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Lipids in digestive gland of Littorina saxatilis rudis (Maton) and in daughter sporocysts of Microphallus similis (Jäg. 1900)
EXPERIMENTAL
37, 157-163
PARASITOLOGY
( 19%)
Lipids in Digestive Gland of Littorina saxatilis rudis (Maton) and in Daughter Sporocysts of Microphallus similis (Jiig. 1900) DONALD
P. MCMANUS,~ IAN MARSHALL,~ AND BRIAN L. JAMES
Department
of Zoology, University (Submitted
College,
for publication
Swansea, United
January
Kingdom
9, 1974)
MCMANUS, DONALD P., MARSHALL, IAN, AND JAMES, BRIAN L. 1975. Lipids in the digestive gland of Littorina saxntilis r&s (Maton) and in the daughter sporocysts of Microphallus similis (Jag. 1900). Experimental Parasitology 37, 157-163. Parasitism by sporocysts was associated with a slight decrease in triglycerides and fatty acids in host digestive gland cells suggesting that the parasite digests, absorbs, and metabolises fats of host origin. An increase in phospholipids and a marked increase in the incorporation of acetate-l-% in parasitised digestive glands may be the result of attempted cell regeneration, The parasite had less monoglycerides, triglycerides, and fatty acids but took up more palmitate-l-l% than the host. Sterols and sterol esters are more concentrated and more readily synthesised in the parasite than in the host. The relatively high level of phospholipids in the former may be related to number of cercariae within the sporocysts. INDEX DESCRIPTORS: Littorina sax&ilk rudis; Microphallus similis sporocysts; Neutral lipids; Sterols; Phospholipids; Acetate-l-Y: Palmitate-1-W.
sporocysts of Microphallus similis (Jag., 1900) was examined using chromatography and spectrophotometry. In addition, the in vitro incorporation of acetate-l-‘% and palmitate-l-l% by these tissues was investigated in an attempt to further elucidate lipid metabolism.
Knowledge of lipids in the sporocysts or rediae of Digenea and the effects of these parasites on the lipid composition of the molluscan host is very limited and is based on histochemical studies by Cheng and Snyder ( 1962), Cheng ( 1963, 1965), James ( 1965) and James and Bowers (1967a, b). The only biochemical studies were carried out by Southgate (1970) on the rediae of Fasciola hepatica and on the digestive gland ,cells of Lymnaea truncatula. In this investigation, the neutral lipid and phospholipid content of healthy and parasitised digestive glands of Littorinz saxatilis rudis (Maton) and of the daughter __-
MATERIALS AND METHODS
Animals Adult Littorina saxatilis rudis were collected from College Rocks, Aberystwyth, Wales throughout the Spring and Summer, 1972. On return to the laboratory, the following tissues were dissected from the molluscs and maintained in an ice-bath prior to lipid extraction: 1. Healthy digestive gland; 2. Parasitised digestive gland (excluding parasites);
1 Present address: Department of Zoology and Applied Entomology, Imperial College of Science and Technology, Prince Consort Road, London SW7 2BB, U.K. 2 Present address: Department of Zoology, University College, Cardiff, U.K. 157 Copyright All rights
0 1975 by Academic it reproduction in any
Press, Inc. form reserved.
1.3
MCMANUS, MARSHALL ATiD JAMES
3. Daughter
sporocysts of Microphalfully formed
Ius similis containing
cercariae. Excess moisture was removed and the fresh weight determined. Tissue from a number of molluscs was pooled in order to obtain sufficient material for analysis.
Extraction and Separution of Lipids Tissue weighing 100 mg was lightly homogenized in chloroform/methanol ( 2 : 1) and the lipids extracted according to the method of Folch et aZ. ( 1957). The washed lipids were evaporated to dryness in tiacuo and the total lipid yields estimated gravimetrically. The neutral lipids and phospholipids were separated by thin-layer chromatography on ready layered silica gel G plates (Type 6060, with fluorescent indicator, Kodak Ltd., Kirkby, Liverpool), at a tcmperature of 15 C. Neutral lipids were first fractionated on preactivated plates (100 C for 30 min) in diethyl ether; benzene; ethanol; glacial acetic acid (40:50:2:0.2) to a distance of 140 mm, followed by development in hexane; diethyl ether (96 :4), in the same direction, to a distance of 180 mm. Phospholipids were fractionated in methanol; dcionised water chloroform; ( 65: 24: 4), in a single development, to a distance of 180 mm. Visualisation was achieved in iodine vapour and identification facilitated by comparison with standards. Phospholipid standards were obtained from Koch Light Laboratories and checked for purity by thin layer chromatography. Neutral lipid standards, purchased from British Drug Houses, were tested similarly.
Estimation of Phosphorus in the Phospholipid Factions Lipid phosphorus was determined spcctrophotometrically by the method of Rouser et al. (1966). The amount of phospholipid was calculated by multiplying the total phosphorus
yield (mg/g wet wt.) by 25 (Subrahmanyam and Venkatesan 1968).
