (Bacto agar, Difco Laboratories) and, after solidification, the agar blocks were incubated at 37 °C in a saline bath. Clumps of parasites were removed from the saline soon after emergence and placed in incubation medium. At the end of incubations, motility of the parasites was confirmed to be at least 95%. In longer incubations of 30 h, 15–20% of sheathed L3 had exsheathed, unrelated to the specific conditions of the incubation, but remained fully motile. No exsheathing occurred during shorter incubations up to 10 h. 2.2. Excretion of ammonia Parasites were suspended in 0.5 or 1 ml of phosphate buffer in a series of capped eppendorf tubes and incubated at 37 °C. At designated times, two of the tubes were removed, centrifuged briefly and the supernatant used to determine the concentration of ammonia or other product in the medium. The phosphate buffer in general use was either 100 mM or 0.8 mM sodium phosphate, pH 7.0 and the incubation temperature was 37 °C, except in specific experiments to test the effects of changing buffer concentration, pH or temperature. L3 were used routinely at a density of 50,000/ml and adult worms were weighed and incubated at 50– 60 mg wet wt/ml. Experiments were carried out in duplicate or triplicate. Specific experimental conditions were designed to evaluate the effects on ammonia excretion by sheathed L3: (1) Parasite density: sheathed L3 were incubated at densities of 5000, 10,000, 50,000, 70,000 or 100,000/ml in 0.8 mM phosphate buffer for 2.5 h (n = 3); (2) Temperature: incubated at 4 °C, 20 °C or 37 °C in 0.8 mM phosphate buffer for 5 h (n = 3); (3) Buffer concentration: incubated in phosphate buffer with concentration of 0.8, 50, 100 or 150 mM for 8 h and 30 h (n = 2); (4) pH: incubated in 100 mM phosphate buffer of pH 2, 4, 7 or 10 for 10 h or 30 h (n = 2); (5) External ammonia concentration: incubated in 100 mM phosphate buffer containing 0, 200 or 600 lM ammonium chloride for 30 h (n = 2); (6) Exsheathing: sheathed, sodium hypochlorite- or CO2exsheathed L3 from the same population were incubated in 100 mM phosphate buffer for 30 h (n = 2).
Ammonia excretion by adult worms (5–6 mg wet wt/ml) was monitored in 2 experiments: in Expt. 1, worms from the same population were incubated in either 0.8 mM or 100 mM phosphate buffer for 6 h (n = 2); in Expt. 2, the incubation was in 0.8 mM buffer for 9 h (n = 3). 2.3. Excretion of other compounds Urea excretion by sheathed or sodium hypochlorite-exsheathed L3 was determined by incubation in 0.8 mM phosphate buffer pH 7.0 for 4 h (n = 3). At each hour, four tubes were removed for urea determination (Section 2.4). Excretions of protein, uric acid and amino acids were monitored in adult worms (6 mg wet wt/ml) and sheathed and sodium hypochlorite-exsheathed L3. Parasites were incubated in 0.8 mM phosphate buffer pH 7.0 at 37 °C for 5 h. At hourly intervals, tubes were removed, centrifuged and the supernatant used for assay of protein, uric acid and amino acids. 2.4. Assays Ammonia concentrations were determined by reacting ammonia with hypochlorite and phenol to produce indophenols, which were monitored spectrophotometrically at 635 nm (Bolleter et al., 1961). The pH of all samples was adjusted to pH 7 before assay. Protein concentrations were determined by the method of Bradford (1976). Uric acid concentration was determined enzymatically by measuring the absorption at 290 nm with and without the addition of 1 U of uricase (Sasaki et al., 1996). Urea concentration was estimated enzymatically using commercial urease. Duplicate 0.5 ml samples, with and without added 1 U urease, were incubated for 30 min at 37 °C, followed by estimation of ammonia concentration in the tube. Urea concentrations were determined from the differences in ammonia concentrations with and without urease. Total amino acids were determined by the ninhydrin method (Magne and Larher, 1992). 3. Results 3.1. Ammonia excretion by L3 T. circumcincta Accumulation of ammonia in phosphate buffer during short (2.5 h) incubations was directly proportional to the number of L3 T. circumcincta per ml of medium (Fig. 1a). Ammonia excretion was also temperature dependent, being very low at 4 °C and greater at 37 °C than at 20 °C (Fig. 1b). There were no consistent effects of either buffer concentration or pH on excretion in shorter experiments (Fig. 1c and d), although differences developed in longer experiments. Ammonia excretion was a little lower in 150 mM buffer in some experiments, such as in the initial period illustrated in Fig. 2. A feature of many experiments was a marked decline in the ammonia concentration in the medium from about 10 h onward (Fig. 2). Exceptions were incubations in 0.8 mM phosphate buffer or in 100 mM buffer at pH other than pH 7, in which the ammonia concentration remained largely unchanged from 10 to 30 h of incubation. Sheathed L3 were also incubated in 100 mM phosphate buffer containing NH4Cl up to 600 lM (Fig. 3). The increment in ammonia concentration above the initial value was similar for the three media, reaching concentrations of 0.6–1.2 mM. After 30 h, the fall in ammonia concentration was about half as great when the initial concentration was 600 lM than in the other two buffers. Excretion of ammonia by L3 exsheathed either with CO2 or sodium hypochlorite was compared with that of sheathed L3 (Fig. 4). The most notable difference was the continuing excretion of
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Fig. 1. Concentrations of ammonia (lM) in phosphate buffer during incubation of sheathed L3 T. circumcincta (a) effect of varying the density of larvae in 0.8 mM phosphate buffer pH 7 during incubation at 37 °C for 2.5 hours; (b) effect of varying the temperature of incubation in 0.8 mM phosphate buffer pH 7: j: 4 °C; d: 20 °C; N: 37 °C; (c) effect of varying the concentration of phosphate buffer pH 7 during incubation at 37 °C: j: 50 mM; N: 100 mM; h: 150 mM; (d) effect of varying the pH of 100 mM phosphate buffer during incubation at 37 °C: j: pH 2; d: pH 4; N: pH 7; h: pH 10.
ammonia into the medium by hypochlorite-exsheathed larvae, contrasting with falling concentrations in the incubation medium of sheathed and CO2-exsheathed L3. 3.2. Ammonia excretion by adult worms Adult worms excreted ammonia into phosphate buffer over a similar time course to L3 (Fig. 5). In one experiment in which worms from the same population were incubated in either 0.8 or 100 mM buffer, buffer strength had no marked effect on ammonia excretion, whereas excretion into 0.8 mM buffer was greater in a second experiment with a different adult worm population. In both cases, there was a noticeably reduced rate of excretion after 4–6 h. 3.3. Excretion of other nitrogenous substances Sheathed L3 increased the urea concentration of the medium to 30 lM over 4 h of incubation (compared with 120 lM ammonia), whereas sodium hypochlorite-exsheathed L3 failed to excrete urea. Adult worms increased the protein content of the buffer much more than either sheathed or exsheathed L3: after 4 h, the protein concentration was 20 lg/ml for adult worms, 1–2 lg/ml for sheathed L3 and undetectable in the buffer in which the exsheathed larvae were incubated. No amino acids or uric acid were detected in media in which adult worms or sheathed L3 had been incubated. 4. Discussion
Fig. 2. Concentrations of ammonia (lM) in the medium during incubation of sheathed L3 T. circumcincta in phosphate buffer at 37 °C for 30 hours: (upper) effect of varying the concentration of phosphate buffer pH 7: d: 0.8 mM; j: 50 mM; N: 100 mM; h: 150 mM (lower) effect of varying the pH of 100 mM phosphate buffer: j: pH 2; d: pH 4; N: pH 7; h: pH 10.
