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Adenosine phosphorylase and other enzymes of purine salvage in Pulmonata snails and their Trematoda parasites
Comp. Biochem. Physiol. Vol. 107B, No. 1, pp. 135-139, 1994
0305-0491/94 $6.00 + 0.00 © 1993 Pergamon Press Ltd
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Adenosine phosphorylase and other enzymes of purine salvage in Pulmonata snails and their Trematoda parasites H. Trembacz and M. M. Je2ewska Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-532 Warsaw, 36 Rakowiecka St., Poland Activities of adenosine and inosine phosphorylases, adenine and hypoxanthine--guanine phosphoribosyltransferases and adenosine deaminase were determined in the hepatopancreas of parasite-free and infected gastropods of nine species, and in the larval forms of three species, of trematode. AdoPho activity exceeded those of all remaining enzymes in stylommatophoran snails but not those of basommatophoran snails and of the trematodes which parasitized them. The HGPRTase activity in snails and trematodes was lowest. The unaggressive trematode species seem to exert a more pronounced effect on the activities of purine salvage enzymes in hepatopanereas of infected snails. Key words: Adenosine phosphorylase; Purine salvage; Pulmonata; Trematoda; Snails; Parasites. Comp. Biochem. Physiol. 107B, 135-139, 1994.
Introduction Detection of the biochemical peculiarities offers an opportunity to expose the investigated organisms to the appropriate chemical agents. The occurrence of adenosine phosphorylase activity in many parasitic organisms, including Schistosoma mansoni (Trematoda) (cf. Trembacz and Je~ewska, 1993), seems to be such a biochemical peculiarity. Bearing in mind that for many digenetic trematodes the basommatophoran snails are obligatory intermediate hosts (Wright, 1966), and that a specific adenosine phosphorylase occurs in the hepatopancreas of the stylommatophoran gastropod Helix pomatia (Trembacz and Je~ewska, 1993), we checked whether this enzymic activity occurs commonly in gastropod species belonging to the Orders Stylommatophora and Basommatophora (Pulmonata). The activities of inosine phosphorylase, and adenosine deaminase, as well as of the adenine and hypoxanthine-guanine phosphoribosyltransferases, known to occur in H. pomatia (cf. Barankiewicz et aL, 1979), were also determined.
Individuals of basommatophoran snails infected with trematodes were examined, separately from uninfected ones, to see whether the infection causes any differences in the activity pattern of hepatopancreatic purine salvage enzymes. The enzymic activities were also determined in larval trematodes isolated from the hepatopancreas to reveal the possible differences between the organisms belonging to two different phyla: Mollusca and Platyhelminthes.
Materials and Methods Chemicals
The sources of chemicals were as follows: 5-phosphoribosyl- 1-pyrophosphate tetrasodium salt (PRPP), phenylmethylsulphonyl fluoride (PMSF), ribose- l-phosphate dicyclohexylammonium salt H20 (R1P), adenosine (Ado) (Sigma, St Louis, MO); 4-(2-hydroxyethyl)-lpiperazinepropane sulphonic acid (HEPPS), inosine, inosine-5'-monophosphate dilitium hexahydrate (IMP), A grade, (Calbiochem, CA); dithiothreitol (DTT), tris(hydroxyCorrespondence to: Maria M. Je~ewska, Institute of Biochemistry Biophysics,Polish Academyof Sciences, methyl)amino-methane (Tris), adenosine-Ymonophosphate (scrva, Heidelberg, Germany); ul. Rakowiecka36, 02-532 Warszawa, Poland Received 25 May 1993; accepted 6 July 1993 hypoxanthine, adenine, adenosine, all 8-'4C 135
136
H. Trembacz and M. M. Je~ewska
labelled (Radiochemical Centre, Amersham, Bucks, U.K.).
Animals Snails in the active period of life were collected in fields and ponds, classified, tested for a trematode infection and used for experiments within three days.
expressed in nkat/g tissue wet weight or in nmol/min/mg protein of the crude hepatopancreas extract (in the case of the parasite--the crude whole body extract). Protein was determined by the method of Bradford (1976) with bovine albumin as a standard.