Uptuke of RadiolocrbelletlAcetate and Palmitate Healthy and parasitisecl digestive glands of L. saratilis ruclis and the sporocysts of hf. sirnilis were incubated aerobically with acetate-l-l’C (specific activity 62 &i/ pmole) or palmitate-1-‘“C (specific activity 55 ,&i/pmolc) in 2 ml incubation vessels. Radioactive acetate and palmitate were obtained from the Radiochemical Centrc, Amersham, U.K. and were dissolved in boiled, filtered sea water, pH 7.4, at a concentration of 2.5 &i/lOO~l of solution. Approximately 100 mg fresh weight of whole tissue was placed in 300 ~1 of labelled solution and incubated for three hours at 23” C, with continuous agitation. The incubation medium was then removed, the tissue rinsed quickly in several changes of filtered sea water, centrifuged and the total lipid extracted, weighed ancl chromatographcd as previously described. Aliquots of total lipid were evaporated to dryness in a tared glass scintillation vial and 10 ml of scintillation fluid (6 g PPO diluted with scintillation grade tolucne to one litre) added. Radioactivity was detcrmined in a Beckman LS 210B liquid scintillation system. Counting efficiency was determined by the Channel Ratio method (approximately 85% ) and the results expressed as disintegrations/min/mg lipid. After visualisation with iodine vapour, individual spots from the chromatograms were cut out and placed in scintillation vials. Toluene scintillant (2 ml) was added and the radioactivity determined. Areas of the chromatograms not responding to the iodine vapour were routinely used in background count controls. RESULTS
Qualitatively, the lipid fractions in the healthy and parasitised digestive glands and in the sporocysts were identical. The
LIPIDS
IN
LI’ITORINA
AND
TABLE A Semiquantitative and Parasitised
Healthy digestive gland Parasitised digestjive gland Sporocysts
Phospholipid
(Green pigment
Monoglycerides
++
++
++
+++ ++++
++ +
++ +
concentration
is indicated
IX!dYCerides
Sterols
Triglyrerides
+++
+
++
++
+++
++
++ ++
+ +
++ +++
+ +
+++ +
++ +++
Fat,ty acids
by an increasing
neutral lipids, in ascending order from the origin, included (i) a green pigment (possible containing chlorophyll or chlorophyll breakdown products), ( ii) monoglycerides, (iii) fatty acids, (iv) diglycerides, (v) sterols, (vi) triglycerides, (vii) a yellowbrown pigment (possibly containing carotenoids), and ( viii) sterol esters. The phospholipids were (i) phosphatidyl serine, (ii) lysophosphatidyl choline, (iii) sphingomyelin, (iv) phosphatidyl inositol, (v) phosphatidyl choline, (vi) phosphatidyl ethanolamine, and (vii) cardiolipin. In addition, spots containing cerebrosides (glycolipids) occurred between phosphatidyl ethanolamine and cardiolipin. Semiquantitative estimates (Table I) based on spot size and density revealed that triglyceride and fatty acid levels were TABLE l’he Lipid
I
Estimation, Hased on Colow Intensity and Spot Size, oj the Ikpid Composition of the Healthy Digestive Gland oj Littorina saxatilis rudis and of the Sporocysts of Microphalhls similis(A
Tisslle
n Increasing
159
MICROPHALLUS
Composition
Lipid
Lipid Digestive
Total lipids Phospholipidsh Neutral lipidsc
Sterol esters
number of crosses.
slightly reduced in the parasitised tissue as compared with the healthy. Most neutral lipid levels (Table I) were lower in the parasite than in the host tissue but sterols and sterol esters were higher. Some of these estimates were confirmed by quantitative analysis (Table II). The parasite contained about twice as much phospholipid as the healthy and 50% more than the parasitised digestive gland but about half the neutral lipid of host tissue. The phospholipids accounted for more than half of the total lipid in the parasite. Phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl inositol were, respectively, the most abundant phospholipids in the tissues (Table III). Every phospholipid (Tables II and III) was more abundant in the parasitised digestive gland II
of the Healthy and Parasitised Digestive Gland of Littorina and of the Sporocysts oj Microphallus similis=
class
Yellowbrown pigment
content
(mg/gm
wet wt.)
gland
Healthy
Parasitised
43.8 (39.3-45.7) 11.5 (11.2S11.6) 32.3
49.1 (48.0&X.0) 16.6 (16.5~16.6) 32.5
saxatilis
Sporocyst,s
39.8 (3X443.6) 23.4 (23.1-23.6) 16.4
a Results are t.he mean of four determinations (the range is given in brackets). * Phosphorus value multiplied by 25 (Subrahmanyam and Venkatesan 196X). e Calculated from the difference between total lipids and phospholipids.
rudis
160
MCMANUS,
MARSHALL
TABLE The l’hospholipid
Composition
AND
JAMES
III
vj ll~r Heulihy mnd Parasitised Diyestive G”luntl oj Littorina and of the Sporoc~/sts of Microphallus similisa Phospholipid Digestive Health)
(mg/gm
saxatilis
wet wt,.) -
.-
hporncysts
gland Parmitised
____ Cardiolipin Phosphatidyl et hanolamine Phosphatidyl choline Phosphatidyl inositol Sphingomyelin Lysophosphatidyl choline PhosphaGdyl serine
0.17 3.7,; t5.88 1.41 0.80 0.02 0.20
rudis:
(0.1 I-0.29) (3.2!)-4.1 1) (5.X0-6.99) (1.39-1.46) (0.07-0.10) (0.01-0.04) (0.1 l-0.26)
0.26 4.Y.5 7.80 2.13 1.12 0.17 0.60
(0.22%0.3.i) (8.62-4.34) (7.42-8.33) (1.81-2.44) (CX-1.48) (0.12-0.26) (0.5X-0.63)
0.40 4.70 14.76 2..i4 0.30 0.02 O..il
~~~
~~~~~
(0.3.?-0.37) (4.22-5.16) (14.25-15.44) (2.18-2.84) (0.17-0.36) (0.01-0.02) (0.31-0.64)
-______. a Results represent
the mean of follr determinations
than in the healthy. The parasite had more ethanolamine, phosphatidyl cardiolipin, phosphatidyl choline, and phosphatidyl inositol than the parasitised host tissue, but less sphingomyelin, lysophosphatidyl choline, and phosphatidyl serine. Particularly noticeable was the phosphatidyl choline level, which was about twice as high in the parasite than in the host. A considerable uptake of labelled substrate occurred in the parasitised host tissue after incubation in acetate-1-“C and in the parasite after incubation in the palmitate-lJ4C (Table IV). In contrast, only a moderate amount of acetate was taken up TABLE
IV
Total Meana Radioactivity Recorded in Lipids Zsolaletl from Healthy and Parasitised Digestive Gland of Lit,torina saxat,ilis rudis and from the Sporocysts of Microphallus similis, ajter Incubation in Each Substrate for Three hours at 23°C Substrate
Activity
(dpm/mg
(t,he range is in brackets).