Ammonia, urea and uric acid are the major nitrogenous excretory products of animals, the principal one depending upon the animal’s environment (Wright, 1995; Singer, 2003). Aquatic animals, both vertebrate and invertebrate, generally excrete ammonia and smaller amounts of urea, as did T. circumcincta incubated in
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Fig. 3. Effect of external ammonia on the excretion of ammonia into the medium during incubation of sheathed L3 T. circumcincta in 100 mM phosphate buffer pH 7 at 37 °C for 30 hours. The concentrations of ammonia (lM) are expressed as increments above the starting concentration of: j: 0 lM; h: 200 lM; d: 600 lM.
Fig. 4. Effect of exsheathing on concentrations of ammonia (lM) in the medium during incubation of L3 T. circumcincta in 100 mM phosphate buffer pH 7 at 37 °C for 30 hours: j: exsheathed with hypochlorite; h: exsheathed with CO2; N: sheathed.
Fig. 5. Concentrations of ammonia (lM) in the medium during incubation of adult T. circumcincta in phosphate buffer pH 7 at 37 °C: j: expt 1, 0.8 mM; h: expt 1, 100 mM; d: expt 2, 100 mM.
phosphate buffer in the present experiments. Rogers (1952), Rothstein (1963, 1970) and Wright (1975) have previously reported similar excretion of ammonia and 20% urea in several free-living
and parasitic nematodes at different life-cycle stages. The source of the ammonia in non-nutrient medium is likely to be the catabolism of amino acids, which principally occurs by transamination, followed by deamination by glutamate dehydrogenase (GDH) or by release of ammonia from glutamate by glutaminase. Other enzymes producing ammonia, including adenylate kinase and serine dehydratase, make appreciable contributions in some invertebrates (Fellows and Hird, 1979). Ammonia can also be generated by many other enzymes, such as asparaginase, which has been identified in the cuticle of Dirofilaria immitis (Tsuji et al., 1999). Urea is unlikely to be produced by the ornithine-urea cycle, as all enzymes are rarely present or only with low activity in helminths (Janssens and Bryant, 1969; Grantham and Barrett, 1986; Mohamed et al., 2005), but instead generated by arginase activity (Campbell, 1963; Senft, 1966). The pattern of excretion of ammonia consistently formed three phases, although quantitatively the amount excreted varied somewhat between parasite batches and experimental days. During the first phase lasting for 4–6 h, the duration of short-term experiments, ammonia accumulated in the incubation medium and excretion was unaffected by external pH and usually also not by osmolarity. The second phase from 6 to 8–10 h typically showed little further net excretion of ammonia, after which there was a progressive net loss of ammonia from the medium in the third phase. Delayed net uptake was sensitive to osmotic and pH gradients between worm and medium and occurred only where these were not extreme. These excretory patterns suggest that parasite metabolism first generates ammonia which passes into the incubation medium, followed by a change in metabolism consistent with ammonia uptake and incorporation under favourable conditions. Short-term excretion of ammonia by sheathed L3 T. circumcincta was linearly related to worm density and was temperature dependent, being very low at 4 °C and much greater at 37 °C than at 20 °C (Fig. 1), due probably to faster metabolic activity at higher temperatures. Exsheathing L3 did not appear to affect their initial ammonia excretion (Fig. 4). During incubations of 8–10 h, the pH of the medium had no apparent effects on ammonia excretion and only high osmotic pressure (150 mM) sometimes affected excretion. As adult worms do not survive as well in vitro as do L3 (Lawton et al., 2002), they were incubated only for short periods, during which they remained actively motile. Ammonia excretion followed a similar time course to that seen in L3, slowing markedly after 4– 6 h of incubation. The final concentration of ammonia was approximately half of that in L3 incubates. While there is probably no reliable way of comparing accurately excretory rates of different lifecycle stages using either numbers of individuals or weight of protein, marked changes in the ratios of different products can readily be seen. A striking difference was apparent between the relative accumulations of ammonia and protein in incubates of 5–6 mg adult worms and 50,000 L3: the adult worms excreted about half as much ammonia as did L3, whereas the protein concentration was 20-fold higher for adult worms. There was no measurable excretion of either uric acid or amino acids by L3 and the small protein loss from the non-feeding, sheathed L3 is likely to result from shedding of cuticular components or secretion. Lack of amino acid excretion over five hours of incubation suggests that the parasites were not stressed by the experimental conditions for that time, based on the conclusions of Wright (1975) that the appearance of amino acids in the medium in which P. redivivus was being incubated was related to environmental stress. Adult worms released greater amounts of protein into the incubation medium (mean 20 lM after 4 h), compared with 1 lM by sheathed L3 and negligible amounts by bleachexsheathed L3. Nematode ES products typically contain numerous proteins, e.g. more than 100 in ES products of adult H. contortus (Yatsuda et al., 2003). Many of these are likely to be of secretory
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origin, rather than excreted, or cuticular components, such as the collagen released by Ascaris suum during development from L3 to L4 in vitro (Rhoads et al., 2001). Longer incubations of L3 for 30 h revealed the different phases and changing rate of excretion with time and delayed effects of buffer concentration and pH on ammonia excretion (Figs. 2 and 3). The most favourable of the media used for the collection of excretory products of L3 would be expected to be 50–100 mM phosphate buffers of pH 7, although they do not supply nutrients required for the increased metabolic rate at 37 °C. In these less stressful environments, the pattern of ammonia excretion was an initial rapid increase, slowing to close to zero net excretion for several hours to 8–10 h, after which there was a progressive net loss of ammonia from the medium. Short-term experiments (Fig. 1) also showed this early excretory pattern and the start of a decline in the ammonia concentration from about 10 h onward (Fig. 2). In contrast, this late decline in external ammonia concentration did not occur in incubations in 0.8 mM phosphate buffer or in 100 mM buffer at pH other than pH 7, in all of which the ammonia concentration remained largely unchanged from 10–30 h of incubation. The enzymes responsible for ammonia incorporation, most likely glutamine synthetase (GS), may be less active under these conditions. To investigate whether the excretion of ammonia was limited or influenced by an existing ammonia gradient across the L3 cuticle, sheathed L3 were also incubated in 100 mM phosphate buffer containing up to 600 lM NH4Cl (Fig. 3). The pattern of excretion was no different over 30 h of incubation, except for a small reduction in the rate of ammonia loss during the last 15 h. During the first 15 h, the increments in ammonia concentration were similar for the three media, suggesting that the slowing of excretion after the fist few hours is not caused by accumulated ammonia but related to metabolic activity of the parasites of ammonia in the medium. The independence of the initial ammonia excretion from the transcuticular gradient also suggests that it does not occur through simple diffusion, which might be expected to be reduced by a high external concentration. Ammonia can cross membranes either by diffusion of the unprotonated NH3 form or by uptake of NH4+ by specific transporters (Kleiner, 1981). Although only 1% is present as ammonia at physiological pH, the conversion of NH4+ to NH3 is instantaneous and is not rate limiting for excretion. Ammonium transporters are present in all classes of organisms (Howitt and Udvardi, 2000; Williams and Miller, 2001; Javelle et al., 2003; Khademi et al., 2004), including Caenorhabditis elegans, which has four homologues of high affinity ammonium transporters in its genome (Howitt and Udvardi, 2000). The apparent re-uptake of ammonia under some conditions is particularly interesting in the contexts of organisms using excreted ammonia as a buffering substance in acid conditions or alternatively absorbing ammonia to use as a possible nutrient source. While it is most likely that the fall in ammonia concentration is due to uptake by the L3, other causes, such as microbial uptake or loss to the environment are possible, but unlikely. There was no visible evidence of microbial contamination and the similarity of time course and rate of fall in concentration supports an active rather than a passive process. In addition, the rate of decline was slower when the maximum concentration was 1.2 mM than 0.6 mM, contrary to what would be expect if ammonia were being released into environment. Ammonia (or ammonium) transport into the L3 would seem to be the most likely cause of the plateau and subsequent decline in ammonia concentration with time. Many pathogenic organisms generate ammonia to counteract acidic conditions (Audia et al., 2001; Cotter and Hill, 2003), including the gastric pathogen Helicobacter pylori, which uses urease to liberate ammonia from urea (Mobley and Hausinger, 1989; Még-