Preparation of enzymic extract
Results and Discussion
In each experiment, one to three specimens of given snail species were used. The weights of the wet hepatopancreatic tissue used to obtain an enzymic extract were 92-680 mg and 0.63-5.7 g in the case of the basommatophoran and stylommatophoran snails, respectively. Snails were killed by cutting the nerve ring (ganglia cerebralia) and the shell (absent in slugs) was removed. The separated hepatopancreas was weighed, and homogenized in 100 mM HEPPS--KOH buffer, pH 8.0, containing l mM PMSF, 10mM DTT and 20% glycerol, with the use of a Potter-Elvehjem homogenizer in an ice-water bath. The homogenate was used without any treatment or was centrifuged at 20,000g for 30 min in a cold room, and in the resulting supernatant the enzymic activities were determined. Crude extracts were obtained from the trematode larval forms in the same way.
The hepatopancreas of gastropods of all species examined displayed high specific activity of adenosine phosphorylase (Table 1). Therefore, the presence of the AdoPho activity is not specific for the parasitic organisms but widespread in at least the Pulmonata gastropods, being intermediate hosts for many parasites. In the representatives of the terrestrial stylommatophoran snails (Helicidae) and slugs (Limacidae and Arionidae) the AdoPho activity exceeds that of inosine phosphorylase, and is the highest of all enzymic activities determined in these studies. In freshwater basommatophoran snails (Lymnaeidae and Planorbidae), the AdoPho activity sometimes equals that of InoPho (in L. stagnalis) but, in general, it is lower. Moreover, a peculiarly very low activity of hypoxanthine-guanine phosphoribosyltransferase is characteristic of all gastropod species examined (Table 1). The HGPRTase activity is lowest in slugs; in all species it was several times lower than that of adenine phosphoribosyltransferase. Strikingly, the pattern of enzymic activity of purine salvage is rather similar in terrestrial and freshwater gastropods despite their dissimilar nitrogen excretion (urico-, ureoand ammonotelic). The physiological processes of gastropods are, to some extent, correlated with seasonal
Enzymic activity determination Activities of adenosine and inosine phosphorylases (AdoPho and InoPho, respectively), adenine and hypoxanthine-guanine phosphoribosyltransferases (APRTase and HGPRTase, respectively) and adenosine deaminase (ADA) were determined as described earlier (Trembacz and Je~ewska, 1993). Enzymic activities were
Table 1. Specific activity of some enzymes of purine salvage pattern in the hepatopancreas of several species of uninfected gastropods AdoPho Gastropoda, Pulmonata
Activities of InoPho APRTase HGPRTase (nkat/g wet hepatopancreas)
ADA
Stylommatophora Heficidae
Helix pomatia active Helix pomatia hibernating Cepaea sp.
31.3 23.8 20.0
15.5 7.9 13.5
8.5 7.9 3.5
1.9 2.6 1.6
6.3 9.2 19.5
20.3
17.4
5.9
0.2
60.1
39.0
23.9
10.5
0.2
57.2
27.4 15.1 19.8
26,8 72.4 163.6
15.6 10.4 32.7
1.5 2.3 3.2
56.5 50.5 8.7
7.1
57.8
9.8
0.6
44.7
Limacidae
Limax maximus Arionidae Arion sp. Basommatophora Lymnaeidae
Lymnaea stagnalis Galba sp. Radix auricularia Planorbidae Coretus sp.
Enzymes of purine salvage in snails and trematodes
137
Table 2. Effects of parasitic infection on some enzymes of purine salvage pattern in the hepatopancreas of basornmatophoran gastropods
Date 3 June 2 Sept. 24 Sept. 8 Oct. 11 July 2 Sept. 11 July 3 June 3 June 9 Oct. 3 June 24 Sept.
Snail species Lymnaea stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis Radix limosa R. limosa Galba sp. Galba sp. Galba sp. Radix auricularia R. auricularia R. auricularia Coretus sp. Coretus sp. Coretus sp. Coretus sp.
