Carotenoids found in Littorina littorea and their relationship to parasitic infection by larva trematodes

JOURNAL

OF

INVERTEBRATE

Carotenoids

PATHOLOGY

34,

276-284 (1979)

Found in Littorina Parasitic Infection

littorea and Their Relationship by Larva Trematodes’

EUGENIA T. ZAVRAS~ AND Department

of Biology,

University

of Bridgeport,

to

HUGO A. JAMES Bridgeport,

Connecticut

06602

Received December 15. 1978 Littorina littorea from Long Island Sound feed primarily on algae: Chlorophyceae (three species) and Rhodophyceae (two species). Carotenoids from the algae accumulate in tissues of the snail in either an unchanged or a metabolized state. p-Carotene, the major pigment of green and red algae, was isolated from the foot, hepatopancreas, and nephridium of these snails. Six oxygenated carotenoids, not completely identified, were isolated from the same tissues. The snails show a variation in foot color from white to brown to red. L. littorea is parasitized by trematode larvae of Cryptocotyle lingua and Cercariu parlsicaudata from which p-carotene and one oxygenated carotenoid were isolated. Contrary to previous work, there is no relation between foot color of the snail and parasitic infection. Neither age nor sex appears to have any relation to foot color. Although carotenoid pigments are known to cause the variation in foot color, the reasons or factors for their accumulation in the snail tissue have not been established. Some hypothetical explanations are discussed. KEY WORDS: Littorina littorea; Cryptocotyle lingua: Cercaria parvicaudata; algae; carotenoid identification.

INTRODUCTION

The carotenoids of many animals have been studied and identified, and possible functions have been ascribed to them. They have been implicated in protective coloration, enzyme activation (Chessman et al., 1967), photoreception, trapping and transferring of energy (Dingle and Lucy, 1965; Govindjee and Govindjee, 1974), and respiration as final electron acceptors during oxygen tension in molluscan neurons (ZsNagy, 1971). In molluscs, carotenoids have been extracted from the mussel Mytilus californianus (see Scheer, 1940), several nudibranchs (see McBeth, 1972), the tissues of the marine snail Cerithidia californicu (see Nakadal, 1960a), the eggs, yolk gland, and liver of the gastropod Pomuceu (see Villela, 1956), the gonads of limpets (Goodwin and Taha, 1950), the digestive gland and gonads ’ This work was supported by the Biology Department of the University of Bridgeport. r Present address: Department of Zoology, Morrill Science Center, University of Massachusetts, Amherst, Mass. 01003.

of Nussarius obsoletus (Hoskin and Cheng, 1975), the digestive gland of L. littoreu and Gibbula umbilicalis (see Marshall, 1974), the neurons of Aplysia (see Arvanitaki and Chalazonitis, 1960), and the brain of a fresh water pulmonate snail (Benjamin and Walker, 1972). Some specimens of the marine prosobranch L. littorea (Linnaeus) have pigmented feet, while others are unpigmented. L. littorea also serves as the intermediate host of Cryptocotyle lingua and for some other trematodes (Rees, 1936; Robson and Williams, 1970; Werding, 1969). Some trematode larvae are pigmented, and carotenoids have been reported from several such larvae (Hoskin and Cheng, 1975; Marshall, 1974; Nakadal, 1960b). The pigmentation in the foot of L. littorea has been attributed by Willey and Gross (1957) to the destruction of the snail’s digestive gland by the larvae of Cryptocotyle lingua and accumulation of the possibly carotenoid pigments of the digestive gland in the foot. Preliminary studies by James (1974) indicated that parasitism does not explain the color in the foot of L. littoreu. 276

0022-201 l/79/060276-09$01.00/O Copyright All rights

0 1979 by Academic Press. Inc. of reproduction in any form reserved.

