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Thermotolerance of L3Ostertagia (Teladorsagia) circumcincta and some of its enzymes
Veterinary Parasitology 146 (2007) 77–82 www.elsevier.com/locate/vetpar
Thermotolerance of L3 Ostertagia (Teladorsagia) circumcincta and some of its enzymes Lisa R. Walker, David C. Simcock, Jennifer D. Neale, Heather V. Simpson, Simon Brown * Laboratory for Biochemical Parasitology, Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11222, Palmerston North, New Zealand Received 25 October 2005; received in revised form 9 February 2007; accepted 9 February 2007
Abstract Parasitic nematodes of ruminants can be expected to experience temperatures in excess of 40 8C in faeces on pasture and, perhaps, in the host. L3 Ostertagia (Teladorsagia) circumcincta survived for at least 90 min at 45 8C in vitro in water, but the larvae were inactivated rapidly by only slightly higher temperatures. The glycolytic enzymes hexokinase and pyruvate kinase were inactivated in a similar temperature range, whereas malate dehydrogenase maintained its activity at temperatures in excess of 50 8C. These data imply that the loss of glycolytic activity might explain the loss of larval motility at temperatures between 45 8C and 50 8C. # 2007 Elsevier B.V. All rights reserved. Keywords: Thermotolerance; Parasite (Ostertagia circumincta); Parasite (Teladorsagia circumcincta); Enzyme
1. Introduction Parasitic nematodes must be able to withstand moderately high temperatures in both free living and parasitic stages. During a long hot summer afternoon, the temperature in a faecal pellet can exceed 40 8C (Berbigier et al., 1990; Rossanigo and Gruner, 1994). In the upper few cm of soil, where sheep parasitic nematodes have been reported (Callinan and Westcott, 1986), the temperature can exceed the ambient air temperature by several degrees celsius. However, such periods of relatively high temperature are quite brief and the diurnal temperature range declines with increasing soil depth (Monteith and Unsworth, 1990). In contrast, the temperature in the ovine abomasum and * Present address: School of Human Life Sciences, University of Tasmania, Locked Bag 1320, Launceston, Tasmania 7250, Australia. Tel.: +61 3 6324 5467; fax: +61 3 6324 3995. E-mail address: [email protected] (S. Brown). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.02.009
rumen is consistently high (38–41 8C (McCrabb and Bortolussi, 1996; Piccione et al., 2002)). This means that sheep parasitic nematodes present in the soil or on the pasture must be able to withstand transient high temperatures, and those ingested by the animal must be adapted to sustained high temperature. It has been reported that third stage larvae (L3) of Ostertagia (Teladorsagia) circumcincta kept in ovine faeces survived for several weeks at temperatures ranging from 50 to 35 8C (Pandey et al., 1993) and eggs developed in distilled water at temperatures ranging from 4 to 35 8C (Pandey, 1972; Young et al., 1980). Rossanigo and Gruner (1995) showed that the highest yield of L3 O. circumcincta was obtained from eggs cultured in ovine faeces at about 25 8C and L3 of the greatest length were also obtained at about this temperature (Rossanigo and Gruner, 1996). However, O. circumcincta experience temperatures even higher than this in many places in both the free living
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(temperature ranging from below zero to more than 40 8C) and parasitic (above about 38 8C) stages. Here, we report results of observations on the effect of elevated temperature (40 8C) on the motility of sheathed L3 O. circumcincta and on the activity of three enzymes that appear to be important in the metabolism of the nematode in this life cycle stage. We also compare the thermal stability of these O. circumcincta enzymes with those in the parasitic nematode Haemonchus contortus and the non-parasitic soil nematode Caenorhabditis elegans. 2. Materials and methods 2.1. Nematodes Pure strains of Ostertagia (Teladorsagia) circumcincta and Haemonchus contortus were maintained by regular passage through sheep to provide L3. Larvae were concentrated by centrifugation at approximately 1000 g, washed and then resuspended in the appropriate medium. Adult H. contortus were obtained from the abomasal contents of donor sheep according to the method described by Merkelbach et al. (2002). Caenorhabditis elegans was maintained on a lawn of Escherichia coli OP50 on agar plates at 20 8C (Brenner, 1974) and the large number of nematodes required for the preparation of homogenates were prepared according to the methods of Sulston and Brenner (1974). These nematodes represented a mixed population of different life cycle stages. 2.2. Motility assays To ensure maximal motility, larvae were Baermannised before being resuspended in water at about 3000 mL 1. Larvae (75 mL 1) were incubated for the appropriate period of time in 1 mL of temperatureequilibrated water and those larvae that were not immotile during the observation period were counted on a McMaster slide. Larval recovery experiments were carried out by incubating the L3 at a defined temperature for 2.5– 10 min and then incubating the tube at 4 8C, 25 8C or 37 8C for 1 h after which the motility was monitored in the standard way. 2.3. Enzyme activity measurements All enzymes were assayed spectrophotometrically at 340 nm at 30 8C (unless otherwise stated) using an