Infection with* E Er Er Er O O O O M T N Ns
AdoPho 16.0 14.8 28.4 28.2 28.1 28.9 22.3 33.8 31.8 28.4 16.8 28.3 6.5 14.6 7.4 9.0 11.6 14.5 13.8 7.5 5.9 6.6 14.0
Activities of InoPho APRTase HGPRTase (nmol/min/mg protein of crude extract) 80.2 68.9 27.7 32.8 53.2 23.0 62.1 48.1 30.7 27.7 20.6 61.3 67.7 70.9 63.3 62.5 81.4 140.4 95.5 52.6 78.4 24.9 192.3
10.2 14.5 16.2 15.5 12.4 11.5 9.1 22.6 36.3 16.2 12.0 46.9 50.9 10.1 7.7 7.6 12.3 29.5 25.8 10.4 7.9 8.4 16.2
1.3 0.9 1.6 1.6 1.3 1.4 1.5 1.2 5.7 1.6 2.6 1.8 7.5 2.2 1.2 1.0 0.3 2.9 3.7 0.1 0.5 0.1 1.1
ADA 53.5 20.4 59.6 85.4 22.0 15.5 16.6 42.9 23.6 59.6 46.1 15.5 24.9 46.9 21.2 14.2 7.2 7.1 22.1 20.0 7.8 25.8 16.1
*O: Opisthioglyphae ranae (Plagiorhiidae, Epithelioeystida); E: Echinoparyphium aconiatum; M: Moliniella anceps (both Echinostomatidae); T: Trichobilharzia ocellata (sehistosomatidae); N: Notocotylus ephemera (Notocotylidae); (E, M, T, N: Anepitheliocystida); r, s: rediae or sporocysts, respectively, were found in the hepatopancreas.
changes in the environment (Krishna, 1985); for example, the deposits of the end-products of nitrogen metabolism by H. pomatia are most abundant after rainfall (Je~ewska, 1969). Changes in the physiological activities could be accompanied by alterations of the metabolic rate and thus by some fluctuations of the enzymic activities. In this connection we compared enzymic activities in the hepatopancreas of parasite-free specimens of L. stagnalis, collected at different sites on 6 June and 11 July (summer), and on 2 and 24 September and 8 October (autumn). In the obtained hepatopancreatic extracts (Table 2) only the activity (in nmol/min per mg protein) of HGPRTase remained constant (1.2-1.6) whereas activities of AdoPho, InoPho, APRTase and ADA varied within the wide ranges of 16.0-33.8, 27.7-80.2, 10.2-22.6 and 22.0-59.6, respectively. Differences in these activities between uninfected R. auricularia and Coretus sp. individuals, collected on different dates (Table 2) may also relate to seasonal changes in the environment. Anyway, the intraspecies fluctuations of various enzymic activities often exceed the interspecies differences (Table 1). Some specimens of basommatophoran gastropods were found to be infected with trematodes identified as belonging to families detailed in Table 2. Three families of the Super-
order Anepitheliocystida were represented as follows: Echinostomatidae by Echinoparyphium aconiatum and Moliniella anceps, Schistosomatidae by Trichobilharzia ocellata and Notocotylidae by Notocotylus ephemera. Each larval form of these trematode species occurred in another host gastropod species. Two subsequent trematode species belonged to the Plagiorchiidae (Superorder Epitheliocystida): Opisthioglyphae ranae, found in three gastropod species and Plagiorchis neomidis, identified in only one host species. Among the above trematode species, the two belonging to the Echinostosomatidae, during their larval development, go through a stage of aggressive rediae which ingest the snail hepatopancreatie ceils. The remained four trematode species identified are unaggressive but their sporoeysts cause degenerative changes of the hepatopancreatic tissue (Wright, 1966); in the case of P. neomidis after the isolation of sporocysts we obtained so little hepatopancreatic tissue that the enzymic activities could be determined only in the parasite (Table 3). Coloured rediae and large sporocysts were found only in the hepatopancreas of snails collected in autumn (Table 2). Effects of trematode infection on hepatopancreatic enzymic activities of host snails seems to be more evident in the case of unaggressive trematodes. The infection of L. stagnalis by
H. Trembacz and M. M. Je~ewska
138
Table 3. Activityof some enzymesof the purine salvage patterns in larval forms of trematodes Activities of AdoPho InoPho APRTase HGPRTase ADA Trematoda-Digenea (nmol/min/mg protein of crude extract) Anepithelioeystida Echinostomatidae Echinoparyphium aconiatum i