TREMATODES

MATERIALS

AND

CAROTENOIDS

AND METHODS

L. Iittorea and several species of green, brown, and red algae were collected throughout the year from two locations on Long Island Sound: Bluewater Hill Beach at Hillspoint Road, Westport, Connecticut, and Seaside Park, Bridgeport, Connecticut. Over 2000 L. littorea with unpigmented (white) and pigmented (shades of brown and red) feet were collected at random from both sites at low tide. Foot color, size, and sex of the snails were recorded. Each group of snails (pigmented and unpigmented) was crushed, dissected, and examined for larval stages of C. lingua and other trematodes. The intestinal contents were examined for identification of the major algae upon which the snails feed. The different snail tissues were kept in the freezer until sufficient material was collected for pigment extraction. The feet of at least 300 pigmented (brown and red) snails were combined, weighed, and ground in acetone in a Vitris 23 homogenizer. The homogenate was centrifuged, and the supernatant dried with anhydrous sodium sulfate and evaporated under dry nitrogen in the dark. The extract was separated into its several lipid fractions by means of thin-layer chromatography (tic), using silica gel 60 (Merck) preparative plates of 2-mm thickness. The plates were developed in a solvent system of petroleum ether (bp 4575°C): ethyl ether:acetic acid (90: 10: 1) (Stahl, 1969). All solvents were purified according to the methods of Vogel (1962). The different lipid classes present in the snail extract were identified by spotting alongside the unknown mixture of a standard lipid mixture (Analabs, North Haven, Conn.). Development of the plates and comparison of the Rf values permitted identification. The three brightly colored orange-yellow to pink-violet spots of Rf values 0.81, 0.05, and 0.03 were scraped from the plate and extracted with chloroform or acetone. Saponification of the crude extract with 10% KOH in methanol

IN

Litkvinu

277

(Czeczuga and Czerpak, 1968a) did not affect the position of the colored spots on the plate. The eluted carotenoid pigment fractions were evaporated under dry nitrogen in the dark, and the residues weighed and rechromatographed on silica gel 60 F-254 (Merck) plates of 0.25-mm thickness. The plates were developed in six solvent systems (Table 3). The Rf values were recorded. The visible spectra of all carotenoid fractions were recorded in both hexane and ethanol in a Gilford 240 spectrophotometer and the extinction coefficient (Eiq cm) was calculated. In addition carotenoids were isolated from the hepatopancreas, the deep purplecolored nephridium of the snail, and the orange sporocysts of Cercaria parvicaudata (see Stunkard and Shaw, 1931; Stunkard, 1950), found in the digestive gland of some snails. Examination of the snail intestinal contents indicates that L. fittorea feeds mainly on the Chlorochyceae Ulva lactuca, Enteromorpha intestinalis, and E. compressa, and on the Rhodophyceae Agardhiella tenera and Polysiphonia spp. The pigments were extracted from these five species of algae by the method of Davies (1965). To extract pigments completely, the algae were soaked in 95% ethanol in the cold for 24 hr. Carotenoids were isolated from the extract by the methods described above. RESULTS

L. fittorea from Long Island Sound is parasitized by larvae of at least two trematodes: C. lingua sporocysts, redia, and cercariae, and the orange sporocysts and xiphidiocercariae of C. parvicaudata. The size of the snails examined ranged between 1.06 and 3.32 cm. The snails were grouped according to foot pigmentation, sex, and parasitic infection (Table 1). From these results it appears that there is no correlation between infection and foot pigmentation. Also foot color appears unrelated to the age or sex of the snail. Although

ZAVRAS

278

AND JAMES

TABLE CORRELATION

BETWEEN

FOOT PIGMENTATION PARASITIC INFECTION

1

IN Litturina littorea WITH TREMATODE

(SIZE RANGE LARVAE

1.06-3.32 cm)

AND

Infected No. of animals

Uninfected

Cryptocotyle

lingua

Cercaria

parvicaudafu

Pigmented 1678

Females Males

1117 561

843 (75.6%) 441 (78.7%)

245 (21.9%) 117 (20.8%)

29 (2.5%) 3 (0.5%)

Nonpigmented 436

Females Males

246 190

233 (94.8%,) 188 (99.0%)

7 (2.8%) 0 (0.0%)

