Variations in aminotransferase activity and total free amino acid level in the body fluid of the snail Lymnaea luteola during different larval trematode infections

JOURNAL

OF INVERTEBRATE

Variations Level

19, 36-41 (1972)

PATHOLOQY

in Aminotransferase in the Body

Fluid

Digerent

Activity of the Snail

Larval

and Total Lymnaea

Trematode

Free Amino

Acid

ZuteoZa during

Infections1

L. MANOJUR, P. VENKATESWARA RAO, AND K. S. SWAMI Department of Zoology, Sri Venkateswara University,

Tir-upati, A.P. India

Received May 10, 1971 The common freshwater snail Lymnaea Zuleola was found to shed three types of trematode cercariae: the xiphidiocercaria of Prosthogonimus sp., the furcocercaria of Schietosoma incognitum, and an amphistome cercaria, Cercaria pigmentala. Total free amino acid content of the body fluids of snails drops significantly in all three types of infection. The total aminotransferase activity in the body fluid rises significantly in xiphidio- and furcocercarial infections and drops in pigmented cercarial infections. Aspartate aminotransferase (GOT) level increases significantly in all types of infection but not alanine aminotransferase (GPT). Alanine aminotransferase activity shows no significant rise in furcocercarial infection and actually drops in pigmented cercarial infections. These differences in the aminotransferase levels have been explained in terms of the biochemical requirements of the parasites harbored by the .&ails.

A number of enzymes have been studied in connection with the diagnosis of diseases, since characteristic serum enzyme patterns have been found to be associatedwith known diseases.The serum enzyme pattern is often distorted by changes in the enzyme pattern of affected organs. The best example of such a distortion is the relative change in the activities of the two commonly studied aminotransferases, aspartate aminotransferase (GOT) and alanine aminotransferase (GPT). The ratio of GOT: GPT in the serum is often taken as an index for liver diseses. Evidence for hepatic damage has been presented as distortions in serum aminotransferase activity patterns in vertebrates during protozoan and helminthis infections (von Brand, 1966). Similar information is lacking in invertebrate hosts. Recently, Mengebier and Wood (1969) have studied the effect of sporozoan infection on the serum phosphounder P.L. 480. Academic

Praaa,Inc.

MATERIAUJ

AND

METHODS

In the course of a survey conducted at Rayalaseema, specimens of Lymnaea luteola from different parts of Chandragiri taluq of

1 This research wsa supported in part by a grant from the U.S. Department of Agriculture

Copyright Q 1972by

hexose isomerase, alkaline phosphatase, and lactate dehydrogenaselevels of the American oyster, Crassostreavirginica. The hepatopancreas of a snail is damaged extensively during larval trematode infections (Cheng and Snyder, 1962; Cheng, 1963b, c; Cheng and Burton, 1965; James, 1965; James and Bowers, 1967). The free amino acid pool of molluscan hosts is reported to show striking changes upon infection (Cheng, 1963c; Senft, 1967; Richards, 1969; Feng et al., 1970). In view of theEe reports, it was felt that a study of transaminaEe activity and total free amino acid levels in the body fluid of the snail hmnaea lu&ola during different trematode infections may reveal the extent of tissue damage done to the host and also help to explain some of the changesin host’s metabolism induced by these larval trematodes.