rediae (2 Sept.) rediae (24 Sept.) rediae (8 Oct.) Notocotylidae
1.2 3.0 2.1
26.8 50.4 59.7
4.2 1.9 3.2
6.4 2.4 4.2
2.5 3.5 26.8
10.I 2.1
214.3 10.0
5.6 3.2
1.1 1.9
90.9 0.0
3.5
3.4
Notocotylus ephemera2
sporocysts (24 Sept.) cercariae (24 Sept.) Epitheliocystida Plagiorchiidae Plagiorchis neomidis3
sporocysts (9 Oct.) 32.1 195.1 55.4 Intermediate host: 1--L. stagnalis; 2--Coretus sp.; 3--R. auricularia. E. aconiatum exerted a slight (if any) effect, even when the hepatopancreas contained the aggressive rediae (Table 2). On the other hand, the infection with unaggressive O. ranae caused a decrease in the AdoPho activity in L. stagnalis (only in autumn), L. limosa and Galba sp., and an increase in the H G P R T a s e activity in both the former species, but a drop in the latter species. A higher activity of HGPRTase was found also in R. auricularia and Coretus sp. infected by T. ocellata and N. ephemera, respectively, both being unaggressive species. The majority of changes (decrease or increase) concerned the ADA activity in snails infected with unaggressive, as well as by aggressive, trematodes. The APRTase activity seems to be less vulnerable to parasite infection. Such a diversity of the effects of trematode infection on the enzymic activities in host snails has been described (Krishna, 1985), also, in the case of carbohydrate and protein metabolism, as well as of the ornithine-urea cycle enzymes (Kondaiah and Venkateswara Rao, 1984). There are great differences in the pattern of activities of purine salvage enzymes between the larval forms of trematodes and the snail hepatopancreas (Tables 2 and 3). In the rediae of E. aconitum, the AdoPho, APRTase and ADA activities were many times lower than those in the hepatopancreas of parasite-free host L. stagnalis, whereas the HGPRTase activity was three times higher, and that of InoPho was similar. In turn, in the sporocysts of N. ephemera the AdoPho, InoPho, H G P R T a s e and ADA activities were higher, and that of APRTase a little lower, as compared with the respective activities in the hepatopancreas of host Coretus sp. In the system of the sporocysts of P. neomidis and the hepatopancreas of its host R. auricularia, the situation was different; in sporocysts, the AdoPho, InoPho and also
APRTase activities were higher than in the snail hepatopancreas, whereas the ADA activity was lower and that of H G P R T a s e similar. Thus, the metabolic relationships in the host snail-parasite system are very differentiated and complex. This is in accordance with the theoretical models of various host-parasite relationships (Renaud and De Meeus, 1991). In the free-living cercariae of N. ephemera, shed by a specimen of Coretus sp., all enzymic activities, except for that of HGPRTase, were lower, compared with N. ephemera sporocysts (Table 3); the AdoPho activity was also present in this stage of trematode development. The role of adenosine phosphorylase occurring in freeliving snails and larval forms of trematodes (free-living or developing in host tissues) remains obscure. It is stressed that in mammals, being the final hosts for many trematode species causing serious diseases, the specific adenosine phosphorylase has never been found, although a decomposition of adenosine to adenine has sometimes been reported. Acknowledgements--The authors are greatly indebted to
Prof. Bo~enaGrabda-Kazubska (Institute of Parasitology, Polish Academyof Sciences)for kind donation of specimens of basommatophoran snails and their classification,identification of trematodes and very helpful discussion.
References Barankiewicz J., Kadlubowska H. and Jezewska M. M. (1979) Adenine cycle in hepatopancreocytes of Helix pomatia (Gastropoda). Acta Biochim. Polon. 26, 11-18. Bradford M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dyebinding. Analyt. Biochem.72, 248-254. JeC~ewskaM. M. (1969)The nephridialexcretionof guanine, xanthine and uric acid in slugs (Limacidae) and snails (Helicidae). Acta Biochim. Polon. 16, 313-320.