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raud et al., 1992). The lack of reuptake of ammonia at pH 2 and 4 by T. circumcincta L3 could be evidence of such use of ammonia to buffer an acid environment (Fig. 2), as normally present in the uninfected abomasum (Lawton et al., 1996). The parasitic stages of T. circumcincta are not tolerant of low pH in vitro (Lawton et al., 2002), so to survive in vivo, they must avoid exposure to acid, either by protecting themselves with gastric mucus, inhibition of parietal cells (Merkelbach et al., 2002) or perhaps through locally generating ammonia. Active buffering of external acid by ammonia may not be occurring, since in the present experiments, the ammonia concentration also remained high at pH 10. Lack of reuptake of ammonia in either low or high pH may indicate low GS activity at non-optimal pH, rather than a specific effect of external acidity. Prolonged exposure to extreme osmotic gradients, e.g. in 0.8 mM phosphate buffer, also impaired uptake and metabolism of ammonia. The relationship of these phases of ammonia excretion to parasite metabolism is interesting, particularly the apparent equilibrium after 4–6 h and subsequent uptake of ammonia from nonnutrient medium after reaching a maximum concentration of only 0.6–1.2 mM. It suggests that ammonia is produced from body protein or amino acids over the first few hours followed by a period when excreted ammonia becomes a source of nitrogen as enzymes capable of assimilating and metabolising ammonia are upregulated. Ammonia may therefore either be absorbed or excreted according to availability of different nitrogenous compounds as nutrients and the breakdown of stored material. Within the sheep, T. circumcincta could absorb ammonia from the different fluids to which they may be exposed, including abomasal fluid, interstitial fluid or fluid present in the mucus gel. In sheep, ammonia concentrations have been reported as around 5 mM in rumen fluid, 1 mM in abomasal fluid (Harrop, 1974; Harrop and Phillipson, 1974), 200 lM in arterial blood and 487 lM in portal blood (Parker et al., 1995). The values for abomasal fluid are similar to those reached when there is a pre-existing ammonia content in the incubation medium (Fig. 3), supporting the possibility of in vivo ammonia absorption and incorporation into amino acids by parasitic stages of T. circumcincta. Ammonia is assimilated principally into glutamate, either by the universal enzyme GDH or by the glutamine synthetase (GS)glutamate synthase (GOGAT) pathway, which is present in a few invertebrates, but not generally in animals. GDH activity in the aminating direction is usually limited by a high Km for ammonium, e.g. the Km for ammonium of H. contortus GDH is 42 mM (Rhodes and Ferguson, 1973). As the GS-GOGAT pathway requires ATP, but has a higher affinity for ammonia, is the preferred pathway in plants and prokaryotes if ATP is readily available (Helling, 1994, 2002). GOGAT activity has been identified in the mosquito Aedes aegypti (Scaraffia et al., 2005), the silkworms Bombyx mori (Hirayama et al., 1998) and Samia cynthia ricini (Osanai et al., 2000) and in the Spodoptera frugiperda Sf9 insect cell line (Doverskog et al., 2000). Gene sequences for NADH-GOGAT are present in the genome of C. elegans (Vanoni and Curti, 1999) and when more nematode species are investigated, GOGAT may be found to be more widespread in nematodes. In summary, these experiments suggest that excretory rates may be very low in dormant L3 or rapidly growing and reproducing parasitic stages of nematodes and that ammonia may either absorbed or excreted according to the external concentration and metabolic requirements for nitrogen. In the non-feeding L3, all excretion is via the cuticle or specialised excretory cell (Buechner et al., 1999; Buechner, 2002), whereas in adult worms, both cuticular and intestinal exchange is likely to contribute to excreted urea and ammonia. In relation to overall nitrogen metabolism, potential targets in the parasites are likely to be found in ammonium transporters or enzymes of glutamate metabolism. Trans-
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porters may be similar to the high affinity ammonia permeases of plants, bacteria and yeasts which scavenge ammonia from the environment and recover ammonia lost by diffusion when growing on sources of nitrogen other than ammonia (Marini et al., 1997). Glutamate metabolism is likely to be central to generating ammonia (Bidigare and King, 1981; Batrel and Le Gal, 1984) as well as assimilating it via GDH or GS-GOGAT. Acknowledgments We are grateful for the financial support of Meat and Wool New Zealand and the E. and C. Thoms Bequest. The Faculty of Medicine and Health Sciences, UNIMAS is thanked for personal support for N. Muhamad. References Armour, J., Jarrett, W.F.H., Jennings, F.W., 1966. Experimental Ostertagia circumcincta infections in sheep: development and pathogenesis of a single infection. American Journal of Veterinary Research 27, 1267–1278. Audia, J.P., Webb, C.C., Foster, J.W., 2001. Breaking the acid barrier: an orchestrated response to proton stress by enteric bacteria. International Journal of Medical Microbiology 291, 97–106. Batrel, Y., Le Gal, Y., 1984. Nitrogen metabolism in Arenicola marina characterization of a NAD dependent glutamate dehydrogenase. Comparative Biochemistry and Physiology 78B, 119–124. Bidigare, R.R., King, F.D., 1981. The measurement of glutamate dehydrogenase activity in Praunus flexuosus and its role in the regulation of ammonium excretion. Comparative Biochemistry and Physiology 70B, 409–413. Bolleter, W.T., Bushman, C.J., Tidwell, P.W., 1961. Spectrophotometric determination of ammonia as indophenol. Analytical Chemistry 33, 592–594. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Buechner, M., 2002. Tubes and the single C. elegans excretory cell. Trends in Cell Biology 12, 479–484. Buechner, M., Hall, D.H., Bhatt, H., Hedgecock, E.M., 1999. Cystic canal mutants in Caenorhabditis elegans are defective in the apical membrane domain of the renal (excretory) cell. Developmental Biology 214, 227–241. Campbell, J.W., 1963. Urea formation and urea cycle enzymes in the cestode, Hymenolepsis diminuta. Comparative Biochemistry and Physiology 8, 13–27. Cotter, P.D., Hill, C., 2003. Surviving the acid test: responses of gram-positive bacteria to low pH. Microbiology and Molecular Biology Reviews 67, 429–453. Cox, G.N., Pratt, D., Hageman, R., Boisvenue, R.J., 1990. Molecular cloning and primary sequence of a cysteine protease expressed by Haemonchus contortus adult worms. Molecular and Biochemical Parasitology 41, 25–34. Doverskog, M., Jacobsson, U., Chapman, B.E., Kuchel, P.W., Häggström, L., 2000. Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective 1H/15N NMR in vitro assay. Journal of Biotechnology 79, 87–97. Fellows, F.C.I., Hird, F.J.R., 1979. Nitrogen metabolism and excretion in the freshwater crayfish Cherax destructor. Comparative Biochemistry and Physiology 64B, 235–238. Gamble, H.R., Mansfield, L.S., 1996. Characterization of excretory–secretory products from larval stages of Haemonchus contortus cultured in vitro. Veterinary Parasitology 62, 291–305. Grantham, B.D., Barrett, J., 1986. Amino acid catabolism in the nematodes Heligmosomoides polygyrus and Panagrellus redivivus 1. Removal of the amino group. Parasitology 93, 481–493. Harrop, C.J.F., 1974. Nitrogen metabolism in the ovine stomach 4. Nitrogenous components of the abomasal secretions. Journal of Agricultural Science 83, 249– 257. Harrop, C.JF., Phillipson, A.T., 1974. Nitrogen metabolism in the ovine stomach 3. Urea in the abomasal secretions. Journal of Agricultural Science 83, 237– 247. Helling, R.B., 1994. Why does Escherichia coli have two primary pathways for synthesis of glutamate? Journal of Bacteriology 176, 4664–4668. Helling, R.B., 2002. Speed versus efficiency in microbial growth and the role of parallel pathways. Journal of Bacteriology 184, 1041–1045. Hirayama, C., Saito, H., Konno, K., Shinbo, H., 1998. Purification and characterization of NADH-dependent glutamate synthase from the silkworm fat body (Bombyx mori). Insect Biochemistry and Molecular Biology 28, 473–482. Howitt, S.M., Udvardi, M.K., 2000. Structure, function and regulation of ammonium transporters in plants. Biochimica et Biophysica Acta 1465, 152–170. Irwin, J.A., Morrissey, P.E.W., Ryan, J.P., Walshe, A., O’Neill, S.M., Carrington, S.D., Matthews, E., Fitzpatrick, E., Mulcahy, G., Corfield, A.P., Dalton, J.P., 2004. Glycosidase activity in the excretory–secretory products of the liver fluke, Fasciola hepatica. Parasitology 129, 465–472. Janssens, P.A., Bryant, C., 1969. The ornithine-urea cycle in some parasitic helminths. Comparative Biochemistry and Physiology 30, 261–272.
