Effects of copper and zinc on the life cycle of Notocotylus attenuatus (Digenea: Notocotylidae)

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EFFECTS OF COPPER AND ZINC ON THE LIFE CYCLE OF NOTOCOTYLUS A TTENUA TUS (DIGENEA: NOTOCOTYLIDAE) N.

A. EVANS

Department of Applied Biology, University of Cambridge, Pembroke Street, Cambridge, CB2 3DX, England* (Received 23 October 1981)

Abstract-Eves N. A. 1982. Effects of copper and zinc on the life cycle of Notocotyhs attenuutus (Digenea : Notocotylidae). ~nternafton~~Journal for Parasitology 12: 363-369. The toxicity of copper and zinc to cercariae and metacercarial cysts of Noto~otyi~ ~tten~~t~s was extremely iow. The infectivity of cysts was not reduced even after exposure to high metat concentrations (IO-0 p.p.m.1 for six weeks whilst the encystment of cercariae was significantly impaired only by 10.0 p.p.m. of copper and by concentrations of zinc greater than 4.0 p.p.m. Metal-induced death of cercariae occurred most frequently during their attempts to encyst. Cercarial shedding from the first intermediate host Lymnueuperegru was, in contrast, substantially reduced by even low concentrations (0.1 p.p.m.) of both metals. Water hardness had a low influence on copper and zinc toxicity. INDEX KEY WORDS: Noto~oty~~ Qtten~Qtus; Lymnaea peregra; toxicity; copper; encystment; cercariai shedding: metacercariai cyst. INTRODUCTION

zinc; cercariae;

MATERIALS AND METHODS Solutions. Stock solutions of copper and zinc containing 100 p.p.m. of metal ion were prepared by dissolving copper suiphate (CuSO,.SH,O) or zinc chloride (ZnC12) in distilled water. These were diluted in soft and hard synthetic freshwater media (H.M.S.O., 1969) having a total hardness of 25 or 250 p.p.m. (measured as CaCO,) respectively, to host-parasite relationships. The present study set out provide a series of test solutions with metal concentrations to investigate the effects of copper and zinc, two ranging from 0.1 to 10.0 p.p.m. During long term experiimportant heavy metal pollutants of aquatic eco- ments the test solutions were renewed daily. Occasionally systems in indust~~ised countries, upon transmi~ion the concentration of both soluble and total metaf in the stages of the digenean, ~o~oco~yl~s a~t~nu~tus. The solutions was monitored and the results compared with life cycle of N. izttentrattcs, a common parasite of calculated concentrations. To measure the concentration of soluble metal the test solution was filtered before acidifiwildfowl, does not involve a second intermediate cation and analysis in an Evans Electroselenium 240 atomic host because metacercarial cysts are formed upon solid objects in the environment. Furthermore there absorption spectrophotometer. The concentration of total metal was obtained by acidification of the solution before is no free-living miracidial stage, the first interanalysis. mediate host becoming infected after ingestion of Ex~~~mentQ~ procedures. Specimens of Lymnuea infective parasite eggs. peregra infected with Noiocot~&s attenuatus were collected Three effects of copper and zinc were investigated. from a tributary of the River Great Ouse at Thornborough, Buckinghamshire. The effect of copper and zinc upon cerFirstly their effect upon cercarial encystment. carial encystment was investigated by introducing recently Secondly their effect tipon the viability of metaceremerged cercariac into a range of test solutions in 25 ml carial cysts and finally their effect upon cercarial glass vials and recording the proportion which formed shedding from the first intermediate host Lymnuea normal cysts. Cercariae killed during encystment were peregw Experiments were conducted in both soft processed for electron microscopy to determine precisely and hard water because water hardness is an importhe stage at which death had occurred. The parasites were tant determinant of metal toxicity. fiied for 1 h in I % glutaraldehyde buffered to pfi 7.2-7.4 with 0.05~ phosphate buffer and made up to a total molarity of 0.27~ with sucrose. Specimens were post-fixed *Present Address: Department of Zoology, King’s College, in buffered 1% osmium tetroxide for 2 h, dehydrated University of London, Strand, London, WCZR 2LS, through a graded series of ethanols and propylene oxide England. and embedded in Epon 812. Sections were collected on ?kE TRANSMISSION of digenean parasites in aquatic habitats may be affected in several ways by poor water quality. For instance, the presence of pollutants could reduce the survival of hosts and parasite stages or alternatively could influence the course of