6 (2.4%) 2 (1.0%)

the infection with C. parvicaudata is low (about 1.8%), this is the first report of this parasite from Long Island Sound. The pigment fractions from the different tissues of L. littorea and from C. par~~icaudata were obtained by preparative chromatography; the characteristics of these fractions are listed in Table 2. In partition, using hexane/95% methanol, all pigments exhibit an epiphasic behavior remaining in the hexane phase. Saponification with 10% alcoholic KOH did not affect the positions of the pigment fractions on the chromatogram, indicating that the chemical nature of the pigment was not affected by the treatment. Fraction I, the orange-yellow pigment with an Rf value of 0.81-0.83 (petroleum ether [bp 45-75”Cl:ethyl ether:acetic acid, 9O:lO: l), appears to be present in all tissues. Its visible spectrum shows maximum absorption at 426, 451, and 476 nm in hexane and 429,450, and 478 nm in ethanol. The E:qcm in both solvents is shown in Table 2. Comparison with standard crystalline p-carotene (Matheson, Coleman and Bell, Norwood, Ohio) indicates that the extracted pigment is p-carotene. Cochromatography with standard p-carotene in five different solvent systems (Table 3) confirms that fraction I is p-carotene. Other carotenoids extracted have not been completely identified, but show low Rf values in a solvent system of petroleum ether (bp 45-75”C):ethyl ether:acetic acid (90: 10: l), which indicates either more polar

carotenoids or oxygenated carotenoids. The Rf values and absorption maxima of these fractions are shown in Table 2. Rechromatography of all these fractions in two different solvent systems did not give a further separation. These Rf values are shown in Table 3. The visible spectra of the yellow and violet fractions extracted from the nephridium were not recorded because these pigments are present only in trace amounts. The yellow and pink-violet fractions extracted from the foot may possibly be ketocarotenoids, as characterized by their single absorption maxima, at 451 nm in hexane, 450 nm in ethanol for the former, and 550 nm in hexane, 450 nm in ethanol for the latter. The distribution of the carotenoids in the different tissues of L. littorea and C. parvicaudata sporocysts per 100 g wet weight is shown in Table 4. p-carotene is the major pigment in the digestive gland, nephridium, and C. par\~icaudata. In the foot, ficarotene and one of the oxygenated carotenoids are the major pigments; the yellow oxygenated carotenoid (Fraction II) is in higher concentration than the pcarotene (Fraction I). Only one carotenoid was isolated from all species of green and red algae. It is orange-yellow in color and shows the same absorption maxima in hexane and in ethanol as the p-carotene isolated from L. littorea tissues. Cochromatography with standard p-carotene shows that this pigment is p-carotene.

in parentheses

= inflection

Pink-red

II

” Values

Orange-yellow

Sporocysts

Red-brown

III

II III I

Yellow

II

Orange-yellow Yellow Violet

Orange-yellow

Pink-violet

III

I

Yellow

II

I

gland

Orange-yellow

possible

0.03

0.04 0.02 0.81

0.83

0.02

0.06

0.83

0.03

0.05

0.81

or impurity

426,45 1,476 429.450.478 360,415G420.451 400,450,(490)”

426,45 1,476 429,450,478 (420),” 451 450,(480)” 450, (480)” 450, (500)” 460 426,45 1,476 429,450,478 420,450,471-472 420.450 420,460 420,450 426,45 1,476 429,450,478 -

shoulder

2

Hexane Ethanol Hexane Ethanol

-

Hexane Ethanol Hexane Ethanol Hexane Ethanol Chloroform Hexane Ethanol Hexane Ethanol Hexane Ethanol Hexane Ethanol

AND

Epiphasic

Epiphasic

-

Epiphasic

2.58

2.58

x lo:’

x IO1

2.57

2.59

x 10”

x 1W

p-carotene

Unidentified

Unidentified Unidentified p-Carotene

p-Carotene

Unidentified

-

x lo:’

Ketocarotenoid (9 Ketocarotenoid (‘2

p-Carotene

-

2.58

-

x IO1

Identification

Epiphasic

x 1tY

2.57

450 nm

IN A SOLVENT

Unidentified

-

x 10’

1 Pnl

SPOROCYSTS

-

2.59

2.56

451 nm

El”’

ptrn’iccludata (9O:lO:l)

Epiphasic

Epiphasic

Epiphasic

Epiphasic

Epiphasic

ACID

Crrcari~~

Partition behavior (hexane/95’% methanol)

ETHER:ACETIC

TISSUES

Solvent

Litrorina lirrorw (bp 45-~~“C):&HYL

absorption (nm)

FROM ETHER

Maximum

FRACTIONS

OF PETROLEUM

indicating

Color of spots

SYSTEM

OF CAROTENOID

I

Fraction

CHROMATOGRAM

Nephridium

Digestive

Foot

Tissue

THIN-LAYER

TABLE

280

ZAVRAS

AND TABLE

THIN-LAYER

CHROMATOGRAM

OF CAROTENOID

JAMES 3

FRACTIONS FROM SPOROCVSTS

Littorinu

littorea

TISSUES AND C’rrcuria

parvicaudata Tissue

Fraction

Foot Digestive gland Nephridium Sporocysts

1

No. and color of spots

R,

1 Orange-yellow

0.94

I

p-carotene

0.52

Ketocarotenoid

(?I

0.78

Same

Ketocarotenoid

(?)