Javelle, A., Andre, B., Marini, A.-M., Chalot, M., 2003. High-affinity ammonium transporters and nitrogen sensing in mycorrhizas. Trends in Microbiology 11, 53–55. Khademi, S., O’Connell, J., Remis, J., Robles-Colmenares, Y., Miercke, L.J.W., Stroud, R.M., 2004. Mechanism of ammonia transport by Amt/MEP/Rh: structure of 0 AmtB at 1.35 Å A. Science 305, 1587–1594. Kleiner, D., 1981. The transport of NH3 and NH4+ across biological membranes. Biochimica et Biophysica Acta 639, 41–52. Lawton, D.E.B., Reynolds, G.W., Hodgkinson, S.M., Pomroy, W.E., Simpson, H.V., 1996. Infection of sheep with adult and larval Ostertagia circumcincta: effects on abomasal pH and serum gastrin and pepsinogen. International Journal for Parasitology 26, 1063–1074. Lawton, D.E.B., Wigger, H., Simcock, D.C., Simpson, H.V., 2002. Effect of Ostertagia circumcincta excretory/secretory products on gastrin release in vitro. Veterinary Parasitology 104, 243–255. Magne, C., Larher, F., 1992. High sugar content of extracts interferes with colorimetric determination of amino acids and free proline. Analytical Biochemistry 200, 115–118. Marini, A.M., Soussi-Boudekou, S., Vissers, S., Andre, B., 1997. A family of ammonium transporters in Saccharomyces cerevisiae. Molecular and Cellular Biology 17, 4282–4293. Mégraud, F., Nemam-Simha, V., Brugmann, D., 1992. Further evidence of the toxic effect of ammonia produced by Helicobacter pylori urease on human epithelial cells. Infection and Immunity 60, 1858–1863. Merkelbach, P., Scott, I., Khalaf, S., Simpson, H.V., 2002. Excretory/secretory products of Haemonchus contortus inhibit aminopyrine accumulation by rabbit gastric glands in vitro. Veterinary Parasitology 104, 217– 228. Mobley, H.L.T., Hausinger, R.P., 1989. Microbial ureases: significance, regulation, and molecular characterization. Microbiological Reviews 53, 85–108. Mohamed, S.A., Fahmy, A.S., Mohamed, T.M., Hamdy, S.M., 2005. Urea cycle of Fasciola gigantica: purification and characterization of arginase. Comparative Biochemistry and Physiology 142B, 308–316. Osanai, M., Okudaira, M., Naito, J., Demura, M., Asakura, T., 2000. Biosynthesis of Lalanine, a major amino acid of fibroin in Samia cynthia ricini. Insect Biochemistry and Molecular Biology 30, 225–232. Parker, D.S., Lomax, M.A., Seal, C.J., Wilton, J.C., Lomax, M.A., Seal, C.J., Wilton, J.C., 1995. Metabolic implications of ammonia production in the ruminant. Proceedings of the Nutrition Society 54, 549–563. Redmond, D.L., Knox, D.P., Newlands, G.F., Smith, W.D., 1997. Molecular cloning and characterisation of a developmentally regulated putative metallopeptidase present in a host protective extract of Haemonchus contortus. Molecular and Biochemical Parasitology 85, 77–87. Rhodes, M.B., Ferguson, D.L., 1973. Haemonchus contortus: enzymes III glutamate dehydrogenase. Experimental Parasitology 34, 100–110. Rhoads, M.L., Fetterer, R.H., Urban, J.F., 2001. Cuticular collagen synthesis by Ascaris suum during development from third to fourth larval stage: identification of a potential chemotherapeutic agent with a novel mechanism of action. Journal of Parasitology 87, 1144–1149. Rogers, W.P., 1952. Nitrogen catabolism in nematode parasites. Australian Journal of Scientific Research B 5, 210–222. Rothstein, M., 1963. Nematode biochemistry—III. Excretion products. Comparative Biochemistry and Physiology 9, 51–59. Rothstein, M., 1970. Nitrogen metabolism in the aschelminthes. In: Campbell, J.W. (Ed.), Comparative Biochemistry of Nitrogen Metabolism. Academic Press, New York, pp. 91–102. Sajid, M., McKerrow, J.H., 2002. Cysteine proteases of parasitic organisms. Molecular and Biochemical Parasitology 120, 1–21. Sasaki, T., Kawamura, M., Ishikawa, H., 1996. Nitrogen recycling in the brown planthopper, Nilaparvata lugens: involvement of yeast-like endosymbionts in uric acid metabolism. Journal of Insect Physiology 42, 125–129. Scaraffia, P.Y., Isoe, J., Murillo, A., Wells, M.A., 2005. Ammonia metabolism in Aedes aegypti. Insect Biochemistry and Molecular Biology 35, 491– 503. Senft, A.W., 1966. Studies in arginine metabolism by schistosomes—I. Arginine uptake and lysis by Schistosoma mansoni. Comparative Biochemistry and Physiology 18, 209–216. Simpson, H.V., Simpson, B.H., Simcock, D.C., Reynolds, G.W., Pomroy, W.E., 1999. Abomasal secretion in sheep receiving adult Ostertagia circumcincta that are prevented from contact with the mucosa. New Zealand Veterinary Journal 47, 20–24. Singer, M.A., 2003. Do mammals, birds, reptiles and fish have similar nitrogen conserving systems? Comparative Biochemistry and Physiology 134B, 543– 558. Sommerville, R.I., 1954. The histotrophic phase of the nematode parasite, Ostertagia circumcincta. Australian Journal of Agricultural Research 5, 130–140. Tsuji, N., Morales, T.H.V., Ozols, V., Carmody, A.B., Chandrashekar, R., 1999. Identification of an asparagine amidohydrolase from the filarial parasite Dirofilaria immitis. International Journal for Parasitology 29, 1451–1455. Vanoni, M.A., Curti, B., 1999. Glutamate synthase: a complex iron–sulfur flavoprotein. Cellular and Molecular Life Sciences 55, 617–638. Williams, L.E., Miller, A.J., 2001. Transporters responsible for the uptake and partitioning of nitrogenous solutes. Annual Review of Plant Physiology and Plant Molecular Biology 52, 659–688.
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Yatsuda, A.P., Krijgsveld, J., Cornelissen, A.W.C.A., Heck, A.J.R., de Vries, E., 2003. Comprehensive analysis of the secreted proteins of the parasite Haemonchus contortus reveals extensive sequence variation and differential immune recognition. Journal of Biological Chemistry 278, 16941–16951. Young, C.J., McKeand, J.B., Knox, D.P., 1995. Proteinases released in vitro by the parasitic stages of Teladorsagia circumcincta, an ovine abomasal nematode. Parasitology 110, 465–471.