Enzymes of purine salvage in snails and trematodes Kondaiah S. and Venkateswara Rao P. (1984) Impact of larval trematode infection on the nitrogen excretion and omithine---urea cycle enzymes in the pulmonate snail host, Lymnaea luteola. J. Invert. Path. 44, 249-228. Krishna G. V. R. 0985) Host-parasite relationship of larval trematodes. Ind. Rev. Life Sci. 5, 205-228. Renaud F. and De Meeus T. (1991) A simple model of host-parasite evolutionary relationships. Para-
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sitism: compromise or conflict? J. theor. Biol. 152, 319-327. Trembacz H. and Jeiewska M. M. (1993) Specific adenosine phosphorylase from hepatopancreas of gastropod Helix pomatia. Comp. Biochem. Physiol. 104Bt 481-487. Wright C. A. (1966) The pathogenesis of Helminthes in the Mollusca. Helminth. Abstr. 35, 207-224.
0305-0491/94 $6.00 + 0.00 © 1993 Pergamon Press Ltd
Printed in Great Britain
Adenosine phosphorylase and other enzymes of purine salvage in Pulmonata snails and their Trematoda parasites H. Trembacz and M. M. Je2ewska Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-532 Warsaw, 36 Rakowiecka St., Poland Activities of adenosine and inosine phosphorylases, adenine and hypoxanthine--guanine phosphoribosyltransferases and adenosine deaminase were determined in the hepatopancreas of parasite-free and infected gastropods of nine species, and in the larval forms of three species, of trematode. AdoPho activity exceeded those of all remaining enzymes in stylommatophoran snails but not those of basommatophoran snails and of the trematodes which parasitized them. The HGPRTase activity in snails and trematodes was lowest. The unaggressive trematode species seem to exert a more pronounced effect on the activities of purine salvage enzymes in hepatopanereas of infected snails. Key words: Adenosine phosphorylase; Purine salvage; Pulmonata; Trematoda; Snails; Parasites. Comp. Biochem. Physiol. 107B, 135-139, 1994.
Introduction Detection of the biochemical peculiarities offers an opportunity to expose the investigated organisms to the appropriate chemical agents. The occurrence of adenosine phosphorylase activity in many parasitic organisms, including Schistosoma mansoni (Trematoda) (cf. Trembacz and Je~ewska, 1993), seems to be such a biochemical peculiarity. Bearing in mind that for many digenetic trematodes the basommatophoran snails are obligatory intermediate hosts (Wright, 1966), and that a specific adenosine phosphorylase occurs in the hepatopancreas of the stylommatophoran gastropod Helix pomatia (Trembacz and Je~ewska, 1993), we checked whether this enzymic activity occurs commonly in gastropod species belonging to the Orders Stylommatophora and Basommatophora (Pulmonata). The activities of inosine phosphorylase, and adenosine deaminase, as well as of the adenine and hypoxanthine-guanine phosphoribosyltransferases, known to occur in H. pomatia (cf. Barankiewicz et aL, 1979), were also determined.
Individuals of basommatophoran snails infected with trematodes were examined, separately from uninfected ones, to see whether the infection causes any differences in the activity pattern of hepatopancreatic purine salvage enzymes. The enzymic activities were also determined in larval trematodes isolated from the hepatopancreas to reveal the possible differences between the organisms belonging to two different phyla: Mollusca and Platyhelminthes.
Materials and Methods Chemicals
The sources of chemicals were as follows: 5-phosphoribosyl- 1-pyrophosphate tetrasodium salt (PRPP), phenylmethylsulphonyl fluoride (PMSF), ribose- l-phosphate dicyclohexylammonium salt H20 (R1P), adenosine (Ado) (Sigma, St Louis, MO); 4-(2-hydroxyethyl)-lpiperazinepropane sulphonic acid (HEPPS), inosine, inosine-5'-monophosphate dilitium hexahydrate (IMP), A grade, (Calbiochem, CA); dithiothreitol (DTT), tris(hydroxyCorrespondence to: Maria M. Je~ewska, Institute of Biochemistry Biophysics,Polish Academyof Sciences, methyl)amino-methane (Tris), adenosine-Ymonophosphate (scrva, Heidelberg, Germany); ul. Rakowiecka36, 02-532 Warszawa, Poland Received 25 May 1993; accepted 6 July 1993 hypoxanthine, adenine, adenosine, all 8-'4C 135
136
H. Trembacz and M. M. Je~ewska
labelled (Radiochemical Centre, Amersham, Bucks, U.K.).