363

N. A

364

copper grids, stained with uranyl acetate and lead citrate and viewed in an AEI EM6B or JEOL 1OOCX electron microscope operating at 80 kV. The toxicity of copper and zinc to metacercarial cysts was investigated as follows. Two-day-old cysts were introduced to test solutions for either 1 or 6 weeks. Control cysts were kept for the same time periods in soft or hard water only. Parasite viability was assessed by administering 30 cysts from each treatment to ‘I-day-old chicks. The chicks were necropsied 6 days post infection and the number of adult worms recovered from the caeca and large intestine recorded. To investigate the effect of copper and zinc upon cercarial shedding, infected specimens of L. peregru were first isolated in crystallising dishes containing hard or soft water for 6 days to obtain an estimate of the daily shedding rate for each snail. The snails were then introduced to the appropriate test solutions and observed for a further 6 days. Three snails were used in each treatment and they were fed on lettuce during the course of the experiment. Experimental temperature was maintained at 18?2”C. RESULTS

Effects of copper and zinc upon encystment of cercariae In hard and soft water controls and in concentrations of heavy metal below 1.0 p.p.m., all cercariae

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EVANS

encysted successfully (Fig. 1). The proportion encysting was significantly different from the controls in 6 treatments only and these were: 10.0 p.p.m. copper in soft water; 4.0, 5.0 and 10-Op.p.m. zinc in hard water and 5.0 and 10.0 p.p.m. zinc in soft water (in each case p(x2>3.84)<0.05; D.F. = I). Zinc thus appeared to be slightly more toxic than copper to cercariae. Water hardness had little influence upon metal toxicity. In test solutions causing significant cercarial mortality it was noted that death usually occurred during the parasites’ attempts to encyst. Occasionally the parasites were killed at a very early stage of encystment before the formation of even the outermost layers of the cyst wall (Fig. 2A). More frequently however, death occurred at a later stage of encystment, during the formation of the inner cyst wall (Figs. 2B and C). Southgate (1971) observed that the inner layer of the cyst wall of N. attenuatus is derived from the granules of sub-tegumental “keratin cells” and that after extrusion from the parasite these keratin-like granules dissociate and lamellae within them unfold to contribute material to the cyst. The ultrastructural observations of the present study

(A)

(C)

Concentration

of

metal,

p.pm

FIG. 1. The percentage (f S.E.M.) of N. attenuatus cercariae completing encystment in differing concentrations of copper in hard (A) and soft (B) water, and in differing concentrations of zinc in hard (C) and soft (D) water.

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Toxicity

of copper

and zinc to N. aftenuatus

365

FIG. 2. Electron micrographs of parasites killed by copper and zinc. A: Section through the ventral surface of a cercaria which has been killed at an early stage of encystment by 10.0 p.p.m. of zinc in soft water. The tegument is packed with cystogenous granules [“type 3” granules of Southgate (1971)] and some have been extruded from the parasite surface. B: Section through the dorsal surface of a parasite killed by 10.0 p.p.m. zinc in soft water. The outer (cwl and cwla) and middle (cw2) layers of the cyst wall are completely formed but the inner layer (cw3) is only partially formed and complete unrolling of the keratin-like granules (arrows) has not occurred. C: Section through the dorsal surface of a parasite killed by 10.0 p.p.m. copper in soft water. Explanation and 1et:ering as in Fig. 2B.

366

N.

A. EVANS

indicate that complete extrusion and unrolling of the “keratin-like” granules had not occurred in those parasites killed by copper or zinc. Effects of copper and zinc upon ~eta~ercaria~ cysts

Although metaeercariaf viability was reduced, relative to the controls, by several treatments (see Fig. 3) none of these reductions were in fact significant (in each case p(x2<364)>0.05; D.F. = 1). The recovery of adult parasites was generally higher from the younger cysts (Figs. 3A and B) than from the older cysts (Figs. 3C and II), thus suggesting that the infectivity of N. ~tfe~~at~~ metacercariae is agedependent. Effects of copper and zinc upon cercarial shedding

The addition of even low concentrations (0.1 p.p.m.) of copper and zinc in both hard and soft water reduced the rate of cercarial shedding (Fig. 4). In copper concentrations of O-5 p.p.m. in soft and hard water cercarial shedding was terminated within