Ultrospec III (LKB Instruments) or a CE 599 (Cecil Instruments Limited, Cambridge, England) and data were collected by computer using software (Brown and Dykstra, 1999) and hardware developed in-house (Brown and Pedley, unpublished). It is inappropriate to give a detailed description of the hardware here, but it has been tested exhaustively and the readings given by the spectrophotometer and the computer are identical and can be compared by the user at any time. Enzyme activity was determined in crude homogenates of L3 prepared by manually grinding frozen larvae, without further preparation. Hexokinase (HK, E.C.2.7.1.1) was assayed using a coupled reaction with glucose 6-phosphate dehydrogenase in a medium containing 5 mM glucose, 1 mM ATP, 200 mM NAD+, 1 mM MgCl2, 50 mM KH2PO4 pH 7.5 in the presence of 1 U glucose 6-phosphate dehydrogenase and 50 mg homogenate protein. Pyruvate kinase (PK, E.C.2.7.1.40) was assayed using a coupled reaction with lactate dehydrogenase in a medium containing 5 mM phosphoenolpyruvate, 1 mM ADP, 10 mM MgCl2, 200 mM NADH, 50 mM KH2PO4 pH 7.5 in the presence of 1 U lactate dehydrogenase and 50 mg homogenate protein. Malate dehydrogenase (MDH, E.C.1.1.1.37) was assayed in a medium containing 5 mM oxaloacetate, 200 mM NADH, 50 mM KH2PO4 pH 7.5 in the presence of 50 mg homogenate protein. Note that the MDH activity probably represented a mixture of at least two distinct enzymes, one located in the cytosol and another in the mitochondria, and no attempt was made to distinguish between them. The mammalian enzymes used as controls were porcine heart MDH, rabbit muscle PK and yeast HK, each of which was purchased from Sigma–Aldrich Pty Ltd., Castle Hill, NSW, Australia. In each case, the conditions were selected to ensure maximal activity at 30 8C. The protein concentration of larval homogenates was determined using the Bradford method (Bradford, 1976). No detergents were employed to solubilise the nematodes as they can interfere with protein assays (Sapan et al., 1999). 2.4. Homogenate incubations Larval homogenates were incubated for the required period (between 5 min and 60 min) in the assay medium (without substrates or effectors) in a heating block at the specified temperature (between 30 8C and 708) before being cooled to the assay temperature (30 8C).
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Fig. 1. Development of immotility of L3 O. circumcincta during incubation in water at various temperatures. (A) The time course of the developing immotility ((*) 40 8C, (&) 458, (~) 46 8C, (~) 47 8C, (^) 48 8C, (&) 508). (B) The proportion of immotile larvae after 30 min incubation. (C) Relationship between the temperature at which 50% of the larvae were immotile and incubation time (derived from A). The LT50 values were estimated by fitting a delayed gamma distribution (Young et al., 1980) to all of the available data by least squares nonlinear regression (in all cases r2 0.9). In each plot, the error bars represent 1 S.E.M. and the straight line in C was obtained using weighted linear regression.
3. Results
3.2. Glycolytic and tricarboxylic acid cycle enzyme activity
3.1. Motility of L3 O. circumcincta Sheathed larvae incubated at 40 8C for up to 90 min did not become significantly immotile, but there was a gradual decline in motility during the observation period at 45 8C and higher temperatures until even 2.5 min at 50 8C rendered the larvae completely immotile during the observation period (Fig. 1A). The larvae were exquisitely sensitive to temperatures greater than about 46 8C (Fig. 1B). From these data it can be estimated that temperature at which 50% of the larvae would be immotile (LT50) declined roughly linearly with incubation time (Fig. 1C) and, extrapolating from these data, that larvae exposed to about 48 8C would instantly become immotile (Fig. 1C). No systematic effort was made to estimate changes in the degree of motility of the larvae, although this also appeared to decline during incubation at temperatures above about 45 8C. Larvae rendered immotile by incubation at 50 8C did not recover during subsequent incubation at 4 8C, 25 8C or 37 8C.
In order to assess the importance of fermentative metabolism in thermotolerance, the activity of two glycolytic enzymes, hexokinase (HK) and pyruvate kinase (PK), and one tricarboxylic acid (TCA) cycle enzyme, malate dehydrogenase (MDH), was measured following 10 min incubation of the L3 homogenate at various temperatures. These enzymes are metabolically important in many species and appear to be significant to L3 O. circumcincta (Walker et al., 2005). The MDH activity measured in L3 O. circumcincta was the highest of any of the activities we have characterized to date. Incubation of larval homogenate temperatures up to 50 8C for 10 min reduced the activity of MDH by no more than about 10%, but above this temperature the activity declined steadily (Fig. 2). No MDH activity could be detected following incubation at 70 8C for 10 min (Fig. 2). In contrast, purified pig heart MDH activity in these conditions was significantly reduced following incubation at 40 8C for 10 min and almost completely inactivated at 44 8C (data not shown). The
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Fig. 2. Relative activity of malate dehydrogenase (*), pyruvate kinase (&) and hexokinase (~) in homogenate of L3 O. circumcincta incubated for 10 min at different temperatures. The data are from one representative experiment in which 100% was 5000 nmoles min1 mg 1, 300 nmoles min 1 mg 1, 10 nmoles min 1 mg 1 protein for MDH, PK and HK, respectively.