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Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta H.V. Simpson a,*, N. Muhamad b, L.R. Walker a, D.C. Simcock c, S. Brown d, K.C. Pedley c a
Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand Faculty of Medicine and Health Sciences, UNIMAS, University Malaysia Sarawak (UNIMAS), 93150 Kuching, Sarawak, Malaysia Institute of Food, Nutrition & Human Health, Massey University, Private Bag 11-222, Palmerston North, New Zealand d School of Life Sciences, University of Tasmania, Locked Bag 1320, Launceston, Tasmania 7250, Australia b c
a r t i c l e
i n f o
Article history: Received 7 February 2009 Received in revised form 30 March 2009 Accepted 7 May 2009 Available online 20 May 2009 Keywords: Nematode Teladorsagia (Ostertagia) circumcincta Nitrogen excretion Ammonia
a b s t r a c t Excretion of nitrogenous substances by Teladorsagia circumcincta was investigated during incubation of L3 in phosphate buffer for up to 30 h and adult worms for 4–6 h. Ammonia was the main excretory product, with about 20% urea. For the first 4–6 h, ammonia excretion by L3 was temperature dependent, directly proportional to the number of larvae, but independent of the pH or strength of the phosphate buffer. Later, ammonia excretion slowed markedly in L3 and adults and reversed to net uptake in L3 by 30 h. An initial external ammonia concentration of 600 lM did not alter the pattern or magnitude of excretion. Re-uptake of ammonia did not occur at extremes of pH or low buffer strength and was slightly reduced at the highest external concentrations. Ammonium transporters and enzymes of glutamate metabolism, including glutamate dehydrogenase, glutamine synthetase and possibly glutamate synthase, are worthy of further investigation as anthelmintic targets. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Novel control measures are being sought to supplement or extend the life of currently used anthelmintic drenches to which nematode parasites of livestock are becoming increasingly resistant. Understanding the biology of these parasites has therefore taken on renewed importance in the search for new anthelmintic targets in the worms. Rapid growth and the demands of egg-laying must require very active energy and nitrogen metabolism in nematodes, yet surprisingly little is known about the sources of nitrogen, nutrient uptake, excretion and the key metabolic enzymes in species parasitising the sheep abomasum. Sources of nutrients for Teladorsagia circumcincta have not been clearly identified either for the free-living larval or parasitic stages. It is generally accepted that the free-living stages feed on microbes or their components in the sheep faeces. Transition to parasitism, first during development in the gastric gland lumen and subsequently in mucus overlying the abomasal mucosa (Sommerville, 1954; Armour et al., 1966), may require changes in both nutrient uptake and metabolism. Alternative sources of nitrogen for parasitic stages are mucins, epithelial cells or cell debris, substances in leaked interstitial fluid or components of the abomasal digesta. Nitrogen could be available as ammonia, urea or protein, or as peptides or amino acids after breakdown by the proteolytic enzymes present on the intestinal brush border and/or secreted by T. circumcincta (Young et al., 1995). Both proteases (Cox et al., 1990; * Corresponding author. Fax: +64 6 350 5636. E-mail address: [email protected] (H.V. Simpson). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.05.003
Redmond et al., 1997; Sajid and McKerrow, 2002) and glycosidases (Gamble and Mansfield, 1996; Irwin et al., 2004) have been identified in abomasal nematodes and their secretions. The nitrogenous compounds excreted by abomasal parasites are assumed to be similar to those excreted by other nematodes, but the relationship of these end-products to worm metabolism or to the changing local environment are largely unknown. The chemicals released during in vitro incubations are known as excretory/ secretory (ES) products and may be excreted metabolic end-products, components of the gut cells or cuticle (Rhoads et al., 2001), secretions from glands or material passing out of the intestine of adult worms (Rothstein, 1963; Wright, 1975; Wright and Newall, 1976). Adult worm ES products contain numerous proteins, e.g. incubates of adult Haemonchus contortus in RPMI contained in excess of 100 proteins on 2D gel electrophoresis (Yatsuda et al., 2003). Some of these may be involved in causing host pathophysiology, modifying the host response or supplying essential nutrients and have potential as protective vaccine candidates. Both free-living and parasitic nematodes predominantly excrete ammonia, with urea as a minor component not usually greater than about 20% of the total nitrogen excretion (Rogers, 1952; Rothstein, 1963, 1970; Wright, 1975). Significant amounts of amino acids are occasionally excreted and may result from stress, rather than reflecting changes in metabolism. Wright (1975) reported this to be the case for the free-living nematode Panagrellus redivivus. The present study addressed the excretion of nitrogenous substances of adult and L3 T. circumcincta during in vitro incubation under various conditions, particularly changing external pH and concentration of the medium. As expected from studies of other
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nematodes, ammonia was the main excreted compound, but an unexpected finding was the apparent re-uptake of ammonia during several hours of incubation in phosphate buffer. 2. Materials and methods All chemicals were supplied by Sigma Chemical Co. (Mo, USA). 2.1. Parasites L3 T. circumcincta were cultured from the faeces of sheep infected with a pure strain of parasite and stored at 4 °C in RO water until used. Prior to each experiment, L3 were Baemannised in RO water to remove inactive worms, counted and suspended in incubation medium. L3 were used sheathed or after exsheathing with sodium hypochlorite or CO2. L3 were incubated at 37 °C in 0.2% sodium hypochlorite for 15 min, filtered, washed and resuspended in medium. CO2 exsheathing was carried out at 40 °C by bubbling CO2 through the suspension for 20 s, shaking for 2 min and exsheathing overnight. Adult worms were recovered from the abomasa of infected sheep using the technique of Simpson et al. (1999). Briefly, abomasal contents were mixed 2:1 with 3% agar (Bacto agar, Difco Laboratories) and, after solidification, the agar blocks were incubated at 37 °C in a saline bath. Clumps of parasites were removed from the saline soon after emergence and placed in incubation medium. At the end of incubations, motility of the parasites was confirmed to be at least 95%. In longer incubations of 30 h, 15–20% of sheathed L3 had exsheathed, unrelated to the specific conditions of the incubation, but remained fully motile. No exsheathing occurred during shorter incubations up to 10 h. 2.2. Excretion of ammonia Parasites were suspended in 0.5 or 1 ml of phosphate buffer in a series of capped eppendorf tubes and incubated at 37 °C. At designated times, two of the tubes were removed, centrifuged briefly and the supernatant used to determine the concentration of ammonia or other product in the medium. The phosphate buffer in general use was either 100 mM or 0.8 mM sodium phosphate, pH 7.0 and the incubation temperature was 37 °C, except in specific experiments to test the effects of changing buffer concentration, pH or temperature. L3 were used routinely at a density of 50,000/ml and adult worms were weighed and incubated at 50– 60 mg wet wt/ml. Experiments were carried out in duplicate or triplicate. Specific experimental conditions were designed to evaluate the effects on ammonia excretion by sheathed L3: (1) Parasite density: sheathed L3 were incubated at densities of 5000, 10,000, 50,000, 70,000 or 100,000/ml in 0.8 mM phosphate buffer for 2.5 h (n = 3); (2) Temperature: incubated at 4 °C, 20 °C or 37 °C in 0.8 mM phosphate buffer for 5 h (n = 3); (3) Buffer concentration: incubated in phosphate buffer with concentration of 0.8, 50, 100 or 150 mM for 8 h and 30 h (n = 2); (4) pH: incubated in 100 mM phosphate buffer of pH 2, 4, 7 or 10 for 10 h or 30 h (n = 2); (5) External ammonia concentration: incubated in 100 mM phosphate buffer containing 0, 200 or 600 lM ammonium chloride for 30 h (n = 2); (6) Exsheathing: sheathed, sodium hypochlorite- or CO2exsheathed L3 from the same population were incubated in 100 mM phosphate buffer for 30 h (n = 2).