Animals Snails in the active period of life were collected in fields and ponds, classified, tested for a trematode infection and used for experiments within three days.
expressed in nkat/g tissue wet weight or in nmol/min/mg protein of the crude hepatopancreas extract (in the case of the parasite--the crude whole body extract). Protein was determined by the method of Bradford (1976) with bovine albumin as a standard.
Preparation of enzymic extract
Results and Discussion
In each experiment, one to three specimens of given snail species were used. The weights of the wet hepatopancreatic tissue used to obtain an enzymic extract were 92-680 mg and 0.63-5.7 g in the case of the basommatophoran and stylommatophoran snails, respectively. Snails were killed by cutting the nerve ring (ganglia cerebralia) and the shell (absent in slugs) was removed. The separated hepatopancreas was weighed, and homogenized in 100 mM HEPPS--KOH buffer, pH 8.0, containing l mM PMSF, 10mM DTT and 20% glycerol, with the use of a Potter-Elvehjem homogenizer in an ice-water bath. The homogenate was used without any treatment or was centrifuged at 20,000g for 30 min in a cold room, and in the resulting supernatant the enzymic activities were determined. Crude extracts were obtained from the trematode larval forms in the same way.
The hepatopancreas of gastropods of all species examined displayed high specific activity of adenosine phosphorylase (Table 1). Therefore, the presence of the AdoPho activity is not specific for the parasitic organisms but widespread in at least the Pulmonata gastropods, being intermediate hosts for many parasites. In the representatives of the terrestrial stylommatophoran snails (Helicidae) and slugs (Limacidae and Arionidae) the AdoPho activity exceeds that of inosine phosphorylase, and is the highest of all enzymic activities determined in these studies. In freshwater basommatophoran snails (Lymnaeidae and Planorbidae), the AdoPho activity sometimes equals that of InoPho (in L. stagnalis) but, in general, it is lower. Moreover, a peculiarly very low activity of hypoxanthine-guanine phosphoribosyltransferase is characteristic of all gastropod species examined (Table 1). The HGPRTase activity is lowest in slugs; in all species it was several times lower than that of adenine phosphoribosyltransferase. Strikingly, the pattern of enzymic activity of purine salvage is rather similar in terrestrial and freshwater gastropods despite their dissimilar nitrogen excretion (urico-, ureoand ammonotelic). The physiological processes of gastropods are, to some extent, correlated with seasonal
Enzymic activity determination Activities of adenosine and inosine phosphorylases (AdoPho and InoPho, respectively), adenine and hypoxanthine-guanine phosphoribosyltransferases (APRTase and HGPRTase, respectively) and adenosine deaminase (ADA) were determined as described earlier (Trembacz and Je~ewska, 1993). Enzymic activities were
Table 1. Specific activity of some enzymes of purine salvage pattern in the hepatopancreas of several species of uninfected gastropods AdoPho Gastropoda, Pulmonata
Activities of InoPho APRTase HGPRTase (nkat/g wet hepatopancreas)
ADA
Stylommatophora Heficidae
Helix pomatia active Helix pomatia hibernating Cepaea sp.
31.3 23.8 20.0
15.5 7.9 13.5
8.5 7.9 3.5
1.9 2.6 1.6
6.3 9.2 19.5
20.3
17.4
5.9
0.2
60.1
39.0
23.9
10.5
0.2
57.2
27.4 15.1 19.8
26,8 72.4 163.6
15.6 10.4 32.7
1.5 2.3 3.2
56.5 50.5 8.7
7.1
57.8
9.8
0.6
44.7
Limacidae
Limax maximus Arionidae Arion sp. Basommatophora Lymnaeidae
Lymnaea stagnalis Galba sp. Radix auricularia Planorbidae Coretus sp.
Enzymes of purine salvage in snails and trematodes
137
Table 2. Effects of parasitic infection on some enzymes of purine salvage pattern in the hepatopancreas of basornmatophoran gastropods
Date 3 June 2 Sept. 24 Sept. 8 Oct. 11 July 2 Sept. 11 July 3 June 3 June 9 Oct. 3 June 24 Sept.