Fig. 4. Relative activity of malate dehydrogenase in homogenates prepared from L3 (*) and adult (*) H. contortus, C. elegans (~) and L3 O. circumcincta (&) following incubation at 50 8C for up to 1 h. The data are from one representative experiment in which 100% was at least 1000 nmol min 1 mg 1 protein.
activity of PK declined steadily approximately linearly with temperature between (30 8C and 50 8C) and HK activity dropped steeply between 44 8C and 45 8C. At 50 8C both HK and PK were essentially inactive (Fig. 2). Prolonged incubation of the O. circumcincta homogenate at 50 8C had little effect on the MDH activity, which fell about 20% over 60 min, but incubation of homogenate at 42 8C caused a steady decline in the activity of PK, which was essentially inactive after 60 min (Fig. 3). The activity of MDH in homogenate prepared from L3 or adult H. contortus or from a mixed population of C. elegans did not decline substantially during incubation at 50 8C for 60 min (Fig. 4). In contrast, MDH prepared from porcine heart showed only 10% of
its initial activity following incubation for only 10 min at 50 8C and was essentially inactive after 60 min (data not shown).
Fig. 3. Relative activity of O. circumcincta malate dehydrogenase (*) and pyruvate kinase (&) following incubation at 50 8C or 42 8C, respectively, for up to 60 min. The data are from one representative experiment in which 100% was 5000 and 300 nmoles min 1 mg 1 protein for MDH and PK, respectively.
4. Discussion The data reported here show that L3 O. circumcincta remain motile after 90 min at 45 8C (Fig. 1A), but are exquisitely sensitive to only slightly higher temperatures (Fig. 1B). The activity of MDH was relatively unaffected by temperatures up to 50 8C, but PK and HK were less thermotolerant and were inactive after 10 min at 50 8C (Fig. 2). The apparent thermostability of MDH from O. circumcincta was also apparent in other species of parasitic nematode, in adults and in the soil nematode C. elegans (Fig. 4). It is possible that the MDH activity observed with C. elegans might have been contaminated by protein from the E. coli on which the nematodes were grown. However, this seems unlikely because the C. elegans were washed extensively before use and Krijgsveld et al. (2003), who fed 15N-labelled E. coli to C. elegans and then analysed the appearance of label in C. elegans homogenates by 2D electrophoresis and mass spectrometry, did not report the presence of any bacterial proteins. However, even if there were substantial E. coli-derived MDH in the C. elegans homogenate, that enzyme is relatively temperature sensitive (50% inactivation within 3 min at 60 8C (Goward et al., 1994)), and so it is unlikely that it would have made a significant contribution to the temperature stability data shown in Fig. 4. The data reported here show that L3 O. circumcincta become immotile during the observation period over a
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very narrow temperature range between 45 8C and 50 8C (Fig. 1B). The regression line shown in Fig. 1C implies that half of the larvae would become immotile instantly at 48 8C, but even a few minutes at 50 8C certainly renders all the larvae immotile (Fig. 1A). These data are consistent with a need for the larvae to be resistant to temperatures above 40 8C and with the observation that L3 O. circumcincta kept in water at 35 8C remained motile for almost 8 weeks (Pandey et al., 1993). We have made no attempt to estimate the lifespan of L3 O. circumcincta at temperatures between 40 8C and 45 8C, although they are likely to be even shorter. However, the larvae are unlikely to experience prolonged periods at such high temperatures because of the diurnal temperature cycle. While this thermotolerance is not unique among nematodes (Nicholas, 1975), it is extraordinary. Even Alvinella pompejana, the ‘most thermotolerant eukaryote on earth’ (Desbruye`res et al., 1998), which lives near hydrothermal deep sea vents at temperatures of 2 to 105 8C, is unlikely to survive long-term at temperatures above 31 8C (Desbruye`res et al., 1998). Other invertebrates from the same environment become inactive between 40 8C and 50 8C (Lee, 2003). Interestingly, the rate of O. circumcincta development in ovine faecal culture has been reported to be reduced at temperatures above or below about 25 8C (Rossanigo and Gruner, 1995), similar to observations reported for O. ostertagi maintained in water (Pandey, 1972). Presumably, O. circumcincta eggs would not have hatched at temperatures of more than 40 8C, although the larvae survived. Hexokinase activity declined steeply between 45 8C and 46 8C (Fig. 2), in a range similar to the observed loss of motility (Fig. 1B), whereas PK activity declined over a much wider temperature range (Fig. 2). Since the activity of PK was much (about 30-fold) greater than that of HK, it is tempting to speculate that there is a link between the thermal inactivation of HK and the loss of larval motility. If such a connection does exist, further work would be required to confirm it. Nevertheless, both HK and PK activity was largely eliminated after 10 min incubation at 45 8C, which is consistent with a significant impact on glycolytic activity in the larvae and, therefore, on the supply of energy required for motility. This speculation is also supported by the observation that L3 O. circumcincta remain motile at 30 8C in anaerobic conditions and in the presence of cyanide (Simcock et al., 2006), consistent with the postulated dependence of motility on glycolytic metabolism. The temperature stability of MDH was substantially greater than that of HK and PK tested in similar