Ammonia excretion by adult worms (5–6 mg wet wt/ml) was monitored in 2 experiments: in Expt. 1, worms from the same population were incubated in either 0.8 mM or 100 mM phosphate buffer for 6 h (n = 2); in Expt. 2, the incubation was in 0.8 mM buffer for 9 h (n = 3). 2.3. Excretion of other compounds Urea excretion by sheathed or sodium hypochlorite-exsheathed L3 was determined by incubation in 0.8 mM phosphate buffer pH 7.0 for 4 h (n = 3). At each hour, four tubes were removed for urea determination (Section 2.4). Excretions of protein, uric acid and amino acids were monitored in adult worms (6 mg wet wt/ml) and sheathed and sodium hypochlorite-exsheathed L3. Parasites were incubated in 0.8 mM phosphate buffer pH 7.0 at 37 °C for 5 h. At hourly intervals, tubes were removed, centrifuged and the supernatant used for assay of protein, uric acid and amino acids. 2.4. Assays Ammonia concentrations were determined by reacting ammonia with hypochlorite and phenol to produce indophenols, which were monitored spectrophotometrically at 635 nm (Bolleter et al., 1961). The pH of all samples was adjusted to pH 7 before assay. Protein concentrations were determined by the method of Bradford (1976). Uric acid concentration was determined enzymatically by measuring the absorption at 290 nm with and without the addition of 1 U of uricase (Sasaki et al., 1996). Urea concentration was estimated enzymatically using commercial urease. Duplicate 0.5 ml samples, with and without added 1 U urease, were incubated for 30 min at 37 °C, followed by estimation of ammonia concentration in the tube. Urea concentrations were determined from the differences in ammonia concentrations with and without urease. Total amino acids were determined by the ninhydrin method (Magne and Larher, 1992). 3. Results 3.1. Ammonia excretion by L3 T. circumcincta Accumulation of ammonia in phosphate buffer during short (2.5 h) incubations was directly proportional to the number of L3 T. circumcincta per ml of medium (Fig. 1a). Ammonia excretion was also temperature dependent, being very low at 4 °C and greater at 37 °C than at 20 °C (Fig. 1b). There were no consistent effects of either buffer concentration or pH on excretion in shorter experiments (Fig. 1c and d), although differences developed in longer experiments. Ammonia excretion was a little lower in 150 mM buffer in some experiments, such as in the initial period illustrated in Fig. 2. A feature of many experiments was a marked decline in the ammonia concentration in the medium from about 10 h onward (Fig. 2). Exceptions were incubations in 0.8 mM phosphate buffer or in 100 mM buffer at pH other than pH 7, in which the ammonia concentration remained largely unchanged from 10 to 30 h of incubation. Sheathed L3 were also incubated in 100 mM phosphate buffer containing NH4Cl up to 600 lM (Fig. 3). The increment in ammonia concentration above the initial value was similar for the three media, reaching concentrations of 0.6–1.2 mM. After 30 h, the fall in ammonia concentration was about half as great when the initial concentration was 600 lM than in the other two buffers. Excretion of ammonia by L3 exsheathed either with CO2 or sodium hypochlorite was compared with that of sheathed L3 (Fig. 4). The most notable difference was the continuing excretion of
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Fig. 1. Concentrations of ammonia (lM) in phosphate buffer during incubation of sheathed L3 T. circumcincta (a) effect of varying the density of larvae in 0.8 mM phosphate buffer pH 7 during incubation at 37 °C for 2.5 hours; (b) effect of varying the temperature of incubation in 0.8 mM phosphate buffer pH 7: j: 4 °C; d: 20 °C; N: 37 °C; (c) effect of varying the concentration of phosphate buffer pH 7 during incubation at 37 °C: j: 50 mM; N: 100 mM; h: 150 mM; (d) effect of varying the pH of 100 mM phosphate buffer during incubation at 37 °C: j: pH 2; d: pH 4; N: pH 7; h: pH 10.
ammonia into the medium by hypochlorite-exsheathed larvae, contrasting with falling concentrations in the incubation medium of sheathed and CO2-exsheathed L3. 3.2. Ammonia excretion by adult worms Adult worms excreted ammonia into phosphate buffer over a similar time course to L3 (Fig. 5). In one experiment in which worms from the same population were incubated in either 0.8 or 100 mM buffer, buffer strength had no marked effect on ammonia excretion, whereas excretion into 0.8 mM buffer was greater in a second experiment with a different adult worm population. In both cases, there was a noticeably reduced rate of excretion after 4–6 h. 3.3. Excretion of other nitrogenous substances Sheathed L3 increased the urea concentration of the medium to 30 lM over 4 h of incubation (compared with 120 lM ammonia), whereas sodium hypochlorite-exsheathed L3 failed to excrete urea. Adult worms increased the protein content of the buffer much more than either sheathed or exsheathed L3: after 4 h, the protein concentration was 20 lg/ml for adult worms, 1–2 lg/ml for sheathed L3 and undetectable in the buffer in which the exsheathed larvae were incubated. No amino acids or uric acid were detected in media in which adult worms or sheathed L3 had been incubated. 4. Discussion
Fig. 2. Concentrations of ammonia (lM) in the medium during incubation of sheathed L3 T. circumcincta in phosphate buffer at 37 °C for 30 hours: (upper) effect of varying the concentration of phosphate buffer pH 7: d: 0.8 mM; j: 50 mM; N: 100 mM; h: 150 mM (lower) effect of varying the pH of 100 mM phosphate buffer: j: pH 2; d: pH 4; N: pH 7; h: pH 10.
Ammonia, urea and uric acid are the major nitrogenous excretory products of animals, the principal one depending upon the animal’s environment (Wright, 1995; Singer, 2003). Aquatic animals, both vertebrate and invertebrate, generally excrete ammonia and smaller amounts of urea, as did T. circumcincta incubated in
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Fig. 3. Effect of external ammonia on the excretion of ammonia into the medium during incubation of sheathed L3 T. circumcincta in 100 mM phosphate buffer pH 7 at 37 °C for 30 hours. The concentrations of ammonia (lM) are expressed as increments above the starting concentration of: j: 0 lM; h: 200 lM; d: 600 lM.
Fig. 4. Effect of exsheathing on concentrations of ammonia (lM) in the medium during incubation of L3 T. circumcincta in 100 mM phosphate buffer pH 7 at 37 °C for 30 hours: j: exsheathed with hypochlorite; h: exsheathed with CO2; N: sheathed.