by the healthy host tissue and by the parasite, and some palmitate by the host tissue. After incubation in palmitate (Table V), most label remained in the fatty acid fractions but some was incorporated into the yellow-brown pigment and into the phospholipids. The three tissues were remarkably similar in this respect. After incubation in acetate-l-*% (Table V), activity was evenly distributed between neutral and phospholipids in the host tissue but predominantly in the neutral lipids in the parasite. The proportion of label incorporated into the sterols and sterol esters was appreciably more in the parasite (about 26%‘) than in the host (680/, ). Considerable activity also occurred in the yellow-brown pigment and in the mono-, di-, and triglycerides. Most activity (Table V) in the phospholipids occurred in the most abundant (Table III) namely, phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol.
lipid) DISCUSSION
ljigestive Healthy Acetate-l-K PalmiWe-1-W
64,313 142,497
gland
Sporocysts
Parasitised 215,241 124,140
a Mean from t,hree experi1nent.s.
46,023 456 e ,.i6 .i
The principle effects of parasitism on the lipid composition and metabolism of the host, revealed in this paper, indicated a decrease in triglyceride and fatty acids (Table I), an increase in each phospholipid (Tables II and III) and a markedly
LIPIDS
IN
LITTORINA
AND
TABLE
161
MICROPHALLUS
V
Total C’ount and ‘j[, 12atlioaclizrity LZecorded in the Lipid I+actiuns Separated jru/t~ Healthy and Parasitised Digestive Gland oj Littorina saxatilis rudis and from the Sporocysts of Microphallus similis, after Incubation in Radioactive Acetate OT Palmitate
The Mcaw
-
Lipid fractions
Acetat,e-1-W Digestive gland Healthy
Palmit,ate-1-W Sporocysts
Parasitised
Digestive gland Healthy
Sporocysts
Parasitised
Neutral lipids
B .e .$ 8 G c k?
Total count (dpm) Originb Green pigment Monoglycerides Fatty acids Diglycerides Sterols Triglycerides Yellow-brown pigment Sterol esters
28,697
45.38 1.91 4.8.5 6.53 2.64 4.73 7.63 25.31 1.OO
98,683
53.00 1.91 3.85 5.61 2.91 6.20 5.10 20.61 1.53
22,011 27.19 2.31 9.22 9.12 2.13 1739 6.63 17.59 8.22
67,624 4.29 0.26 0.57 91.60 0.18 0.14 0.28 2.42 0.26
39,073
3.25 0.23 0.58 87.01 0.08 0.17 0.37 8.10 0.21
210,614 5.38 0.57 1.25 86.52 0.12 0.23 0.33 5.26 0.34
Phospholipids 31,338 94,677 24,199 58,678 61,118 199,283 2.41 3.76 3.83 0.43 0.15 0.60 2.79 5.90 1.82 0.42 0.59 0.08 h .z 1.60 0.69 1.56 0.32 0.57 0.05 .$ 1.Ofl 0.63 1.61 0.27 0.15 0.46 7.88 5.76 5.70 0.48 0.36 0.80 2 3 12.63 17.06 7.88 1.18 1.18 1.13 E 3 .3 “2 11.95 6.89 1.19 1.15 0.67 Ls 2.0.3 4.86 2.4.; 0.40 0.13 0.59 2.70 7.94 2.00 0.20 0.47 0.08 52.13 49.32 69.83 95.04 94.12 96.67 - __a Mean from three separate experiments. b Phospholipids. c Glycolipids. d Occur at solvent front,. Total count (dpm) Origin Phosphatidyl serine Lysophosphatidyl choline Sphingomyelin Phosphatidyl inositol Phosphatidyl choline Phosphatidyl ethanolamine Cerebrosidesc Cardiolipin Neutral lipidsd
increased uptake of acetate-l-14C (Table IV). This supports the suggestion, made on histochemical evidence ( Cheng 1963, 1965; James and Bowers 1967a, b), that the parasite digests, absorbs and metabolises triglycerides and fatty acids of host origin. The avid uptake of palmitate-l-l% (Table IV) by the parasite also supports this suggestion. The accumulation of phospholipid in the parasitised host may be the result of an attempt at regeneration by the ,cell membranes which have been broken down as a result of parasitism (James 1965). The increase in uptake of acetate-l-14C also sug-
gests this. Significantly, proportionally more label is included in phosphatidyl choline (Table V) in the parasitised host than in the healthy. The parasite had generally less neutral lipid (Table II) but probably more sterols and sterol esters (Table I) than the host. The latter conclusion, which was based on semiquantitative measurements (Table I ), is supported by the proportionately high incorporation of label in sterols and sterol esters by the parasite (Table V), after incubation in acetate-l-l%. This suggests that these parasites synthesise sterols and sterol esters. In contrast, Meyer and Meyer
162
MCMANUS,
MARSHALL
(1972) believed that cle WOO fatty acid and sterol biosynthesis does not occur in the Platyhelminthes. Their arguments are attractive but should be treated with caution because they are based on only five species, namely larval and adult Spirolnetrn mnnsonoicles ( Meyer et al. 1966), adult Hymenolepis diminuta (Ginger and Fairbairn 1966; Jacobsen and Fairbairn 1967), adult Schistosoma mansoni (Smith and Brooks 1969; Smith et al. 1970; Meyer et al. 1970)) adult Dugesia dorotocephala (Meyer et al. 1970), and larval and adult Conuoluta roscofensis (Meyer and Meyer 1972). In our opinion, more work is required before valid gcneralisations can be made. The high phospholipid content of the parasite, as compared with the host (Tables II and III), may result from the large number of cercariae which occur within the sporocysts. In spite of this, relatively less label (Table V) was incorporated in the phospholipid of the parasite after incubation in acetate-l-‘%. This may be due to the fact that the sporocysts contained only fully formed cercariae, undergoing no further development. Alternatively, the host may be actively regenerating broken down tissue. It is possible that the palmitate-1-“C taken up by the host and parasite is oxidatively degraded, as nearly all label remains in the fatty acid fraction (Table V), but much may remain unmetabolised. However, the low neutral lipid content (Tables I and II) together with the high incorporation of label in neutral lipids (Table V) may suggest a rapid rate of catabolism by the parasite. The incorporation of label in the yellowbrown spot suggests that the pigment is associated with lipids and/or proteins, since animals cannot synthesise carotenoids. The incorporation of label into the green pigment may represent contamination from the origin or monoglyceride fraction as some streaking occurred at this point in the chromatograms.