36

AMINOTRANSFEFMSE

Andhra Pradesh, India, were brought to the laboratory in perforated wide-mouth polythene bottles. They were isolated in about 25 ml of dechlorinated tap water in specimen tubes for 24 hr and observed periodically. Those which shed cercariae were considered infected and others normal. The snails from different regions were observed to be shedding different types of cercariae. The commonly encountered cercariae of these parts are the xiphidiocercaria of Prosthogonimus sp., the furcocercous cercaria of Schistosoma incognitum, and an amphistome cercaria, Cerrw-ia pigment&a. The snails with single infections were maintained in a cold room (23 f 2°C) in separate, well-aerated aquaria with 2-inch column of dechlorinated tap water and fed on half-boiled leaves of Amaranthus viredis. The normal snails also were similarly maintained and examined periodically for infection. Well fed and actively moving snails in the weight range of 400450 mg were blotted free of water and used for study. The snails were exsanguinated and the body fluid collected simultaneously from all snails using an improvised stand into separate ice-jacketed, wide-mouth fusion tubes and centrifuged at 2000 rpm for 5 min. Body fluids with suspected cercarial contaminations were discarded. The exsanguinated noninfected snails were teased in gastropod ringer (Hughes and Kerkut, 1956) under a stereomicroscope to confirm the absence of infection. The body fluids were pooled in each case from 3 snails and appropriately diluted when necessary. All operations were done in a walk-in cooler at 16 f 2’C. Aminotransferase activity was assayed calorimetrically by the simplified procedure of Rietmann and Frankel as given by Bergmeyer (1965). The incubation mixture contained 100 pmoles of phosphate buffer, pH 7.4, either 100 /Imoles of L-aspartic acid or 200 pmoles of ncalanine, 2 pmoles of a-oxoglutarate in a total volume of 1 ml. The reaction was started by adding 0.2 ml of body fluid appropriately diluted with phosphate

37

IN &'7l7l&?a

buffer of pH 7.4. The concentration of the red phenylhydraaone complex was measured at 540 MI.1 and the value read from a standard curve prepared with sodium pyruvate. The enzyme activity was expressed as micromoles of pyruvate per milligram of protein per hour. Protein in the body fluid was estimated by the method of Lowry et al. (1951), using bovine albumin as the st.andard. Total free amino acid content was determined in deproteinized body fluid by the ninhydrin method (Colowick and Kaplan, 1957). Tyrosine was used as the standard and the free amino acid content expressed as micrograms of tyrosine per milliliter of body fluid. L-Aspartic acid, cr-oxoglutaric acid, sodium pyruvate, and methyl Cellosolve (ethylene glycolmonoethyl ether) were supplied by E. Merck A. G., Darmstadt, Germany, and t,he rest by the British Drug House, Poole, England. Optical densities were measured with a Hilger and Watts (England) spectrophotometer. RESULTS

From the results presented in Table 1: it appears that the total free amino acid pool of infected snails is reduced. The drop in the body fluid free amino acid level, although significant in all the types of infections, is most acute (66.5%) in snails infected wit,h Cercuria pigmentuta. The total body fluid aminotransferase (BFAT) activity increases signi&antly in Prosthogonimus and S. incognitum infections (289.5 % and 30.08 70, respectively), whereas it drops in C. pigmentata infections (15.26 %). Of the two body fluid aminotransferases studied, aspartate aminotransferase (BFGOT) activity in normal snails is lower than alanine aminotransferase (BFGPT) activity, thereby giving a De Ritis quotient of 0.8 (Table 1). But in all the types of infections studied, it is the BFGOT activity level that is significantly elevated (Table l), thereby raising the De Ritis quotient to values above 1.0. It is interesting to note

3.439 f

Alanine iuninotranaferase activity (GPT) (runolee/mg of protein/h@ 0.819

0.317

1.096

34.04

20.515 P < 0.001)

6.532 f 1.460 (+89.90/ P < 0.001) 2.683

17.526 f 1.746 (+536.8%, P < 0.001)

24.058 f 2.450 (+289.5%, P < 0.901)

231.78 f (-41.3%,

Prodhoganimus sp.

3.612 f 1.006 (+5%, not eignificant) 1.226

4.428 rt 0.170 (+60.9%, P < 0.001)

8.039 f 0.9431 (+30.08%, P < 0.01)

337.72 f 33.347 (-14.50/,, P < 0.01)

Schidosoma incognihm

Infected with

Each value ie the mean f SD of 10 observations. The valuee given in parentheses are percentage deviation and negative sign indicating decrease) and student’s 1 test.