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conditions. Relatively thermostable MDH activity has been observed in other parasites, such as Ascaris suum (Zee and Zinkham, 1968), A. lumbricoides (Barrett and Fairbairn, 1971) and Toxocara canis (Andrade et al., 1983). In Ascaris spp. (Zee and Zinkham, 1968; Barrett and Fairbairn, 1971), the mitochondrial MDH (mMDH) was inactivated at lower temperatures than the cytosolic MDH (cMDH). This prompts the tentative hypothesis that the initial small (10%) decline in activity of MDH at about 50 8C could represent the inactivation of mMDH and the remainder might represent the inactivation of cMDH. This is not the only thermostable enzyme in A. suum, since Z˙o´ltowska (2001) reported a similar difference in thermostability of intestinal and muscle a-amylase. In contrast, Kapur et al. (1984, 1985) assayed a variety of enzyme activities in H. contortus homogenate and reported no specific thermotolerant MDH activity. It is not clear how these experiments were carried out, but it is possible that the temperatures reported represent those at which the activity was assayed rather than those at which the homogenate was incubated. It should be noted that the solutes present in the homogenate could have affected the apparent thermostability of MDH activity reported here (see Nash and Wiskich (1982), for example) and so the enzyme(s) need not be intrinsically thermotolerant. While this cannot be discounted two counterarguments can be made. First, during the high temperature incubations, the endogenous solutes were diluted at least 104-fold compared with those likely to apply in vivo; and, secondly, the observed thermotolerance compares well with that reported by Zee and Zinham (1968) for the purified A. suum enzyme(s) and by Andrade et al. (1983) for the partially purified T. canis cMDH. Infective larvae of O. circumcincta can tolerate temperatures up to about 45 8C for at least 90 min, but at higher temperatures the larvae become increasingly immotile. The relative temperature-sensitivity of glycolytic enzymes imply that any tissue having a high energy demand (such as nervous tissue and muscle) would be particularly affected by any reduction in the activity of the pathway. Of course, pathways other than those investigated here might be even more temperature sensitive. The temperature range tolerated by O. circumcincta is consistent with that of the environment(s) in which the larvae are found and it is possible that the seasonal variation in larval availability reported for various nematode parasites (Gordon, 1948; Vlassoff et al., 2001) is related to the thermotolerance of the larvae.
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Acknowledgements This work was supported by Meat and Wool Innovation Ltd. and DCS was supported by a personal fellowship from AGMARDT. The authors would like to thank Laura Green for generously providing C. elegans. References Andrade, C.M., Ferreira, M.F.A., Ribiero, L.P., 1983. Cytosolic malate dehydrogenase in muscle extracts of Toxocara canis. Comp. Biochem. Physiol. 75B, 147–152. Barrett, J., Fairbairn, D., 1971. Effects of temperature on the kinetics of malate dehydrogenases in the developing eggs and adult muscle of Ascaris lumbricoides (Nematoda). J. Exptl. Zool. 176, 169–178. Berbigier, P., Gruner, L., Mambrini, M., Sophie, S.A., 1990. Faecal water content and egg survival of goat gastro-intestinal strongyles under dry tropical conditions in Guadeloupe. Parasitol. Res. 76, 379–385. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Brenner, S., 1974. The genetics of Caenorhabditis elegans. Genetics 77, 71–94. Brown, S., Dykstra, R., 1999. Computerised data acquisition and analysis in the biochemistry teaching laboratory. Biochem. Educ. 27, 93–96. Callinan, A.P.L., Westcott, J.M., 1986. Vertical distribution of trichostrongylid larvae on herbage and in soil. Int. J. Parasitol. 16, 241– 244. Desbruye`res, D., Chevaldonne´, P., Alayse, A.-M., Jollivet, D., Lallier, F.H., Jouin-Toulmond, C., Zal, F., Sarradin, P.-M., Cosson, R., Caprais, J.-C., Arndt, C., O’Brien, J., Guezennec, J., Hourdez, S., Riso, R., Gaill, F., Laubier, L., Toulmond, A., 1998. Biology and ecology of the ‘‘Pompeii worm’’ (Alvinella pompejana Desbruyeres and Laubier), a normal dweller of an extreme deepsea environment: a synthesis of current knowledge and recent developments. Deep Sea Res. II 45, 383–422. Gordon, H.M., 1948. The epidemiology of parasitic diseases, with special reference to studies with nematode parasites of sheep. Aust. Vet. J. 24, 17–45. Goward, C.R., Miller, J., Nicholls, D.J., Irons, L.I., Scawen, M.D., O’Brien, R., Chowdhry, B.Z., 1994. A single amino acid mutation enhances the thermal stability of Escherichia coli malate dehydrogenase. Eur. J. Biochem. 224, 249–255. Kapur, J., Sood, M.L., Soni, G.L., 1984. Effect of pH and temperature on four dehydrogenase enzymes of Haemonchus contortus. Acta Vet. Hung. 32, 165–170. Kapur, J., Sood, M.L., Soni, G.L., 1985. Haemonchus contortus: effect of pH and temperature on some dehydrogenases. Acta Vet. Hung. 33, 19–23. Krijgsveld, J., Ketting, R.F., Mahmoudi, T., Johansen, J., Artal-Sanz, M., Verrijzer, C.P., Plasterk, R.H.A., Heck, A.J.R., 2003. Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics. Nat. Biotechnol. 21, 927–931. Lee, R.W., 2003. Thermal tolerances of deep-sea hydrothermal vent animals from the Northeast Pacific. Biol. Bull. 205, 98–101.