AND JAMES
In conclusion, it should be emphasised that the above suggestions remain speculative until further work is carried out, particularly with gas-liquid chromatography. It is known from work with other helminth parasites (Overturf and Dryer 1968; Daugherty 1957; Ginger and Fairbairn 1966 and Jacobsen and Fairbairn 1967) that lipid composition and metabolism varies considerably with such factors as the diet of the host and the physiology of the parasite but much variation is still unexplained. ACKNOWLEDGMENTS We are very grateful to Professor E. W. KnightJones for the provision of excellent working facilities and to the Science Research Council for financial support (D. P. h1. and I. hf.). REFEREXES T. C. 1963. Biochemical requirements of larval trematodes. Annals of the New York A&em y of Sciences 113, 289-321. C~exc, T. C. 1965. Histochemical observations on changes in the lipid composition of the American oyster Crassostrea virginica (Gmelin) parasitised by the trematode Bucephulus sp. CHEW,
Journal
of
Invertebrate
Pathology
7, 39%
407. CHEXG,
T.
C.,
AND
SNYDER,
R.
W.,
JH.
1962.
Studies on host-parasite relationships bctween larval trematodes and their hosts. 111. Certain aspects of lipid metabolism in Helisoma trivolcis (Say) infected with the larvae of Glypthelmins pennsylvaniensis Cheng and related phenomena. American Microscopical
Society 81, 327-331. DAUGHERTY, J. W. 1957. The active absorption of cc&in metabolites by helminths. American
Journal
of
Tropicul Medicine and Hygiene 6,
464-472. FOLCH, J., LEES, Al., AND SLOANE-STANLEY, G. H. 1957. A simple method for isolation and purification of total lipids from animal tissues.
Journul 509.
of
Biological
Chemistry
226, 497-
L. D., AND FAIRBAIRN, D. 1966. Lipid metabolism in helminth parasites. II. The major origins of the lipids of Hymenolepb diminuta ( Cestoda). journal of Parasitology 52, 1097-1107. JACOBSEN, N. S., AND FAIRBAIRN, D. 1967. Lipid metabolism in helminth parasites. III. BioGINGER,
LIPIDS
IN
LITTORINA
synthesis and interconversions of fatty acids by Hymenolepk diminuta (Cestoda). JOUTnal of Parasitology 53, 355-361. JAMES, B. L. 1965. The effects of parasitism by larval Digenea on the digestive gland of the Littorina saxatilis intertidal prosobranch, ( Olivi) spp. tenebrosa (Montagu). Parasitology 55, 93-115. JAMES, B. L., AND BOWERS, E. A. 1967a. Histochemical observations on the occurrence of carbohydrates, lipids and enzymes in the daughter sporocysts of Cercaria bwephalopsis haimeanu ( Lacaze-Duthiers 1854) (Digenea : Bucephalidae). Parasitology 57, 7986. JAMES, B. L., AND BOWERS, E. A. 1967b. The effects of parasitism by the daughter sporocysts of Cercaria bucephalopsis haimeanu ( Lacaze-Duthiers 1854) on the digestive tubules of the cockle, Car&urn edule I,. Parasitology 57, 67-77. MEYER, F., KIMURA, S., AND MUELLER, J. F. 1966. Lipid metabolism in the larval and adult forms of the tapeworm Spirometra mansonoides. The Journal of Biological Chemistry 241,4224-4232. MEYER, F., AND MEYER, H. 1972. Loss of fatty acid biosynthesis in flatworms. In “Comparative Biochemistry of Parasites” (H. Van den Bossche, Ed.), Academic Press, New York and London.
AND
MICROPHhLLUS
163
MEYER, F., MEYER, H., AND BUEDING, E. 1970. Lipid metabolism in the parasitic and free living flatworms, Schistosoma mansoni and Dugesia dorotocephalu. Bicchimicu et Biophysica Acta 210, 257-266. OVERTURF, M., AND DRYER, R. L. 1968. Lipid metabolism in the adult cestode Hymenobpis dimz’nuta. Comparative Biochemistry and Physiology 27, 145-175. ROUSER, G., SIAKOTOS, A. N., AND FLEISCHER, S. 1966. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1, 85-86. ShmH, T. M., AND BROOKS, T. J., JR. 1969. In vitro studies on fatty acid metabolism in the blood fluke Schistosomu munsoni. Federation Proceedings 28, 688. SIWTH, T. M., BROOKS, T. J., JR., AND LOCKARD, V. G. 1970. Short communication: In ktro studies on cholesterol metabolism in the blood fluke Schistosoma mansoni. Lipids 5, 854. SOUTHGATE, V. R. 1970. Observations on the effect of the rediae of Fasciokz hepatica on the lipid composition of Lymnaea truncatula. Parasitology 61, 293-299. SUBRAH~IANYAM, D., AND VENKATESAN, S. 1968. On the phospholipids of Ascaris lumbricoides. Comparutiue Biochemistry and Physiology 25,733-737.