0.54

Same

Unidentified

0.71

Same

Unidentified

0.82 1 Orange-yellow

0.57 0.92

Orange-yellow 1 Orange-yellow Foot

II III

Digestive

gland

II III

Sporocysts

II

Foot

II

0.95

3 Yellow 2 Pink-violet 3 Yellow 2 Red-brown 2 Pink-red

0.78 0.39

Digestive

gland

11

1 Pink-violet 2 Yellow

III Sporocysts

II

Red-brown 1 Pink-red

&Carotene p-carotene p-carotene &Carotene

Unidentified Ketocarotenoid

(?)

0.94

Methylene dichloride: ethyl acetate (4:1, v/v) (Stahl, 1%9) Same

Ketocarotenoid

(?)

0.50

Same

Unidentified

0.79

Same

Unidentified

< III

Identification

A. Benzene B. Benzene:hexane (2: 1, v/v) (Czeczuga and (Czerpak, 1%8a) Petroleum ether (bp 45-70”C):benzene (1:l. v/v) (Stahl, 1969) Undecane: methylene dichloride (4: 1, v/v) (Stahl, 1%9) Hexane:ethyl acetate (2:l. v/v) (Foppen, 1971) Benzene:ethyl ether: methanol (17:2: 1, v/v/v) (Czeczuga and Czerpak. 1968a) Same

Orange-yellow I

System of solvents

0.42

DISCUSSION

Contrary to the reports of Willey and Gross (1957), this study shows that the foot pigmentation of L. littorea from Long Island Sound cannot be the result of infection with trematode larvae (Table 1). Although 23.5% of the snails with pigmented feet were parasitized, it does not indicate that the pigment is due to the parasitic infection, since 76.5% of the snails with pigmented feet were clearly not parasitized. Only 3.4% of the snails with unpigmented feet were parasitized, but a much smaller number of unpigmented than pigmented animals were collected in the area. It appears that animals exhibiting foot pigmentation are dominant in the area. Since animals were

Unidentified

collected at random all year round, dominance cannot be due to seasonal variation. Also early and late stages of infection were considered but no correlation could be made with foot color. It is clear that the majority of the snails exhibiting foot pigmentation (light brown, dark brown, or red) were not infected with larval stages of C. lingua or C. parvicaudata, and therefore the foot pigment cannot be due to the parasitic infection. Marshall (1974) suggested that the destruction of the digestive gland by the trematode larvae brings on the release of carotenoids. The presence of pigmented larval stages indicates absorption of the released carotenoids by the parasite. If the

TREMATODES

AND

CAROTENOIDS

TABLE

IN

281

Lirrorinn

4

APPROXIMATE PERCENTAGE OF CAROTENOIDS IN Litrorina

lirforea

parvicaudatu

TISSUES AND Cercaria

SPOROCYSTS

No. of animals 300 100 200 54 17

Tissue Pigmented foot Nonpigmented foot Digestive gland Nephridium Sporocyst

Weight (g) 30

Carotenoid fractions (g)