McCrabb, G.J., Bortolussi, G., 1996. Placental growth and the ability of sheep to thermoregulate in hot environment. Small Rumin. Res. 20, 121–127. 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. Vet. Parasitol. 104, 217–228. Monteith, J.L., Unsworth, M.H., 1990. Principles of Environmental Physics. Edward Arnold, London, 291 p. Nash, D., Wiskich, J.T., 1982. Effects of various solutes on the thermostability of isocitrate dehydrogenase and malate dehydrogenase of isolated plant mitochondria. Aust. J. Plant Physiol. 9, 715–723. Nicholas, W.L., 1975. The Biology of Free-living Nematodes. Oxford University Press, Oxford, p. 219. Pandey, V.S., 1972. Effect of temperature on development of the free-living stages of Ostertagia ostertagi. J. Parasitol. 58, 1037– 1041. Pandey, V.S., Chaer, A., Dakkak, A., 1993. Effect of temperature and relative humidity on survival of eggs and infective larvae of Ostertagia circumcincta. Vet. Parasitol. 49, 219–227. Piccione, G., Caola, G., Refinetti, R., 2002. Effect of shearing on the core body temperature of three breeds of Mediterranean sheep. Small Rumin. Res. 46, 211–215. Rossanigo, C.E., Gruner, L., 1994. Relative effect of temperature and moisture on the development of strongyle eggs to infective larvae in bovine pats in Argentina. Vet. Parasitol. 55, 317–325. Rossanigo, C.E., Gruner, L., 1995. Moisture and temperature requirements in faeces for the development of free-living stages of gastrointestinal nematodes of sheep, cattle and deer. J. Helminthol. 69, 357–362. Rossanigo, C.E., Gruner, L., 1996. The length of strongylid nematode infective larvae as a reflection of developmental conditions in faeces and consequences on their viability. Parasitol. Res. 82, 304– 311. Sapan, C.V., Lundblad, R.L., Price, N.C., 1999. Colorimetric protein assay techniques. Biotechnol. Appl. Biochem. 29, 99–108. Simcock, D.C., Brown, S., Neale, J.D., Przemeck, S.M.C., Simpson, H.V., 2006. L3 and adult Ostertagia (Teladorsagia) circumcincta exhibit cyanide sensitive oxygen uptake. Exp. Parasitol. 112, 1– 7. Sulston, J.E., Brenner, S., 1974. The DNA of Caenorhabditis elegans. Genetics 77, 95–104. Vlassoff, A., Leathwick, D.M., Heath, A.C.G., 2001. The epidemiology of nematode infections of sheep. N. Z. Vet. J. 49, 213–221. Walker, L.R., Simcock, D.C., Brown, S., Simpson, H.V., 2005. Energy metabolism in Ostertagia circumcincta: glycolysis. N. Z. J. Zool. 32, 278. Young, R.R., Anderson, N., Overend, D., Tweedie, R.L., Malafant, K.W.J., Preston, G.A.N., 1980. The effect of temperature on times to hatching of eggs of the nematode Ostertagia circumcincta. Parasitology 81, 477–491. Zee, D.S., Zinkham, W.H., 1968. Malate dehydrogenase in Ascaris suum: characterization, onotgeny, and genetic control. Arch. Biochem. Biophys. 126, 574–584. Z˙o´ltowska, K., 2001. Purification and characterization of a-amylases from the intestine and muscle of Ascaris suum (Nematoda). Acta Biochim. Polon. 48, 763–774.
Thermotolerance of L3 Ostertagia (Teladorsagia) circumcincta and some of its enzymes Lisa R. Walker, David C. Simcock, Jennifer D. Neale, Heather V. Simpson, Simon Brown * Laboratory for Biochemical Parasitology, Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11222, Palmerston North, New Zealand Received 25 October 2005; received in revised form 9 February 2007; accepted 9 February 2007
Abstract Parasitic nematodes of ruminants can be expected to experience temperatures in excess of 40 8C in faeces on pasture and, perhaps, in the host. L3 Ostertagia (Teladorsagia) circumcincta survived for at least 90 min at 45 8C in vitro in water, but the larvae were inactivated rapidly by only slightly higher temperatures. The glycolytic enzymes hexokinase and pyruvate kinase were inactivated in a similar temperature range, whereas malate dehydrogenase maintained its activity at temperatures in excess of 50 8C. These data imply that the loss of glycolytic activity might explain the loss of larval motility at temperatures between 45 8C and 50 8C. # 2007 Elsevier B.V. All rights reserved. Keywords: Thermotolerance; Parasite (Ostertagia circumincta); Parasite (Teladorsagia circumcincta); Enzyme
1. Introduction Parasitic nematodes must be able to withstand moderately high temperatures in both free living and parasitic stages. During a long hot summer afternoon, the temperature in a faecal pellet can exceed 40 8C (Berbigier et al., 1990; Rossanigo and Gruner, 1994). In the upper few cm of soil, where sheep parasitic nematodes have been reported (Callinan and Westcott, 1986), the temperature can exceed the ambient air temperature by several degrees celsius. However, such periods of relatively high temperature are quite brief and the diurnal temperature range declines with increasing soil depth (Monteith and Unsworth, 1990). In contrast, the temperature in the ovine abomasum and * Present address: School of Human Life Sciences, University of Tasmania, Locked Bag 1320, Launceston, Tasmania 7250, Australia. Tel.: +61 3 6324 5467; fax: +61 3 6324 3995. E-mail address: [email protected] (S. Brown). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.02.009