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2 and 3 days respectively as a result of metal-induced death of the snail hosts. In all other test solutions the snails survived the 6 day exposure period although their activity was reduced. The effect of both metals upon cercarial shedding was not substantially influenced by water hardness. The fate of copper and zinc in test solutions

Losses of dissolved metal occurred within 10 min in all test solutions and continued for at least a further 6 h (Table 1). The initial rate of loss was greatest from the high (10~0 p.p.m.) metal concentrations. After 14 h the concentration of soluble forms of the metais was largely independent of water hardness. The observed losses of dissolved metal were presumably due to their precipitation as insoluble inorganic complexes and adsorption to the glass walls of the container rather than to evaporation because the measured total metal concentrations remained similar to calculated concentrations in the majority of cases.

q cu

- (B)

n

Zn

~~ 0

Concentration

12. 1982

of

metal,

0.1

IO

10.0

ppm.

FIG. 3. The percentage recovery of N. attenuafus from chicks after administration of metacercarial cysts exposed to differing metal concentrations for I week in hard (A) and soft (B) water and for 6 weeks in hard fC) and soft (D) water.

Toxicity of copper and zinc to N. alfenuam

I.J.P. VOL.12. 1982

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Time, days FIG. 4. Shedding rate of N. attenuafus cercariae from L. peregra in differing concentrations of copper and zinc in hard (A, B) and soft (C, D) water: f l), control; (m), 0.5 p.p.m. Cu; (A), 0.1 p.p.m. Cu; (0), I.0 p.p.m. Zn; (C!), 0.5 p.p.m. Zn; (A), 0.1 p.p.m. Zn. Solid arrows indicate time of transfer of snails to test solutions. TABLEI-THE Time after manufacture of test solution (a) Copper 10 min

6h

14 h (b) Zinc 10 min

6h

14 h

FATE

OF SOLUBLE

Calculated concentration of metal (p.p.m.)

Measured concentration of solubfe metal (p.p.m.) In hard In soft water water

IN TEST

SOLUTIONS*

Measured concentration of total metal (p.p.m.) In hard In soft water water

0.36 0.57 3.10 0.20 0.43 0.82 o-34 0.35 I.14

0.42 0.81 880 0.28 0.38 4.15 0.30 0.42 240

1.05

I-02

9.52 O-47 1.01 9.70

8.96 0.48 0.95 9.39

0.5 1.0 10.0 0.5 1.0 to.0 0.5

0.43 0.75 7.81 0.36 0.57 4-45 0.34 0.61 4.20

040 0.79 8.10 0.32 0.49 5.73 0.31 o-53 5.22

0.54 0.95 9.30 0.51 O-98 9.00 0.47 0.96 9.14

0.48

I .o No

OF COPPERANDZINC

0.5 1.0 10.0 O-5 I.0 10.0 0.5 I.0 to.0

10.0 *N.B.

FORMS

organisms present in the test solutions.

0.48 1.00 8.91 0.51

0.50 I-00 8.83 0.49

1.06 9.32 0.49 0.96 9-56 0.50 o-93 9.20

N. A.

368 DISCUSSION

The toxicity of copper and zinc to the cercariae and in particular, the metacercarial cysts of N. a~tenuatus was extremely low. The viability of metacercariae was unaffected by exposure to metal solutions for 6 weeks, suggesting that the thick and chemically complex cyst wall acts as an effective barrier to toxic metal ions. This impermeability may be attributable largely to the keratin-like protein comprising the inner cyst wall. Zinc and copper concentrations as high as 4-O and 10.0 p.p.m. respectively were required to significantly affect cercarial encystment. This relative insusceptibility of N. attenuates cercariae is surprising but may be partly explained by the rapid rate of encystment which occurred in the small experimental containers as a result of the high rate of contacts between the cercariae and a suitable solid surface. The consequent shortening of the cercarial life span could have reduced the toxicity of copper and zinc because, once encysted, the parasites are protected from the metals’ effects. Where mortality of cercariae did occur the period of maximum vulnerability was during encystment. Encystment is accompanied by intense secretory activity from the parasite tegument (Southgate, 1971) and it is possible that at this time tegumental permeability is increased thus enhancing the uptake of toxic metal species. Both copper and zinc exert their toxic effects by binding to the active sites on enzyme molecules and therefore could cause death by de-activating any of a wide variety of enzyme systems. In contrast to the insusceptibility of the cercarial and metacercarial stages, the shedding of N. attenuatus cercariae from the first intermediate host was markedly reduced by low concentrations of both metals. In treatments with 0.5 p.p.m. of copper the cessation of cercarial shedding was a consequence of metal-induced snail mortality. In all other treatments the depressed rate of shedding may have been due to observed reductions in snail activity or to a build up of heavy metal concentrations within snail tissues which adversely affected parasite development. Anderson, Nowosielski & Croll (1976) demonstrated that the maximal shedding rate of Trichobilharzia ocelluta cercariae coincided with periods of peak activity in the snail host Lymnnea stag&is and they speculated that muscular movements of the snail might aid the parasites to reach exit points. Thus the lowered activity of L. peregra induced by copper and zinc could possibly account for reductions in the shedding of N. attenuatus cercariae. An alternative explanation of reduced cercarial shedding is the inhibition of parasite development within metal treated snails. Yescott & Hansen (1976) observed reduced shedding of Schistosoma mansoni cercariae from manganese treated Biomphalaria glabrata and considered that this was caused by an inhibition of parasite development. Such inhibition might well be metal