37, 157-163
PARASITOLOGY
( 19%)
Lipids in Digestive Gland of Littorina saxatilis rudis (Maton) and in Daughter Sporocysts of Microphallus similis (Jiig. 1900) DONALD
P. MCMANUS,~ IAN MARSHALL,~ AND BRIAN L. JAMES
Department
of Zoology, University (Submitted
College,
for publication
Swansea, United
January
Kingdom
9, 1974)
MCMANUS, DONALD P., MARSHALL, IAN, AND JAMES, BRIAN L. 1975. Lipids in the digestive gland of Littorina saxntilis r&s (Maton) and in the daughter sporocysts of Microphallus similis (Jag. 1900). Experimental Parasitology 37, 157-163. Parasitism by sporocysts was associated with a slight decrease in triglycerides and fatty acids in host digestive gland cells suggesting that the parasite digests, absorbs, and metabolises fats of host origin. An increase in phospholipids and a marked increase in the incorporation of acetate-l-% in parasitised digestive glands may be the result of attempted cell regeneration, The parasite had less monoglycerides, triglycerides, and fatty acids but took up more palmitate-l-l% than the host. Sterols and sterol esters are more concentrated and more readily synthesised in the parasite than in the host. The relatively high level of phospholipids in the former may be related to number of cercariae within the sporocysts. INDEX DESCRIPTORS: Littorina sax&ilk rudis; Microphallus similis sporocysts; Neutral lipids; Sterols; Phospholipids; Acetate-l-Y: Palmitate-1-W.
sporocysts of Microphallus similis (Jag., 1900) was examined using chromatography and spectrophotometry. In addition, the in vitro incorporation of acetate-l-‘% and palmitate-l-l% by these tissues was investigated in an attempt to further elucidate lipid metabolism.
Knowledge of lipids in the sporocysts or rediae of Digenea and the effects of these parasites on the lipid composition of the molluscan host is very limited and is based on histochemical studies by Cheng and Snyder ( 1962), Cheng ( 1963, 1965), James ( 1965) and James and Bowers (1967a, b). The only biochemical studies were carried out by Southgate (1970) on the rediae of Fasciola hepatica and on the digestive gland ,cells of Lymnaea truncatula. In this investigation, the neutral lipid and phospholipid content of healthy and parasitised digestive glands of Littorinz saxatilis rudis (Maton) and of the daughter __-
MATERIALS AND METHODS
Animals Adult Littorina saxatilis rudis were collected from College Rocks, Aberystwyth, Wales throughout the Spring and Summer, 1972. On return to the laboratory, the following tissues were dissected from the molluscs and maintained in an ice-bath prior to lipid extraction: 1. Healthy digestive gland; 2. Parasitised digestive gland (excluding parasites);
1 Present address: Department of Zoology and Applied Entomology, Imperial College of Science and Technology, Prince Consort Road, London SW7 2BB, U.K. 2 Present address: Department of Zoology, University College, Cardiff, U.K. 157 Copyright All rights
0 1975 by Academic it reproduction in any
Press, Inc. form reserved.
1.3
MCMANUS, MARSHALL ATiD JAMES
3. Daughter
sporocysts of Microphalfully formed
Ius similis containing
cercariae. Excess moisture was removed and the fresh weight determined. Tissue from a number of molluscs was pooled in order to obtain sufficient material for analysis.
Extraction and Separution of Lipids Tissue weighing 100 mg was lightly homogenized in chloroform/methanol ( 2 : 1) and the lipids extracted according to the method of Folch et aZ. ( 1957). The washed lipids were evaporated to dryness in tiacuo and the total lipid yields estimated gravimetrically. The neutral lipids and phospholipids were separated by thin-layer chromatography on ready layered silica gel G plates (Type 6060, with fluorescent indicator, Kodak Ltd., Kirkby, Liverpool), at a tcmperature of 15 C. Neutral lipids were first fractionated on preactivated plates (100 C for 30 min) in diethyl ether; benzene; ethanol; glacial acetic acid (40:50:2:0.2) to a distance of 140 mm, followed by development in hexane; diethyl ether (96 :4), in the same direction, to a distance of 180 mm. Phospholipids were fractionated in methanol; dcionised water chloroform; ( 65: 24: 4), in a single development, to a distance of 180 mm. Visualisation was achieved in iodine vapour and identification facilitated by comparison with standards. Phospholipid standards were obtained from Koch Light Laboratories and checked for purity by thin layer chromatography. Neutral lipid standards, purchased from British Drug Houses, were tested similarly.
Estimation of Phosphorus in the Phospholipid Factions Lipid phosphorus was determined spcctrophotometrically by the method of Rouser et al. (1966). The amount of phospholipid was calculated by multiplying the total phosphorus
yield (mg/g wet wt.) by 25 (Subrahmanyam and Venkatesan 1968).