rumen is consistently high (38–41 8C (McCrabb and Bortolussi, 1996; Piccione et al., 2002)). This means that sheep parasitic nematodes present in the soil or on the pasture must be able to withstand transient high temperatures, and those ingested by the animal must be adapted to sustained high temperature. It has been reported that third stage larvae (L3) of Ostertagia (Teladorsagia) circumcincta kept in ovine faeces survived for several weeks at temperatures ranging from 50 to 35 8C (Pandey et al., 1993) and eggs developed in distilled water at temperatures ranging from 4 to 35 8C (Pandey, 1972; Young et al., 1980). Rossanigo and Gruner (1995) showed that the highest yield of L3 O. circumcincta was obtained from eggs cultured in ovine faeces at about 25 8C and L3 of the greatest length were also obtained at about this temperature (Rossanigo and Gruner, 1996). However, O. circumcincta experience temperatures even higher than this in many places in both the free living
78
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(temperature ranging from below zero to more than 40 8C) and parasitic (above about 38 8C) stages. Here, we report results of observations on the effect of elevated temperature (40 8C) on the motility of sheathed L3 O. circumcincta and on the activity of three enzymes that appear to be important in the metabolism of the nematode in this life cycle stage. We also compare the thermal stability of these O. circumcincta enzymes with those in the parasitic nematode Haemonchus contortus and the non-parasitic soil nematode Caenorhabditis elegans. 2. Materials and methods 2.1. Nematodes Pure strains of Ostertagia (Teladorsagia) circumcincta and Haemonchus contortus were maintained by regular passage through sheep to provide L3. Larvae were concentrated by centrifugation at approximately 1000 g, washed and then resuspended in the appropriate medium. Adult H. contortus were obtained from the abomasal contents of donor sheep according to the method described by Merkelbach et al. (2002). Caenorhabditis elegans was maintained on a lawn of Escherichia coli OP50 on agar plates at 20 8C (Brenner, 1974) and the large number of nematodes required for the preparation of homogenates were prepared according to the methods of Sulston and Brenner (1974). These nematodes represented a mixed population of different life cycle stages. 2.2. Motility assays To ensure maximal motility, larvae were Baermannised before being resuspended in water at about 3000 mL 1. Larvae (75 mL 1) were incubated for the appropriate period of time in 1 mL of temperatureequilibrated water and those larvae that were not immotile during the observation period were counted on a McMaster slide. Larval recovery experiments were carried out by incubating the L3 at a defined temperature for 2.5– 10 min and then incubating the tube at 4 8C, 25 8C or 37 8C for 1 h after which the motility was monitored in the standard way. 2.3. Enzyme activity measurements All enzymes were assayed spectrophotometrically at 340 nm at 30 8C (unless otherwise stated) using an
Ultrospec III (LKB Instruments) or a CE 599 (Cecil Instruments Limited, Cambridge, England) and data were collected by computer using software (Brown and Dykstra, 1999) and hardware developed in-house (Brown and Pedley, unpublished). It is inappropriate to give a detailed description of the hardware here, but it has been tested exhaustively and the readings given by the spectrophotometer and the computer are identical and can be compared by the user at any time. Enzyme activity was determined in crude homogenates of L3 prepared by manually grinding frozen larvae, without further preparation. Hexokinase (HK, E.C.2.7.1.1) was assayed using a coupled reaction with glucose 6-phosphate dehydrogenase in a medium containing 5 mM glucose, 1 mM ATP, 200 mM NAD+, 1 mM MgCl2, 50 mM KH2PO4 pH 7.5 in the presence of 1 U glucose 6-phosphate dehydrogenase and 50 mg homogenate protein. Pyruvate kinase (PK, E.C.2.7.1.40) was assayed using a coupled reaction with lactate dehydrogenase in a medium containing 5 mM phosphoenolpyruvate, 1 mM ADP, 10 mM MgCl2, 200 mM NADH, 50 mM KH2PO4 pH 7.5 in the presence of 1 U lactate dehydrogenase and 50 mg homogenate protein. Malate dehydrogenase (MDH, E.C.1.1.1.37) was assayed in a medium containing 5 mM oxaloacetate, 200 mM NADH, 50 mM KH2PO4 pH 7.5 in the presence of 50 mg homogenate protein. Note that the MDH activity probably represented a mixture of at least two distinct enzymes, one located in the cytosol and another in the mitochondria, and no attempt was made to distinguish between them. The mammalian enzymes used as controls were porcine heart MDH, rabbit muscle PK and yeast HK, each of which was purchased from Sigma–Aldrich Pty Ltd., Castle Hill, NSW, Australia. In each case, the conditions were selected to ensure maximal activity at 30 8C. The protein concentration of larval homogenates was determined using the Bradford method (Bradford, 1976). No detergents were employed to solubilise the nematodes as they can interfere with protein assays (Sapan et al., 1999). 2.4. Homogenate incubations Larval homogenates were incubated for the required period (between 5 min and 60 min) in the assay medium (without substrates or effectors) in a heating block at the specified temperature (between 30 8C and 708) before being cooled to the assay temperature (30 8C).