Percentage carotenoids/lOO g weight

I

II

III

I

II

III

31

40.8

12.2

0.10

0.14

0.04

3.0

0.05

0.06

0.03

29.4 Trace -

0.52 0.57 0.15

0.18 Trace 0.05

0.09 Trace

8.52

4.3

5.5

32 1 2.18

165 5.7 3.2

56.1 Trace 1.1

parasite does not have pigmented larva stages, the released carotenoids circulate in the hemolymph and are deposited in the foot. This study (Table 1) shows that the above scheme does not hold true. Snails with both pigmented and unpigmented feet were found to be parasitized by C. lingua with unpigmented larva stages, and by C. pan%-audata with pigmented larva stages. The possibility that the pigment might be sex related or result from aging has been ruled out (Table 1). Both males and females show pigmentation as do the very young and very old snails. Snails have been collected at random from both areas all year round. With respect to their foot color and their distance from water at low tide, the snails do not show any distinct zonation or segregation pattern. Pigmented and unpigmented snails are found at all levels up from the low-tide mark, and there is not a particular area or rock where one type of snail predominates over the other. Apparently foot pigmentation cannot be correlated with any of the above factors. Also snails kept in the laboratory for about 6 months showed no observable change in foot pigmentation, which implies that some intrinsic factor controls foot pigmentation. The major pigment extracted from the tissues of L. littorea is p-carotene. This pigment has been isolated from the tissues of many molluscs (Hoskin and Cheng,

1975; McBeth, 1972; Marshall, 1974; Nakadal, 1960af. Since p-carotene is a plant pigment not produced by animals, it must come from the algae upon which the snails feed. p-carotene has been isolated from the five species of algae upon which L. littorea feeds. The presence of oxygenated carotenoids in the tissues of L. littorea indicates that part of the p-carotene is metabolized and accumulated by the snail. Many vertebrates and invertebrates metabolize plant carotenoids and store them in their tissues. Nudibranch molluscs cannot metabolize carotenoids (McBeth, 1972) but deposit them unchanged in their tegument. L. littorea appears to have the ability to metabolize carotenoid pigments. The two other carotenoids from the foot are possibly ketocarotenoids because of the single absorption maxima. Many invertebrates are known to contain ketocarotenoids (Czeczuga, 1972; Nakadal. 1960a) and several pathways of degradation of p-carotene to different monoketo- and diketocarotenoids have been postulated (Islet-, 1971; Lee, 1966). It is possible that L. littorea metabolizes p-carotene to ketocarotenoids through one of these described pathways or through a different one. In almost all proposed pathways echinenone seems to be the first or second product in P-carotene metabolism. The

282

ZAVRAS

AND

yellow carotenoid from the foot, which appears in higher concentration than pcarotene (Table 4), with maximum absorption at 451 nm in hexane may possibly be echinenone. Since the absorption maxima of echinenone extracted from different invertebrates in hexane has been reported as 451 (Veerman, 1974), 455-456, and 458 nm (Czeczuga and Czerpak, 1968a, b), and because standard echinenone was not available for positive identification, this carotenoid is considered here as a ketocarotenoid, possibly echinenone. In the other tissues examined, the hepatopancreas and nephridium, pcarotene is the major pigment (Table 4). The other carotenoids in these tissues have not been completely identified. In the nephridium the carotenoids are found as purple granules in distinct transparent spheres. The function of these inclusions in the columnar epithelium of some prosobranchs (Hyman, 1967) is unknown. They may represent excess amount of carotenoids for excretion or stored carotenoids for usage when needed in the form of carotenoproteins. The orange sporocysts of C. par1-~icaudata contain two carotenoids, with p-carotene as the major pigment. Carotenoids have been reported from several species of trematode larvae, sporocysts, rediae, and cercariae occurring in the marine snail Cerithideu culifornicu (Nakadal, 1960b), from Himusthlu quissetensis rediae found in Nussurias obsoletus (Hoskin and Cheng, 1975), and from Himusthlu leptosomu redia and the sporocysts of Cercariu linearis occurring in Littorinu littoreu and Gibbalu umbiliculis (Marshall, 1974). Apparently trematode larvae selectively absorb p-carotene from the host tissues and accumulate it unchanged. A pink-red carotenoid extracted also from C. purvicuudatu sporocyst has not been completely identified (Tables 2, 3). It is possible that it has been also absorbed from the host tissues, but it is more likely