0.8

2.752 i

Aspartate aminotransferase activity (GOT) (rmolea/mg of pmtkdn/hr)

quotient

6.196 i

Total aminotraneferase activity @.uolee/mg of protein/hr)

De Ritie (GOT:GPT)

394.87 f

of

Total free amino acida bg/ml body fluid)

Normal

(positive sign indicating

increase

1.159 f 0.489 (-66.3% P < 0.001) 3.519

4.079 f 0.179 (+48.2%, P < 0.001)

5.236 f 0.5724 (-15.260/o, P < 0.05)

125.61 f 12.81 (-66.650/o, P < 0.601)

Corcatia pigmnJal0

TABLE 1 TOTAL FREE AMINO Acme AND ~XINOTRANBFERASE ACTIVITY IN NORMAL htw INFECTED SNAILS

a” kH

iii

5! “0

E

i

I

”F

E 0

AMINOTRANSFERASE

;hat BFGPT activity level increases significantly only in snails infected with Pros!hegonimus (89.9%) while it actually drops (66.3 %) in those infected with C. pigmentata (Table 1).

IN

LyVWUiHl

39

(1968), it is more probable that the body fluid aminotransferase activity pattern is more a reflection of the situation inside the cell. Since snail tissues are rich in these two aminotransferases (Bryant. et al., 1964), a DISCUSSION rise in these enzyme levels in the body fluid In the absence of similar studies on BFAT on infection is not unusual (Table 1). Howactivity in snails, a comparison of our results ever, it is of interest to note the wide with the data from bivalves and brachiopods disparity in percentage deviation (Table 1) (Hammen, 1968) reveals that the general in the two aminotransferase activities during BFAT activity is very high in Lymnuea different infections. In view of this wide Zuteola (Table 1). The De Ritis quotient disparity, an explanation for this rise in (GOT:GPT) is less than 1.0, being 0.8, as body fluid enzyme levels solely based on BFGOT activit.y is lower than BFGPT damage done to the hepatopancreas of the activity as in many of the bivalve malluscs snail by the parasite is not feasible. Other (Hammen, 1968). The l4C incorporation factors may play a role in elevating the studies (Hammen and Wilbur, 1959; Awaaminotransferase levels in the body fluid on para and Campbell, 1964; Bryant et al. 1964) infection. on snail tissue homogenates also suggest Transaminations might be of particular similar high level of activity of these en- importance under conditions that impose a zymes. This may be so, since Simpson et al. heavy drain on the animal’s store of metabo(1959) have found, in their survey of 17 lites (Goddard, 1966). The important role different invertebrates, that most of the free these aminotransferases play has been well L-amino acid nitrogen is distributed in few documented during shell formation (Hamamino acids, particularly in alanine, as- men and Wilbur, 1959) and in relation to partic acid, and glutamic acid, which form a amino acid excretion (Hammen, 1968). Delink with the citric acid cycle. Senft (1967) pletion of the host’s glycogen (Cheng, 1963a) has reported the abundance of aspart,ic acid and amino acids (Targett, 1962; Cheng, and g!utamic acid or their amines in the 1963c; Senft, 1967; Richards, 1969; Feng snail hosts of Schistosoma munsoni. In our et al., 1970) has been report.ed, and to this present investigation the free amino acid extent the parasite must be treated as a content of t,he normal snail body fluid is metabolic burden on the host. Therefore, it. similarly high as compared to that of the might be suggested that the infected snail hemolymph of Crassostrea virginica (Feng raises its intracellular aminotransferase levels to replenish the metabolites lost, to the et al, 1970) and those of Physa gyrina, advantage of the parasites, and this is Helisoma t,rivolvis, and Musculium partumium reflected in the body fluid aminotransferase (Cheng, 1963c). The lower BFGOT activity of normal levels. Transamination not only serves as a Lymnuea ZuteoZarelative to BFGPT activity pathway of conversion of cr-keto acids to might be either a reflection of the intracellular concentration or a consequence of L-amino acids, but also as an alternative differences in intracellular localization, per- means of replenishing the pyruvate pool. meation through cell membrane, and rate of Since there is a much higher increase in BFGOT activity than BFGPT activit.y, inact,ivation and elimination of these two aminotransferases. In view of the results of and as aspartic and glutamic acids seem to be abundant in snails (Senft, 1967), it Awapara and Campbell (1964) and Hammen