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Fig. 1. Development of immotility of L3 O. circumcincta during incubation in water at various temperatures. (A) The time course of the developing immotility ((*) 40 8C, (&) 458, (~) 46 8C, (~) 47 8C, (^) 48 8C, (&) 508). (B) The proportion of immotile larvae after 30 min incubation. (C) Relationship between the temperature at which 50% of the larvae were immotile and incubation time (derived from A). The LT50 values were estimated by fitting a delayed gamma distribution (Young et al., 1980) to all of the available data by least squares nonlinear regression (in all cases r2 0.9). In each plot, the error bars represent 1 S.E.M. and the straight line in C was obtained using weighted linear regression.
3. Results
3.2. Glycolytic and tricarboxylic acid cycle enzyme activity
3.1. Motility of L3 O. circumcincta Sheathed larvae incubated at 40 8C for up to 90 min did not become significantly immotile, but there was a gradual decline in motility during the observation period at 45 8C and higher temperatures until even 2.5 min at 50 8C rendered the larvae completely immotile during the observation period (Fig. 1A). The larvae were exquisitely sensitive to temperatures greater than about 46 8C (Fig. 1B). From these data it can be estimated that temperature at which 50% of the larvae would be immotile (LT50) declined roughly linearly with incubation time (Fig. 1C) and, extrapolating from these data, that larvae exposed to about 48 8C would instantly become immotile (Fig. 1C). No systematic effort was made to estimate changes in the degree of motility of the larvae, although this also appeared to decline during incubation at temperatures above about 45 8C. Larvae rendered immotile by incubation at 50 8C did not recover during subsequent incubation at 4 8C, 25 8C or 37 8C.
In order to assess the importance of fermentative metabolism in thermotolerance, the activity of two glycolytic enzymes, hexokinase (HK) and pyruvate kinase (PK), and one tricarboxylic acid (TCA) cycle enzyme, malate dehydrogenase (MDH), was measured following 10 min incubation of the L3 homogenate at various temperatures. These enzymes are metabolically important in many species and appear to be significant to L3 O. circumcincta (Walker et al., 2005). The MDH activity measured in L3 O. circumcincta was the highest of any of the activities we have characterized to date. Incubation of larval homogenate temperatures up to 50 8C for 10 min reduced the activity of MDH by no more than about 10%, but above this temperature the activity declined steadily (Fig. 2). No MDH activity could be detected following incubation at 70 8C for 10 min (Fig. 2). In contrast, purified pig heart MDH activity in these conditions was significantly reduced following incubation at 40 8C for 10 min and almost completely inactivated at 44 8C (data not shown). The
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Fig. 2. Relative activity of malate dehydrogenase (*), pyruvate kinase (&) and hexokinase (~) in homogenate of L3 O. circumcincta incubated for 10 min at different temperatures. The data are from one representative experiment in which 100% was 5000 nmoles min1 mg 1, 300 nmoles min 1 mg 1, 10 nmoles min 1 mg 1 protein for MDH, PK and HK, respectively.
Fig. 4. Relative activity of malate dehydrogenase in homogenates prepared from L3 (*) and adult (*) H. contortus, C. elegans (~) and L3 O. circumcincta (&) following incubation at 50 8C for up to 1 h. The data are from one representative experiment in which 100% was at least 1000 nmol min 1 mg 1 protein.
activity of PK declined steadily approximately linearly with temperature between (30 8C and 50 8C) and HK activity dropped steeply between 44 8C and 45 8C. At 50 8C both HK and PK were essentially inactive (Fig. 2). Prolonged incubation of the O. circumcincta homogenate at 50 8C had little effect on the MDH activity, which fell about 20% over 60 min, but incubation of homogenate at 42 8C caused a steady decline in the activity of PK, which was essentially inactive after 60 min (Fig. 3). The activity of MDH in homogenate prepared from L3 or adult H. contortus or from a mixed population of C. elegans did not decline substantially during incubation at 50 8C for 60 min (Fig. 4). In contrast, MDH prepared from porcine heart showed only 10% of
its initial activity following incubation for only 10 min at 50 8C and was essentially inactive after 60 min (data not shown).
Fig. 3. Relative activity of O. circumcincta malate dehydrogenase (*) and pyruvate kinase (&) following incubation at 50 8C or 42 8C, respectively, for up to 60 min. The data are from one representative experiment in which 100% was 5000 and 300 nmoles min 1 mg 1 protein for MDH and PK, respectively.