JAMES

that it is a product of metabolism of pcarotene within the sporocyst, since the characteristics of this pigment are different from the characteristics of all other carotenoids found in the host’s tissues. These results as compared with the results of Hoskin and Cheng (1975) and Nakadal (1960b) indicate that some trematode larvae have the ability to metabolize p-carotene as well as accumulate it, in an unchanged form. This selective absorption and accumulation of carotenoids indicates that these pigments may play some important role in the physiology of the organism. The function of carotenoids in animals, especially in molluscs, is not yet clear. In many invertebrates they occur as carotenoproteins (Cheesman et al., 1976) and they may stabilize the protein structure or act as enzyme activators in the maintenance of mucous membranes in Mytilus (Scheer, 1940). The chemical structure of carotenoids, with their many double bonds, indicates that they can be excellent acceptors and donors of electrons, and could facilitate the transfer of electrons from one compound to another. Carotenoids have been implicated in many energy transfer mechanisms in plants and animals (Dingle and Lucy, 1965; Govinjee and Govinjee, 1974). It is also possible that carotenoids are involved in the respiration and electron transport system of invertebrates since many molluscs have pigmented ganglia or neurons. The degree to which molluscs can tolerate anoxic conditions depends on the degree of pigmentation of their nervous system (Zs-Nagy, 1971). Carotenoids can act as final electron acceptors, substituting molecular oxygen in the metabolism when the mollusc is in a low-oxygen environment (Zs-Nagy, 1971). If this is true for the molluscan nervous tissue, it can very well be true for the muscle and other tissues in molluscs. L. littorea lives in the intertidal zone where fluctuations of temperature, salinity, and oxygen exist. Extreme oxygen tensions

TREMATODES

AND

CAROTENOIDS

can occur in such an environment. The animals may be adapted to this environment by possibly utilizing carotenoids obtained from their algae food to survive when the environment cannot provide sufficient oxygen tension. The amount of carotenoids in the foot of these animals seem to be regulated by some other factor, presumably genetic, rather than the amount of food available. If genetically controlled, variation in foot color may indicate the existence of two populations of L. littorecr with respect to carotenoid content. Interbreeding of dark brown and white-footed populations may produce the light brown offspring. This excessive accumulation of pigment in the foot of L. littarea. which is also a mucus-producing structure, may provide the animal with a backup system for survival in extreme conditions of oxygen tension. Most internal parasites are anaerobic or adapted to low oxygen tension. C. par~icaudata destroys the visceral mass of L. littorecr (Robson and Williams, 1971) which probably produces a complete anoxia in that area. The carotenoids may be involved in the electron transport system of the parasite. In this way the parasite which probably is not an obligate anaerobe can survive under the extreme conditions of parasiteinduced anoxia. Since very little is known about the physiology of these larvae, more work is required to elucidate the function of carotenoids in C. parvicaudcrtcr as well as in its host L. littorea. REFERENCES ARVANITAKI, A., AND CHALAZONITIS, N. 1960. Photopotentiels d’activation et d’inhibition de differents somata identifiables (Aplysia). Activation monochromatiques. &,!I. Inst. Owrrno~r.. 1164, l-83. BENJAMIN, P. R.. AND WALKER, T. S. 1972. Two pigments in the brain of a fresh-water pulmonate snail. Camp. Biochern. Physiol. B. 41, 813-821. CHEESMAN, D. F.. LEE. W. L.. AND ‘ZALGALSKY, P. F. 1967. Carotenoproteins in invertebrates. Biol. Ret,.. 42, 131-160. CZECZUGA. B. 1972. Astaxanthin-The carotenoid predominant in Eylais hamotu (Koenike. 1897) (Hydracarina, Arachnoidea). Camp. Biochem. Ph~.\io~. 8. 42, 137- 141.