40

MANOEAR,

VJINKATESWARA

might be suggested that, on infection, reactions involving oxaloacetate teem to gain more importance than others involving pyruvate. This change from pyruvate-oriented to oxaloacetate-oriented metabolism might have something to do with replenishment of host’s glycogen store. The unequal rise in the total aminotransferase activity in the body fluid of snails with different infections might be due to more than one reason. It may reflect different extents to which the parasites damage the hepatopancreas of the host or, alternately, the extents to which they prove to be metabolic burdens to their hosts by draining their stores of glycogen and amino acids. The highest rise in BFAT level observed in Proslhogonimus-infected hosts might either be due to greater damage because of their stylets or to probable higher metabolic rate of this parasite ~d3inferred from the higher drop in free amino acids of the host (Richards, 1969). Of the two amino transferees, BFGOT level increases in all the three types of infection, though to different extents, emphssizing oxaloacetate oriented pathways, perhaps operating at difYerent rates. This Eeems all the more EO in view of the changes in the BFGPT levels. A sign5cant increae in this enzyme level in Prosthogonimus infections and lack of such a change in S. incognitum infections, suggests the subsidiary role playeq by this aminotransferme on infection. The marked drop in BFGPT level in C. pigment&z infections may speak of the sparing effect on alanine, which might be useful to the parasite, sa demonstrated in infected bivalves (Feng et al., 1970). The great diminution of the free amino acid pool (Table 1) of these hosts shedding C. pigmentda with no concomitant increase in total BFAT activity points in the same direction of amino acids being used up sa they are by the parasite (Richards, 1969). It appears from t.he present investigation that, on infection, the host’s metabolism is geared toward meeting the biochemical re-

RAO, AND

quirements parasites.

SWAMI

of the

developing

trematodt

ACKNOWLEDGMENT The authors are highly thankful to Dr. P Padmavathi, Department of Parasitology, Collee of Veterinary Science, A.P. Agricultural Uni’ versity, Tirupati, in helping in the identificatio 4 of the cercariae.

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CHENQ, T. C., AND BURMN, R. W. 1965. Relationships between Bucephalua sp. and Craaso&sea tirginica: histopathology and sites of infection. Cheaupeakc Sti., 6, 3-16. CHENQ, T. C., AND SNYDER, R. W., JR. 1962. Studies on host-parasite relationships between larval trematodes and their hosts. I. A review. II. The utilization of hoets glycogen by the intramolluscan larvae of Glypthdmins pmnsyluanimsia Cheng and associated phenomena. Trans. Amer. Microac. Sot., 81, 20% 228. 9. P., AND &PLAN, N.O. 1967. Metho Eneynol., 3, pp. 403. FENG, S. Y., KHAIRALLAE, E. A., AND CANEONIEE, N. 8. 1970. Haemolymph free amino acids and related nitrogenous compounds of Crtx+ sostrea virginico infected with Bucephalue sp. and Minchinia nelsoni. Camp. Biochem. Phyriol., 34, 64747. COILWICK,

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and quantitative estimation of the free amino acids in the healthy and parasitized digestive gland and gonad of Littorina saxatilis tenebrosa (Mont.) and in the daughter sporocysts of Microphallus pygmaeus (Levinsen, 1881) and Microphallus similis (Jagerskiold, 1900) (Trenatoda: Microphalidae). Cump. Biochem. Physiol., 31, 655-665. SENFT, A. W. 1967. Studies on arginine metabolism by schistosomes. II. Arginine depletion in mammals and snails infected with S. mansoni or S. haematobium. Comp. Biochem. Physiol., 21, 299306. Sr~pson, J. W., ALLEN, K., AND AWAP.~RA, J. 1959. Free amino acids in some aquatic invertebrates. Biol. Bull., 117, 371-381. TARGETT, G. A. T. 1962. A study of the amino acids present in Lymnaea stugnalis, Planorbarks coneus and Auslralorbis glabratus before and after infection with Schistosoma mansoni. Ann. Trop. Med. Parasitol., 56, 210-215. VON BRAND, T. 1966. ‘LBiochemistry of Parasites.” Academic Press, Kew York.