Uptuke of RadiolocrbelletlAcetate and Palmitate Healthy and parasitisecl digestive glands of L. saratilis ruclis and the sporocysts of hf. sirnilis were incubated aerobically with acetate-l-l’C (specific activity 62 &i/ pmole) or palmitate-1-‘“C (specific activity 55 ,&i/pmolc) in 2 ml incubation vessels. Radioactive acetate and palmitate were obtained from the Radiochemical Centrc, Amersham, U.K. and were dissolved in boiled, filtered sea water, pH 7.4, at a concentration of 2.5 &i/lOO~l of solution. Approximately 100 mg fresh weight of whole tissue was placed in 300 ~1 of labelled solution and incubated for three hours at 23” C, with continuous agitation. The incubation medium was then removed, the tissue rinsed quickly in several changes of filtered sea water, centrifuged and the total lipid extracted, weighed ancl chromatographcd as previously described. Aliquots of total lipid were evaporated to dryness in a tared glass scintillation vial and 10 ml of scintillation fluid (6 g PPO diluted with scintillation grade tolucne to one litre) added. Radioactivity was detcrmined in a Beckman LS 210B liquid scintillation system. Counting efficiency was determined by the Channel Ratio method (approximately 85% ) and the results expressed as disintegrations/min/mg lipid. After visualisation with iodine vapour, individual spots from the chromatograms were cut out and placed in scintillation vials. Toluene scintillant (2 ml) was added and the radioactivity determined. Areas of the chromatograms not responding to the iodine vapour were routinely used in background count controls. RESULTS
Qualitatively, the lipid fractions in the healthy and parasitised digestive glands and in the sporocysts were identical. The
LIPIDS
IN
LI’ITORINA
AND
TABLE A Semiquantitative and Parasitised
Healthy digestive gland Parasitised digestjive gland Sporocysts
Phospholipid
(Green pigment
Monoglycerides
++
++
++
+++ ++++
++ +
++ +
concentration
is indicated
IX!dYCerides
Sterols
Triglyrerides
+++
+
++
++
+++
++
++ ++
+ +
++ +++
+ +
+++ +
++ +++
Fat,ty acids
by an increasing
neutral lipids, in ascending order from the origin, included (i) a green pigment (possible containing chlorophyll or chlorophyll breakdown products), ( ii) monoglycerides, (iii) fatty acids, (iv) diglycerides, (v) sterols, (vi) triglycerides, (vii) a yellowbrown pigment (possibly containing carotenoids), and ( viii) sterol esters. The phospholipids were (i) phosphatidyl serine, (ii) lysophosphatidyl choline, (iii) sphingomyelin, (iv) phosphatidyl inositol, (v) phosphatidyl choline, (vi) phosphatidyl ethanolamine, and (vii) cardiolipin. In addition, spots containing cerebrosides (glycolipids) occurred between phosphatidyl ethanolamine and cardiolipin. Semiquantitative estimates (Table I) based on spot size and density revealed that triglyceride and fatty acid levels were TABLE l’he Lipid
I
Estimation, Hased on Colow Intensity and Spot Size, oj the Ikpid Composition of the Healthy Digestive Gland oj Littorina saxatilis rudis and of the Sporocysts of Microphalhls similis(A
Tisslle
n Increasing
159
MICROPHALLUS
Composition
Lipid
Lipid Digestive
Total lipids Phospholipidsh Neutral lipidsc
Sterol esters
number of crosses.
slightly reduced in the parasitised tissue as compared with the healthy. Most neutral lipid levels (Table I) were lower in the parasite than in the host tissue but sterols and sterol esters were higher. Some of these estimates were confirmed by quantitative analysis (Table II). The parasite contained about twice as much phospholipid as the healthy and 50% more than the parasitised digestive gland but about half the neutral lipid of host tissue. The phospholipids accounted for more than half of the total lipid in the parasite. Phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl inositol were, respectively, the most abundant phospholipids in the tissues (Table III). Every phospholipid (Tables II and III) was more abundant in the parasitised digestive gland II
of the Healthy and Parasitised Digestive Gland of Littorina and of the Sporocysts oj Microphallus similis=
class
Yellowbrown pigment
content
(mg/gm
wet wt.)
gland
Healthy
Parasitised
43.8 (39.3-45.7) 11.5 (11.2S11.6) 32.3
49.1 (48.0&X.0) 16.6 (16.5~16.6) 32.5
saxatilis
Sporocyst,s
39.8 (3X443.6) 23.4 (23.1-23.6) 16.4
a Results are t.he mean of four determinations (the range is given in brackets). * Phosphorus value multiplied by 25 (Subrahmanyam and Venkatesan 196X). e Calculated from the difference between total lipids and phospholipids.
rudis
160
MCMANUS,
MARSHALL
TABLE The l’hospholipid
Composition
AND
JAMES
III
vj ll~r Heulihy mnd Parasitised Diyestive G”luntl oj Littorina and of the Sporoc~/sts of Microphallus similisa Phospholipid Digestive Health)
(mg/gm
saxatilis
wet wt,.) -
.-
hporncysts
gland Parmitised
____ Cardiolipin Phosphatidyl et hanolamine Phosphatidyl choline Phosphatidyl inositol Sphingomyelin Lysophosphatidyl choline PhosphaGdyl serine
0.17 3.7,; t5.88 1.41 0.80 0.02 0.20
rudis:
(0.1 I-0.29) (3.2!)-4.1 1) (5.X0-6.99) (1.39-1.46) (0.07-0.10) (0.01-0.04) (0.1 l-0.26)
0.26 4.Y.5 7.80 2.13 1.12 0.17 0.60
(0.22%0.3.i) (8.62-4.34) (7.42-8.33) (1.81-2.44) (CX-1.48) (0.12-0.26) (0.5X-0.63)
0.40 4.70 14.76 2..i4 0.30 0.02 O..il
~~~
~~~~~
(0.3.?-0.37) (4.22-5.16) (14.25-15.44) (2.18-2.84) (0.17-0.36) (0.01-0.02) (0.31-0.64)
-______. a Results represent
the mean of follr determinations
than in the healthy. The parasite had more ethanolamine, phosphatidyl cardiolipin, phosphatidyl choline, and phosphatidyl inositol than the parasitised host tissue, but less sphingomyelin, lysophosphatidyl choline, and phosphatidyl serine. Particularly noticeable was the phosphatidyl choline level, which was about twice as high in the parasite than in the host. A considerable uptake of labelled substrate occurred in the parasitised host tissue after incubation in acetate-1-“C and in the parasite after incubation in the palmitate-lJ4C (Table IV). In contrast, only a moderate amount of acetate was taken up TABLE
IV
Total Meana Radioactivity Recorded in Lipids Zsolaletl from Healthy and Parasitised Digestive Gland of Lit,torina saxat,ilis rudis and from the Sporocysts of Microphallus similis, ajter Incubation in Each Substrate for Three hours at 23°C Substrate
Activity
(dpm/mg
(t,he range is in brackets).