Snail species Lymnaea stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis L. stagnalis Radix limosa R. limosa Galba sp. Galba sp. Galba sp. Radix auricularia R. auricularia R. auricularia Coretus sp. Coretus sp. Coretus sp. Coretus sp.
Infection with* E Er Er Er O O O O M T N Ns
AdoPho 16.0 14.8 28.4 28.2 28.1 28.9 22.3 33.8 31.8 28.4 16.8 28.3 6.5 14.6 7.4 9.0 11.6 14.5 13.8 7.5 5.9 6.6 14.0
Activities of InoPho APRTase HGPRTase (nmol/min/mg protein of crude extract) 80.2 68.9 27.7 32.8 53.2 23.0 62.1 48.1 30.7 27.7 20.6 61.3 67.7 70.9 63.3 62.5 81.4 140.4 95.5 52.6 78.4 24.9 192.3
10.2 14.5 16.2 15.5 12.4 11.5 9.1 22.6 36.3 16.2 12.0 46.9 50.9 10.1 7.7 7.6 12.3 29.5 25.8 10.4 7.9 8.4 16.2
1.3 0.9 1.6 1.6 1.3 1.4 1.5 1.2 5.7 1.6 2.6 1.8 7.5 2.2 1.2 1.0 0.3 2.9 3.7 0.1 0.5 0.1 1.1
ADA 53.5 20.4 59.6 85.4 22.0 15.5 16.6 42.9 23.6 59.6 46.1 15.5 24.9 46.9 21.2 14.2 7.2 7.1 22.1 20.0 7.8 25.8 16.1
*O: Opisthioglyphae ranae (Plagiorhiidae, Epithelioeystida); E: Echinoparyphium aconiatum; M: Moliniella anceps (both Echinostomatidae); T: Trichobilharzia ocellata (sehistosomatidae); N: Notocotylus ephemera (Notocotylidae); (E, M, T, N: Anepitheliocystida); r, s: rediae or sporocysts, respectively, were found in the hepatopancreas.
changes in the environment (Krishna, 1985); for example, the deposits of the end-products of nitrogen metabolism by H. pomatia are most abundant after rainfall (Je~ewska, 1969). Changes in the physiological activities could be accompanied by alterations of the metabolic rate and thus by some fluctuations of the enzymic activities. In this connection we compared enzymic activities in the hepatopancreas of parasite-free specimens of L. stagnalis, collected at different sites on 6 June and 11 July (summer), and on 2 and 24 September and 8 October (autumn). In the obtained hepatopancreatic extracts (Table 2) only the activity (in nmol/min per mg protein) of HGPRTase remained constant (1.2-1.6) whereas activities of AdoPho, InoPho, APRTase and ADA varied within the wide ranges of 16.0-33.8, 27.7-80.2, 10.2-22.6 and 22.0-59.6, respectively. Differences in these activities between uninfected R. auricularia and Coretus sp. individuals, collected on different dates (Table 2) may also relate to seasonal changes in the environment. Anyway, the intraspecies fluctuations of various enzymic activities often exceed the interspecies differences (Table 1). Some specimens of basommatophoran gastropods were found to be infected with trematodes identified as belonging to families detailed in Table 2. Three families of the Super-
order Anepitheliocystida were represented as follows: Echinostomatidae by Echinoparyphium aconiatum and Moliniella anceps, Schistosomatidae by Trichobilharzia ocellata and Notocotylidae by Notocotylus ephemera. Each larval form of these trematode species occurred in another host gastropod species. Two subsequent trematode species belonged to the Plagiorchiidae (Superorder Epitheliocystida): Opisthioglyphae ranae, found in three gastropod species and Plagiorchis neomidis, identified in only one host species. Among the above trematode species, the two belonging to the Echinostosomatidae, during their larval development, go through a stage of aggressive rediae which ingest the snail hepatopancreatie ceils. The remained four trematode species identified are unaggressive but their sporoeysts cause degenerative changes of the hepatopancreatic tissue (Wright, 1966); in the case of P. neomidis after the isolation of sporocysts we obtained so little hepatopancreatic tissue that the enzymic activities could be determined only in the parasite (Table 3). Coloured rediae and large sporocysts were found only in the hepatopancreas of snails collected in autumn (Table 2). Effects of trematode infection on hepatopancreatic enzymic activities of host snails seems to be more evident in the case of unaggressive trematodes. The infection of L. stagnalis by
H. Trembacz and M. M. Je~ewska