Fig. 5. Concentrations of ammonia (lM) in the medium during incubation of adult T. circumcincta in phosphate buffer pH 7 at 37 °C: j: expt 1, 0.8 mM; h: expt 1, 100 mM; d: expt 2, 100 mM.
phosphate buffer in the present experiments. Rogers (1952), Rothstein (1963, 1970) and Wright (1975) have previously reported similar excretion of ammonia and 20% urea in several free-living
and parasitic nematodes at different life-cycle stages. The source of the ammonia in non-nutrient medium is likely to be the catabolism of amino acids, which principally occurs by transamination, followed by deamination by glutamate dehydrogenase (GDH) or by release of ammonia from glutamate by glutaminase. Other enzymes producing ammonia, including adenylate kinase and serine dehydratase, make appreciable contributions in some invertebrates (Fellows and Hird, 1979). Ammonia can also be generated by many other enzymes, such as asparaginase, which has been identified in the cuticle of Dirofilaria immitis (Tsuji et al., 1999). Urea is unlikely to be produced by the ornithine-urea cycle, as all enzymes are rarely present or only with low activity in helminths (Janssens and Bryant, 1969; Grantham and Barrett, 1986; Mohamed et al., 2005), but instead generated by arginase activity (Campbell, 1963; Senft, 1966). The pattern of excretion of ammonia consistently formed three phases, although quantitatively the amount excreted varied somewhat between parasite batches and experimental days. During the first phase lasting for 4–6 h, the duration of short-term experiments, ammonia accumulated in the incubation medium and excretion was unaffected by external pH and usually also not by osmolarity. The second phase from 6 to 8–10 h typically showed little further net excretion of ammonia, after which there was a progressive net loss of ammonia from the medium in the third phase. Delayed net uptake was sensitive to osmotic and pH gradients between worm and medium and occurred only where these were not extreme. These excretory patterns suggest that parasite metabolism first generates ammonia which passes into the incubation medium, followed by a change in metabolism consistent with ammonia uptake and incorporation under favourable conditions. Short-term excretion of ammonia by sheathed L3 T. circumcincta was linearly related to worm density and was temperature dependent, being very low at 4 °C and much greater at 37 °C than at 20 °C (Fig. 1), due probably to faster metabolic activity at higher temperatures. Exsheathing L3 did not appear to affect their initial ammonia excretion (Fig. 4). During incubations of 8–10 h, the pH of the medium had no apparent effects on ammonia excretion and only high osmotic pressure (150 mM) sometimes affected excretion. As adult worms do not survive as well in vitro as do L3 (Lawton et al., 2002), they were incubated only for short periods, during which they remained actively motile. Ammonia excretion followed a similar time course to that seen in L3, slowing markedly after 4– 6 h of incubation. The final concentration of ammonia was approximately half of that in L3 incubates. While there is probably no reliable way of comparing accurately excretory rates of different lifecycle stages using either numbers of individuals or weight of protein, marked changes in the ratios of different products can readily be seen. A striking difference was apparent between the relative accumulations of ammonia and protein in incubates of 5–6 mg adult worms and 50,000 L3: the adult worms excreted about half as much ammonia as did L3, whereas the protein concentration was 20-fold higher for adult worms. There was no measurable excretion of either uric acid or amino acids by L3 and the small protein loss from the non-feeding, sheathed L3 is likely to result from shedding of cuticular components or secretion. Lack of amino acid excretion over five hours of incubation suggests that the parasites were not stressed by the experimental conditions for that time, based on the conclusions of Wright (1975) that the appearance of amino acids in the medium in which P. redivivus was being incubated was related to environmental stress. Adult worms released greater amounts of protein into the incubation medium (mean 20 lM after 4 h), compared with 1 lM by sheathed L3 and negligible amounts by bleachexsheathed L3. Nematode ES products typically contain numerous proteins, e.g. more than 100 in ES products of adult H. contortus (Yatsuda et al., 2003). Many of these are likely to be of secretory
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origin, rather than excreted, or cuticular components, such as the collagen released by Ascaris suum during development from L3 to L4 in vitro (Rhoads et al., 2001). Longer incubations of L3 for 30 h revealed the different phases and changing rate of excretion with time and delayed effects of buffer concentration and pH on ammonia excretion (Figs. 2 and 3). The most favourable of the media used for the collection of excretory products of L3 would be expected to be 50–100 mM phosphate buffers of pH 7, although they do not supply nutrients required for the increased metabolic rate at 37 °C. In these less stressful environments, the pattern of ammonia excretion was an initial rapid increase, slowing to close to zero net excretion for several hours to 8–10 h, after which there was a progressive net loss of ammonia from the medium. Short-term experiments (Fig. 1) also showed this early excretory pattern and the start of a decline in the ammonia concentration from about 10 h onward (Fig. 2). In contrast, this late decline in external ammonia concentration did not occur in incubations in 0.8 mM phosphate buffer or in 100 mM buffer at pH other than pH 7, in all of which the ammonia concentration remained largely unchanged from 10–30 h of incubation. The enzymes responsible for ammonia incorporation, most likely glutamine synthetase (GS), may be less active under these conditions. To investigate whether the excretion of ammonia was limited or influenced by an existing ammonia gradient across the L3 cuticle, sheathed L3 were also incubated in 100 mM phosphate buffer containing up to 600 lM NH4Cl (Fig. 3). The pattern of excretion was no different over 30 h of incubation, except for a small reduction in the rate of ammonia loss during the last 15 h. During the first 15 h, the increments in ammonia concentration were similar for the three media, suggesting that the slowing of excretion after the fist few hours is not caused by accumulated ammonia but related to metabolic activity of the parasites of ammonia in the medium. The independence of the initial ammonia excretion from the transcuticular gradient also suggests that it does not occur through simple diffusion, which might be expected to be reduced by a high external concentration. Ammonia can cross membranes either by diffusion of the unprotonated NH3 form or by uptake of NH4+ by specific transporters (Kleiner, 1981). Although only 1% is present as ammonia at physiological pH, the conversion of NH4+ to NH3 is instantaneous and is not rate limiting for excretion. Ammonium transporters are present in all classes of organisms (Howitt and Udvardi, 2000; Williams and Miller, 2001; Javelle et al., 2003; Khademi et al., 2004), including Caenorhabditis elegans, which has four homologues of high affinity ammonium transporters in its genome (Howitt and Udvardi, 2000). The apparent re-uptake of ammonia under some conditions is particularly interesting in the contexts of organisms using excreted ammonia as a buffering substance in acid conditions or alternatively absorbing ammonia to use as a possible nutrient source. While it is most likely that the fall in ammonia concentration is due to uptake by the L3, other causes, such as microbial uptake or loss to the environment are possible, but unlikely. There was no visible evidence of microbial contamination and the similarity of time course and rate of fall in concentration supports an active rather than a passive process. In addition, the rate of decline was slower when the maximum concentration was 1.2 mM than 0.6 mM, contrary to what would be expect if ammonia were being released into environment. Ammonia (or ammonium) transport into the L3 would seem to be the most likely cause of the plateau and subsequent decline in ammonia concentration with time. Many pathogenic organisms generate ammonia to counteract acidic conditions (Audia et al., 2001; Cotter and Hill, 2003), including the gastric pathogen Helicobacter pylori, which uses urease to liberate ammonia from urea (Mobley and Hausinger, 1989; Még-