by the healthy host tissue and by the parasite, and some palmitate by the host tissue. After incubation in palmitate (Table V), most label remained in the fatty acid fractions but some was incorporated into the yellow-brown pigment and into the phospholipids. The three tissues were remarkably similar in this respect. After incubation in acetate-l-*% (Table V), activity was evenly distributed between neutral and phospholipids in the host tissue but predominantly in the neutral lipids in the parasite. The proportion of label incorporated into the sterols and sterol esters was appreciably more in the parasite (about 26%‘) than in the host (680/, ). Considerable activity also occurred in the yellow-brown pigment and in the mono-, di-, and triglycerides. Most activity (Table V) in the phospholipids occurred in the most abundant (Table III) namely, phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol.
lipid) DISCUSSION
ljigestive Healthy Acetate-l-K PalmiWe-1-W
64,313 142,497
gland
Sporocysts
Parasitised 215,241 124,140
a Mean from t,hree experi1nent.s.
46,023 456 e ,.i6 .i
The principle effects of parasitism on the lipid composition and metabolism of the host, revealed in this paper, indicated a decrease in triglyceride and fatty acids (Table I), an increase in each phospholipid (Tables II and III) and a markedly
LIPIDS
IN
LITTORINA
AND
TABLE
161
MICROPHALLUS
V
Total C’ount and ‘j[, 12atlioaclizrity LZecorded in the Lipid I+actiuns Separated jru/t~ Healthy and Parasitised Digestive Gland oj Littorina saxatilis rudis and from the Sporocysts of Microphallus similis, after Incubation in Radioactive Acetate OT Palmitate
The Mcaw
-
Lipid fractions
Acetat,e-1-W Digestive gland Healthy
Palmit,ate-1-W Sporocysts
Parasitised
Digestive gland Healthy
Sporocysts
Parasitised
Neutral lipids
B .e .$ 8 G c k?
Total count (dpm) Originb Green pigment Monoglycerides Fatty acids Diglycerides Sterols Triglycerides Yellow-brown pigment Sterol esters
28,697
45.38 1.91 4.8.5 6.53 2.64 4.73 7.63 25.31 1.OO
98,683
53.00 1.91 3.85 5.61 2.91 6.20 5.10 20.61 1.53
22,011 27.19 2.31 9.22 9.12 2.13 1739 6.63 17.59 8.22
67,624 4.29 0.26 0.57 91.60 0.18 0.14 0.28 2.42 0.26
39,073
3.25 0.23 0.58 87.01 0.08 0.17 0.37 8.10 0.21
210,614 5.38 0.57 1.25 86.52 0.12 0.23 0.33 5.26 0.34
Phospholipids 31,338 94,677 24,199 58,678 61,118 199,283 2.41 3.76 3.83 0.43 0.15 0.60 2.79 5.90 1.82 0.42 0.59 0.08 h .z 1.60 0.69 1.56 0.32 0.57 0.05 .$ 1.Ofl 0.63 1.61 0.27 0.15 0.46 7.88 5.76 5.70 0.48 0.36 0.80 2 3 12.63 17.06 7.88 1.18 1.18 1.13 E 3 .3 “2 11.95 6.89 1.19 1.15 0.67 Ls 2.0.3 4.86 2.4.; 0.40 0.13 0.59 2.70 7.94 2.00 0.20 0.47 0.08 52.13 49.32 69.83 95.04 94.12 96.67 - __a Mean from three separate experiments. b Phospholipids. c Glycolipids. d Occur at solvent front,. Total count (dpm) Origin Phosphatidyl serine Lysophosphatidyl choline Sphingomyelin Phosphatidyl inositol Phosphatidyl choline Phosphatidyl ethanolamine Cerebrosidesc Cardiolipin Neutral lipidsd
increased uptake of acetate-l-14C (Table IV). This supports the suggestion, made on histochemical evidence ( Cheng 1963, 1965; James and Bowers 1967a, b), that the parasite digests, absorbs and metabolises triglycerides and fatty acids of host origin. The avid uptake of palmitate-l-l% (Table IV) by the parasite also supports this suggestion. The accumulation of phospholipid in the parasitised host may be the result of an attempt at regeneration by the ,cell membranes which have been broken down as a result of parasitism (James 1965). The increase in uptake of acetate-l-14C also sug-
gests this. Significantly, proportionally more label is included in phosphatidyl choline (Table V) in the parasitised host than in the healthy. The parasite had generally less neutral lipid (Table II) but probably more sterols and sterol esters (Table I) than the host. The latter conclusion, which was based on semiquantitative measurements (Table I ), is supported by the proportionately high incorporation of label in sterols and sterol esters by the parasite (Table V), after incubation in acetate-l-l%. This suggests that these parasites synthesise sterols and sterol esters. In contrast, Meyer and Meyer
162
MCMANUS,
MARSHALL
(1972) believed that cle WOO fatty acid and sterol biosynthesis does not occur in the Platyhelminthes. Their arguments are attractive but should be treated with caution because they are based on only five species, namely larval and adult Spirolnetrn mnnsonoicles ( Meyer et al. 1966), adult Hymenolepis diminuta (Ginger and Fairbairn 1966; Jacobsen and Fairbairn 1967), adult Schistosoma mansoni (Smith and Brooks 1969; Smith et al. 1970; Meyer et al. 1970)) adult Dugesia dorotocephala (Meyer et al. 1970), and larval and adult Conuoluta roscofensis (Meyer and Meyer 1972). In our opinion, more work is required before valid gcneralisations can be made. The high phospholipid content of the parasite, as compared with the host (Tables II and III), may result from the large number of cercariae which occur within the sporocysts. In spite of this, relatively less label (Table V) was incorporated in the phospholipid of the parasite after incubation in acetate-l-‘%. This may be due to the fact that the sporocysts contained only fully formed cercariae, undergoing no further development. Alternatively, the host may be actively regenerating broken down tissue. It is possible that the palmitate-1-“C taken up by the host and parasite is oxidatively degraded, as nearly all label remains in the fatty acid fraction (Table V), but much may remain unmetabolised. However, the low neutral lipid content (Tables I and II) together with the high incorporation of label in neutral lipids (Table V) may suggest a rapid rate of catabolism by the parasite. The incorporation of label in the yellowbrown spot suggests that the pigment is associated with lipids and/or proteins, since animals cannot synthesise carotenoids. The incorporation of label into the green pigment may represent contamination from the origin or monoglyceride fraction as some streaking occurred at this point in the chromatograms.