IN

Littorina

283

CZECZUGA, B., AND CZERPAK, R. 1968a. Carotenoids in the carapace of the Orconectes limosus (Raf.) (Crustacea: Decapoda). Eur. J. Biochem.. 5, 429-432. CZECZUGA. B., AND CZERPAK. R. 1968b. The presence of carotenoids in Eylais hamara (Koenike, 1897) (Hydracarina, Arachoidea). Camp. Biochem. Physiol.. 24, 37-46. DAVIES, B. H. 1965. Analysis of carotenoid pigments. In “Chemistry and Biochemistry of Plant Pigments” (T. W. Goodwin. ed.), pp. 489-532. Academic Press, London/New York. DINGI.E, J. T., AND LUCY, J. A. 1965. Vitamin A, carotenoids and cell function. Biol. Rev.. 40, 422-461. FOPPEN, F. H. 1971. Tables for the identification of carotenoid pigments. Chromatogr. Rev., 14, 133-298. GOODWIN, T. W., AND TAHA, M. M. 1950. The carotenoids of the gonads of the limpets Patella taltlgcrtcr and Pate/lo depressu. Biochem. J.. 47, 244-249. GOVINDJEE, AND GOVINDJEE. R. 1974. The absorption of light in photosynthesis. Sci. Amer. 231, 68-82. HOSKIN. G. P., AND CHENG. T. C. 1975. Occurrence of carotenoids in Himusthla yuissetensis rediae and the host Nusswiu.s obsoletrcs. J. Parnsitol., 61, 381-382. HYMAN, L. H. 1967. “The Invertebrates: Mollusca I.” Vol. VI. McGraw-Hill, New York. ISLER, 0. (ed.) 1971. “Carotenoids.” Halstead, New York. JAMES, H. A. 1974. Sex and foot color in the periwinkle snail, Littorina liftorea (L.) infected with larval stages of the heterophyid tremadode, C’lyptocot~le lingua (Creplin). In “Third International Congress of Parasitology,” Vol. 1, p. 341. LEE, W. L. 1966. Pigmentation of the marine isopod Idotheu granctlosa (Rathke). Comp. Biochem. Ph.wio/.. 19, 13-27. MCBETH, J. W. 1972. Carotenoids from nudibranchs. Camp. Biochem. Physiol. B, 41, 55-68. MARSHALL, I. 1974. Carotenoids in Littorinu littorea and Gibbula umbilicalis and in their germinal sacs. Pclrasitology. 69, 9. NAKADAL, A. M. 1960a. Carotenoids and chlorophyllit pigments in the marine snail, Cerithidea californicn Haldeman, intermediate host for several avian trematodes. Biol. Bull.. 119, 98- 108. NAKADAL, A. M. 1960b. Types and sources of pigments in certain species of larval trematodes. J. Parasitol.. 46, 777-786. REES. W. J. 1936. The effect of parasitism by larval trematodes on the tissues of Littorinu littorecr Linne). Proc. 2001. Sot. London, 2, 357-368. ROBSON, E. M.. AND WILLIAMS, I. C. 1970. Relationships of some species of digenea with the marine prosobranch Littorina littorea (L.): The occurrence of larval digenea in L. litforea on the north Yorkshire coast. J. Hehninthol.. 44, 153- 168.

284

ZAVRAS

ROBSON, E. M., AND WILLIAMS, I. C. 1971. Relationship of some species of digenea with the marine prosobranch Litrorina littorea (L.). II. The effect of larval digenea on the reproductive biology of L. littoreu. .I. Helminthol., 45, 1455149. SCHEER. T. 1940. Some features of the metabolism of the carotenoid pigments in the Californian sea mussel (Myfilus caljforniunu~). J. Biol. Chem.. 136, 275-299. STAHL, E. (ed.) 1969. “Thin Layer Chromatography: A Laboratory Handbook.” 2nd ed. Springer-Verlag. New York. STUNKARD, H. W. 1950. Further observations on Cercuria parvicarrdata. Stunkard and Shaw, 1931. Biol. Bull.. 99, 136-142. STUNKARD, H. W., AND SHAW, C. R. 1931. The effect of dilution of sea water on the activity and longevity of certain marine cercariae. with descriptions of two new species. Biol. Bull., 61, 242-271. VEERMAN, A. 1974. The carotenoid pigments of

AND

JAMES Schizonobiu sycophantn Womersley (Atari: Tetranychidae). Camp. Biochrm. Physiol. B. 48, 321-327. VILLELA, G. G. 1956. Carotenoids of some Brazilian freshwater gastropods of the genus Pomaceu. Nuture (London) 178, 93. VOGEI., A. I. 1962. “A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis.” Wiley, New York. WERDING. B. 1969. Morphologie, entwicklung und okologie digener trematoden-larven der strandschnecke Litturincr liftorrrr. Mar. Biol., 3, 306-333. WILLEY. C. H.. AND GROSS, P. R. 1957. Pigmentation in the foot of Litrorina litroreu as a means of recognition of infection with trematode larvae. J. Puru.sifol.. 43, 324-329. ZS-NAGY, I. 1971. The lipochrome pigment of molluscan neurons as a specific electron acceptor. Conrp. B&hem. Physiol. A, 40, 595-602.