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raud et al., 1992). The lack of reuptake of ammonia at pH 2 and 4 by T. circumcincta L3 could be evidence of such use of ammonia to buffer an acid environment (Fig. 2), as normally present in the uninfected abomasum (Lawton et al., 1996). The parasitic stages of T. circumcincta are not tolerant of low pH in vitro (Lawton et al., 2002), so to survive in vivo, they must avoid exposure to acid, either by protecting themselves with gastric mucus, inhibition of parietal cells (Merkelbach et al., 2002) or perhaps through locally generating ammonia. Active buffering of external acid by ammonia may not be occurring, since in the present experiments, the ammonia concentration also remained high at pH 10. Lack of reuptake of ammonia in either low or high pH may indicate low GS activity at non-optimal pH, rather than a specific effect of external acidity. Prolonged exposure to extreme osmotic gradients, e.g. in 0.8 mM phosphate buffer, also impaired uptake and metabolism of ammonia. The relationship of these phases of ammonia excretion to parasite metabolism is interesting, particularly the apparent equilibrium after 4–6 h and subsequent uptake of ammonia from nonnutrient medium after reaching a maximum concentration of only 0.6–1.2 mM. It suggests that ammonia is produced from body protein or amino acids over the first few hours followed by a period when excreted ammonia becomes a source of nitrogen as enzymes capable of assimilating and metabolising ammonia are upregulated. Ammonia may therefore either be absorbed or excreted according to availability of different nitrogenous compounds as nutrients and the breakdown of stored material. Within the sheep, T. circumcincta could absorb ammonia from the different fluids to which they may be exposed, including abomasal fluid, interstitial fluid or fluid present in the mucus gel. In sheep, ammonia concentrations have been reported as around 5 mM in rumen fluid, 1 mM in abomasal fluid (Harrop, 1974; Harrop and Phillipson, 1974), 200 lM in arterial blood and 487 lM in portal blood (Parker et al., 1995). The values for abomasal fluid are similar to those reached when there is a pre-existing ammonia content in the incubation medium (Fig. 3), supporting the possibility of in vivo ammonia absorption and incorporation into amino acids by parasitic stages of T. circumcincta. Ammonia is assimilated principally into glutamate, either by the universal enzyme GDH or by the glutamine synthetase (GS)glutamate synthase (GOGAT) pathway, which is present in a few invertebrates, but not generally in animals. GDH activity in the aminating direction is usually limited by a high Km for ammonium, e.g. the Km for ammonium of H. contortus GDH is 42 mM (Rhodes and Ferguson, 1973). As the GS-GOGAT pathway requires ATP, but has a higher affinity for ammonia, is the preferred pathway in plants and prokaryotes if ATP is readily available (Helling, 1994, 2002). GOGAT activity has been identified in the mosquito Aedes aegypti (Scaraffia et al., 2005), the silkworms Bombyx mori (Hirayama et al., 1998) and Samia cynthia ricini (Osanai et al., 2000) and in the Spodoptera frugiperda Sf9 insect cell line (Doverskog et al., 2000). Gene sequences for NADH-GOGAT are present in the genome of C. elegans (Vanoni and Curti, 1999) and when more nematode species are investigated, GOGAT may be found to be more widespread in nematodes. In summary, these experiments suggest that excretory rates may be very low in dormant L3 or rapidly growing and reproducing parasitic stages of nematodes and that ammonia may either absorbed or excreted according to the external concentration and metabolic requirements for nitrogen. In the non-feeding L3, all excretion is via the cuticle or specialised excretory cell (Buechner et al., 1999; Buechner, 2002), whereas in adult worms, both cuticular and intestinal exchange is likely to contribute to excreted urea and ammonia. In relation to overall nitrogen metabolism, potential targets in the parasites are likely to be found in ammonium transporters or enzymes of glutamate metabolism. Trans-
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porters may be similar to the high affinity ammonia permeases of plants, bacteria and yeasts which scavenge ammonia from the environment and recover ammonia lost by diffusion when growing on sources of nitrogen other than ammonia (Marini et al., 1997). Glutamate metabolism is likely to be central to generating ammonia (Bidigare and King, 1981; Batrel and Le Gal, 1984) as well as assimilating it via GDH or GS-GOGAT. Acknowledgments We are grateful for the financial support of Meat and Wool New Zealand and the E. and C. Thoms Bequest. The Faculty of Medicine and Health Sciences, UNIMAS is thanked for personal support for N. Muhamad. References Armour, J., Jarrett, W.F.H., Jennings, F.W., 1966. Experimental Ostertagia circumcincta infections in sheep: development and pathogenesis of a single infection. American Journal of Veterinary Research 27, 1267–1278. Audia, J.P., Webb, C.C., Foster, J.W., 2001. Breaking the acid barrier: an orchestrated response to proton stress by enteric bacteria. International Journal of Medical Microbiology 291, 97–106. Batrel, Y., Le Gal, Y., 1984. Nitrogen metabolism in Arenicola marina characterization of a NAD dependent glutamate dehydrogenase. Comparative Biochemistry and Physiology 78B, 119–124. Bidigare, R.R., King, F.D., 1981. The measurement of glutamate dehydrogenase activity in Praunus flexuosus and its role in the regulation of ammonium excretion. Comparative Biochemistry and Physiology 70B, 409–413. Bolleter, W.T., Bushman, C.J., Tidwell, P.W., 1961. Spectrophotometric determination of ammonia as indophenol. Analytical Chemistry 33, 592–594. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Buechner, M., 2002. Tubes and the single C. elegans excretory cell. Trends in Cell Biology 12, 479–484. Buechner, M., Hall, D.H., Bhatt, H., Hedgecock, E.M., 1999. Cystic canal mutants in Caenorhabditis elegans are defective in the apical membrane domain of the renal (excretory) cell. Developmental Biology 214, 227–241. Campbell, J.W., 1963. Urea formation and urea cycle enzymes in the cestode, Hymenolepsis diminuta. Comparative Biochemistry and Physiology 8, 13–27. Cotter, P.D., Hill, C., 2003. Surviving the acid test: responses of gram-positive bacteria to low pH. Microbiology and Molecular Biology Reviews 67, 429–453. Cox, G.N., Pratt, D., Hageman, R., Boisvenue, R.J., 1990. Molecular cloning and primary sequence of a cysteine protease expressed by Haemonchus contortus adult worms. Molecular and Biochemical Parasitology 41, 25–34. Doverskog, M., Jacobsson, U., Chapman, B.E., Kuchel, P.W., Häggström, L., 2000. Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective 1H/15N NMR in vitro assay. Journal of Biotechnology 79, 87–97. Fellows, F.C.I., Hird, F.J.R., 1979. Nitrogen metabolism and excretion in the freshwater crayfish Cherax destructor. Comparative Biochemistry and Physiology 64B, 235–238. Gamble, H.R., Mansfield, L.S., 1996. Characterization of excretory–secretory products from larval stages of Haemonchus contortus cultured in vitro. Veterinary Parasitology 62, 291–305. Grantham, B.D., Barrett, J., 1986. Amino acid catabolism in the nematodes Heligmosomoides polygyrus and Panagrellus redivivus 1. Removal of the amino group. Parasitology 93, 481–493. Harrop, C.J.F., 1974. Nitrogen metabolism in the ovine stomach 4. Nitrogenous components of the abomasal secretions. Journal of Agricultural Science 83, 249– 257. Harrop, C.JF., Phillipson, A.T., 1974. Nitrogen metabolism in the ovine stomach 3. Urea in the abomasal secretions. Journal of Agricultural Science 83, 237– 247. Helling, R.B., 1994. Why does Escherichia coli have two primary pathways for synthesis of glutamate? Journal of Bacteriology 176, 4664–4668. Helling, R.B., 2002. Speed versus efficiency in microbial growth and the role of parallel pathways. Journal of Bacteriology 184, 1041–1045. Hirayama, C., Saito, H., Konno, K., Shinbo, H., 1998. Purification and characterization of NADH-dependent glutamate synthase from the silkworm fat body (Bombyx mori). Insect Biochemistry and Molecular Biology 28, 473–482. Howitt, S.M., Udvardi, M.K., 2000. Structure, function and regulation of ammonium transporters in plants. Biochimica et Biophysica Acta 1465, 152–170. Irwin, J.A., Morrissey, P.E.W., Ryan, J.P., Walshe, A., O’Neill, S.M., Carrington, S.D., Matthews, E., Fitzpatrick, E., Mulcahy, G., Corfield, A.P., Dalton, J.P., 2004. Glycosidase activity in the excretory–secretory products of the liver fluke, Fasciola hepatica. Parasitology 129, 465–472. Janssens, P.A., Bryant, C., 1969. The ornithine-urea cycle in some parasitic helminths. Comparative Biochemistry and Physiology 30, 261–272.
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Yatsuda, A.P., Krijgsveld, J., Cornelissen, A.W.C.A., Heck, A.J.R., de Vries, E., 2003. Comprehensive analysis of the secreted proteins of the parasite Haemonchus contortus reveals extensive sequence variation and differential immune recognition. Journal of Biological Chemistry 278, 16941–16951. Young, C.J., McKeand, J.B., Knox, D.P., 1995. Proteinases released in vitro by the parasitic stages of Teladorsagia circumcincta, an ovine abomasal nematode. Parasitology 110, 465–471.