AND JAMES
In conclusion, it should be emphasised that the above suggestions remain speculative until further work is carried out, particularly with gas-liquid chromatography. It is known from work with other helminth parasites (Overturf and Dryer 1968; Daugherty 1957; Ginger and Fairbairn 1966 and Jacobsen and Fairbairn 1967) that lipid composition and metabolism varies considerably with such factors as the diet of the host and the physiology of the parasite but much variation is still unexplained. ACKNOWLEDGMENTS We are very grateful to Professor E. W. KnightJones for the provision of excellent working facilities and to the Science Research Council for financial support (D. P. h1. and I. hf.). REFEREXES T. C. 1963. Biochemical requirements of larval trematodes. Annals of the New York A&em y of Sciences 113, 289-321. C~exc, T. C. 1965. Histochemical observations on changes in the lipid composition of the American oyster Crassostrea virginica (Gmelin) parasitised by the trematode Bucephulus sp. CHEW,
Journal
of
Invertebrate
Pathology
7, 39%
407. CHEXG,
T.
C.,
AND
SNYDER,
R.
W.,
JH.
1962.
Studies on host-parasite relationships bctween larval trematodes and their hosts. 111. Certain aspects of lipid metabolism in Helisoma trivolcis (Say) infected with the larvae of Glypthelmins pennsylvaniensis Cheng and related phenomena. American Microscopical
Society 81, 327-331. DAUGHERTY, J. W. 1957. The active absorption of cc&in metabolites by helminths. American
Journal
of
Tropicul Medicine and Hygiene 6,
464-472. FOLCH, J., LEES, Al., AND SLOANE-STANLEY, G. H. 1957. A simple method for isolation and purification of total lipids from animal tissues.
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Biological
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L. D., AND FAIRBAIRN, D. 1966. Lipid metabolism in helminth parasites. II. The major origins of the lipids of Hymenolepb diminuta ( Cestoda). journal of Parasitology 52, 1097-1107. JACOBSEN, N. S., AND FAIRBAIRN, D. 1967. Lipid metabolism in helminth parasites. III. BioGINGER,
LIPIDS
IN
LITTORINA
synthesis and interconversions of fatty acids by Hymenolepk diminuta (Cestoda). JOUTnal of Parasitology 53, 355-361. JAMES, B. L. 1965. The effects of parasitism by larval Digenea on the digestive gland of the Littorina saxatilis intertidal prosobranch, ( Olivi) spp. tenebrosa (Montagu). Parasitology 55, 93-115. JAMES, B. L., AND BOWERS, E. A. 1967a. Histochemical observations on the occurrence of carbohydrates, lipids and enzymes in the daughter sporocysts of Cercaria bwephalopsis haimeanu ( Lacaze-Duthiers 1854) (Digenea : Bucephalidae). Parasitology 57, 7986. JAMES, B. L., AND BOWERS, E. A. 1967b. The effects of parasitism by the daughter sporocysts of Cercaria bucephalopsis haimeanu ( Lacaze-Duthiers 1854) on the digestive tubules of the cockle, Car&urn edule I,. Parasitology 57, 67-77. MEYER, F., KIMURA, S., AND MUELLER, J. F. 1966. Lipid metabolism in the larval and adult forms of the tapeworm Spirometra mansonoides. The Journal of Biological Chemistry 241,4224-4232. MEYER, F., AND MEYER, H. 1972. Loss of fatty acid biosynthesis in flatworms. In “Comparative Biochemistry of Parasites” (H. Van den Bossche, Ed.), Academic Press, New York and London.
AND
MICROPHhLLUS
163
MEYER, F., MEYER, H., AND BUEDING, E. 1970. Lipid metabolism in the parasitic and free living flatworms, Schistosoma mansoni and Dugesia dorotocephalu. Bicchimicu et Biophysica Acta 210, 257-266. OVERTURF, M., AND DRYER, R. L. 1968. Lipid metabolism in the adult cestode Hymenobpis dimz’nuta. Comparative Biochemistry and Physiology 27, 145-175. ROUSER, G., SIAKOTOS, A. N., AND FLEISCHER, S. 1966. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1, 85-86. ShmH, T. M., AND BROOKS, T. J., JR. 1969. In vitro studies on fatty acid metabolism in the blood fluke Schistosomu munsoni. Federation Proceedings 28, 688. SIWTH, T. M., BROOKS, T. J., JR., AND LOCKARD, V. G. 1970. Short communication: In ktro studies on cholesterol metabolism in the blood fluke Schistosoma mansoni. Lipids 5, 854. SOUTHGATE, V. R. 1970. Observations on the effect of the rediae of Fasciokz hepatica on the lipid composition of Lymnaea truncatula. Parasitology 61, 293-299. SUBRAH~IANYAM, D., AND VENKATESAN, S. 1968. On the phospholipids of Ascaris lumbricoides. Comparutiue Biochemistry and Physiology 25,733-737.