4. Discussion The data reported here show that L3 O. circumcincta remain motile after 90 min at 45 8C (Fig. 1A), but are exquisitely sensitive to only slightly higher temperatures (Fig. 1B). The activity of MDH was relatively unaffected by temperatures up to 50 8C, but PK and HK were less thermotolerant and were inactive after 10 min at 50 8C (Fig. 2). The apparent thermostability of MDH from O. circumcincta was also apparent in other species of parasitic nematode, in adults and in the soil nematode C. elegans (Fig. 4). It is possible that the MDH activity observed with C. elegans might have been contaminated by protein from the E. coli on which the nematodes were grown. However, this seems unlikely because the C. elegans were washed extensively before use and Krijgsveld et al. (2003), who fed 15N-labelled E. coli to C. elegans and then analysed the appearance of label in C. elegans homogenates by 2D electrophoresis and mass spectrometry, did not report the presence of any bacterial proteins. However, even if there were substantial E. coli-derived MDH in the C. elegans homogenate, that enzyme is relatively temperature sensitive (50% inactivation within 3 min at 60 8C (Goward et al., 1994)), and so it is unlikely that it would have made a significant contribution to the temperature stability data shown in Fig. 4. The data reported here show that L3 O. circumcincta become immotile during the observation period over a
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very narrow temperature range between 45 8C and 50 8C (Fig. 1B). The regression line shown in Fig. 1C implies that half of the larvae would become immotile instantly at 48 8C, but even a few minutes at 50 8C certainly renders all the larvae immotile (Fig. 1A). These data are consistent with a need for the larvae to be resistant to temperatures above 40 8C and with the observation that L3 O. circumcincta kept in water at 35 8C remained motile for almost 8 weeks (Pandey et al., 1993). We have made no attempt to estimate the lifespan of L3 O. circumcincta at temperatures between 40 8C and 45 8C, although they are likely to be even shorter. However, the larvae are unlikely to experience prolonged periods at such high temperatures because of the diurnal temperature cycle. While this thermotolerance is not unique among nematodes (Nicholas, 1975), it is extraordinary. Even Alvinella pompejana, the ‘most thermotolerant eukaryote on earth’ (Desbruye`res et al., 1998), which lives near hydrothermal deep sea vents at temperatures of 2 to 105 8C, is unlikely to survive long-term at temperatures above 31 8C (Desbruye`res et al., 1998). Other invertebrates from the same environment become inactive between 40 8C and 50 8C (Lee, 2003). Interestingly, the rate of O. circumcincta development in ovine faecal culture has been reported to be reduced at temperatures above or below about 25 8C (Rossanigo and Gruner, 1995), similar to observations reported for O. ostertagi maintained in water (Pandey, 1972). Presumably, O. circumcincta eggs would not have hatched at temperatures of more than 40 8C, although the larvae survived. Hexokinase activity declined steeply between 45 8C and 46 8C (Fig. 2), in a range similar to the observed loss of motility (Fig. 1B), whereas PK activity declined over a much wider temperature range (Fig. 2). Since the activity of PK was much (about 30-fold) greater than that of HK, it is tempting to speculate that there is a link between the thermal inactivation of HK and the loss of larval motility. If such a connection does exist, further work would be required to confirm it. Nevertheless, both HK and PK activity was largely eliminated after 10 min incubation at 45 8C, which is consistent with a significant impact on glycolytic activity in the larvae and, therefore, on the supply of energy required for motility. This speculation is also supported by the observation that L3 O. circumcincta remain motile at 30 8C in anaerobic conditions and in the presence of cyanide (Simcock et al., 2006), consistent with the postulated dependence of motility on glycolytic metabolism. The temperature stability of MDH was substantially greater than that of HK and PK tested in similar
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conditions. Relatively thermostable MDH activity has been observed in other parasites, such as Ascaris suum (Zee and Zinkham, 1968), A. lumbricoides (Barrett and Fairbairn, 1971) and Toxocara canis (Andrade et al., 1983). In Ascaris spp. (Zee and Zinkham, 1968; Barrett and Fairbairn, 1971), the mitochondrial MDH (mMDH) was inactivated at lower temperatures than the cytosolic MDH (cMDH). This prompts the tentative hypothesis that the initial small (10%) decline in activity of MDH at about 50 8C could represent the inactivation of mMDH and the remainder might represent the inactivation of cMDH. This is not the only thermostable enzyme in A. suum, since Z˙o´ltowska (2001) reported a similar difference in thermostability of intestinal and muscle a-amylase. In contrast, Kapur et al. (1984, 1985) assayed a variety of enzyme activities in H. contortus homogenate and reported no specific thermotolerant MDH activity. It is not clear how these experiments were carried out, but it is possible that the temperatures reported represent those at which the activity was assayed rather than those at which the homogenate was incubated. It should be noted that the solutes present in the homogenate could have affected the apparent thermostability of MDH activity reported here (see Nash and Wiskich (1982), for example) and so the enzyme(s) need not be intrinsically thermotolerant. While this cannot be discounted two counterarguments can be made. First, during the high temperature incubations, the endogenous solutes were diluted at least 104-fold compared with those likely to apply in vivo; and, secondly, the observed thermotolerance compares well with that reported by Zee and Zinham (1968) for the purified A. suum enzyme(s) and by Andrade et al. (1983) for the partially purified T. canis cMDH. Infective larvae of O. circumcincta can tolerate temperatures up to about 45 8C for at least 90 min, but at higher temperatures the larvae become increasingly immotile. The relative temperature-sensitivity of glycolytic enzymes imply that any tissue having a high energy demand (such as nervous tissue and muscle) would be particularly affected by any reduction in the activity of the pathway. Of course, pathways other than those investigated here might be even more temperature sensitive. The temperature range tolerated by O. circumcincta is consistent with that of the environment(s) in which the larvae are found and it is possible that the seasonal variation in larval availability reported for various nematode parasites (Gordon, 1948; Vlassoff et al., 2001) is related to the thermotolerance of the larvae.
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