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Table 3. Activityof some enzymesof the purine salvage patterns in larval forms of trematodes Activities of AdoPho InoPho APRTase HGPRTase ADA Trematoda-Digenea (nmol/min/mg protein of crude extract) Anepithelioeystida Echinostomatidae Echinoparyphium aconiatum i
rediae (2 Sept.) rediae (24 Sept.) rediae (8 Oct.) Notocotylidae
1.2 3.0 2.1
26.8 50.4 59.7
4.2 1.9 3.2
6.4 2.4 4.2
2.5 3.5 26.8
10.I 2.1
214.3 10.0
5.6 3.2
1.1 1.9
90.9 0.0
3.5
3.4
Notocotylus ephemera2
sporocysts (24 Sept.) cercariae (24 Sept.) Epitheliocystida Plagiorchiidae Plagiorchis neomidis3
sporocysts (9 Oct.) 32.1 195.1 55.4 Intermediate host: 1--L. stagnalis; 2--Coretus sp.; 3--R. auricularia. E. aconiatum exerted a slight (if any) effect, even when the hepatopancreas contained the aggressive rediae (Table 2). On the other hand, the infection with unaggressive O. ranae caused a decrease in the AdoPho activity in L. stagnalis (only in autumn), L. limosa and Galba sp., and an increase in the H G P R T a s e activity in both the former species, but a drop in the latter species. A higher activity of HGPRTase was found also in R. auricularia and Coretus sp. infected by T. ocellata and N. ephemera, respectively, both being unaggressive species. The majority of changes (decrease or increase) concerned the ADA activity in snails infected with unaggressive, as well as by aggressive, trematodes. The APRTase activity seems to be less vulnerable to parasite infection. Such a diversity of the effects of trematode infection on the enzymic activities in host snails has been described (Krishna, 1985), also, in the case of carbohydrate and protein metabolism, as well as of the ornithine-urea cycle enzymes (Kondaiah and Venkateswara Rao, 1984). There are great differences in the pattern of activities of purine salvage enzymes between the larval forms of trematodes and the snail hepatopancreas (Tables 2 and 3). In the rediae of E. aconitum, the AdoPho, APRTase and ADA activities were many times lower than those in the hepatopancreas of parasite-free host L. stagnalis, whereas the HGPRTase activity was three times higher, and that of InoPho was similar. In turn, in the sporocysts of N. ephemera the AdoPho, InoPho, H G P R T a s e and ADA activities were higher, and that of APRTase a little lower, as compared with the respective activities in the hepatopancreas of host Coretus sp. In the system of the sporocysts of P. neomidis and the hepatopancreas of its host R. auricularia, the situation was different; in sporocysts, the AdoPho, InoPho and also
APRTase activities were higher than in the snail hepatopancreas, whereas the ADA activity was lower and that of H G P R T a s e similar. Thus, the metabolic relationships in the host snail-parasite system are very differentiated and complex. This is in accordance with the theoretical models of various host-parasite relationships (Renaud and De Meeus, 1991). In the free-living cercariae of N. ephemera, shed by a specimen of Coretus sp., all enzymic activities, except for that of HGPRTase, were lower, compared with N. ephemera sporocysts (Table 3); the AdoPho activity was also present in this stage of trematode development. The role of adenosine phosphorylase occurring in freeliving snails and larval forms of trematodes (free-living or developing in host tissues) remains obscure. It is stressed that in mammals, being the final hosts for many trematode species causing serious diseases, the specific adenosine phosphorylase has never been found, although a decomposition of adenosine to adenine has sometimes been reported. Acknowledgements--The authors are greatly indebted to
Prof. Bo~enaGrabda-Kazubska (Institute of Parasitology, Polish Academyof Sciences)for kind donation of specimens of basommatophoran snails and their classification,identification of trematodes and very helpful discussion.
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