induced

because

many

molluscs

are known

to

EVANS

I.J.P. VOL.

12. 1982

accumulate substantial amounts of heavy metals in their tissues (Bryan, 1976). Furthermore, in a study of metal uptake by Biomphalaria gIabrata, Yager & Harry (1966) have shown that the digestive gland is the major organ for metal accumulation and this is the primary site for multiplication and development of larval digeneans. It is generally agreed that an inverse relationship exists between metal toxicity and water hardness due largely to an increased precipitation from solution of toxic forms of the metals as water hardness increases (Black, Roberts, Johnson, Minicucci, Mancy & Allen, 1973). The low influence of water hardness observed in the present study is therefore surprising but may be partly explained by the fact that the concentrations of soluble forms of the metals fell to broadly similar levels in test solutions irrespective of the hardness of the water with which they were manufactured. The concentrations of copper and zinc in natural waters are extremely variable. FGrstner & Whittman (1979) quote 0.01 and 0.002 p.p.m. as typical background concentrations of soluble zinc and copper although in heavily polluted waters such as mine waste contaminated rivers the concentration of soluble zinc and copper may rise to 2.4 and 0.8 p.p.m. respectively (Brown, 1977). Even these high environmental concentrations would have little effect upon cercarial and metacercarial stages of N. attenuatus but they would severely affect cercarial shedding, assuming that the results of the present laboratory study have a general applicability to field situations. In the absence of information concerning the potential toxicity of heavy metals to N. attenuatus eggs it is considered most likely that in natural environments subjected to copper and zinc pollution, any disruption of the parasite’s life cycle would be due to reduced viability of the first intermediate host and depression of cercarial shedding. Acknowledgemenrs-This work was conducted during tenure of an Imperial Group Research Fellowship Wolfson College, Cambridge.

the at

REFERENCES ANDERSON

P. A.,

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J. W.

& CROLL N.

A.

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Lymnaea sragnalis. Canadian Journal of Zoology 54: 1481-1487.

BLACK

J. A.,

ROBERTS

R.

F.,

JOHNSON

D.

M.,

MINICUCCI

D. D., MANCY K. H. & ALLEN H. E. 1973. The significance of physicochemical variables in aquatic bioassays of heavy metals. In: BioassayTechniquesand Environmental Chemistry (Edited by GLASS G. E.) pp. 259-275. Ann Arbor Science Publishers, Ann Arbor. BROWN B. E. 1977. Effects of mine drainage on the River Hayle, Cornwall. A. Factors affecting concentrations of copper, zinc and iron in water, sediments and dominant invertebrate fauna. Hydrobiologia 52: 22 I-233.

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Toxicity of copper and zinc to N. attenuatus

BRYAN G. W. 1976. Some aspects of heavy metal tolerance in aquatic organisms. In: Effects of Pollutants on Aquatic Organisms (Edited by LOCKWOOD A. P. M.), pp. 7-34. Cambridge University Press, Cambridge. FGRSTNER U. & WHITTMAN G. T. W. 1979. Metal Pollution in the Aquatic Environment. Springer-Verlag, Berlin, Heidelberg and New York. H.M.S.O. 1969. Fish Toxicity Tests. H.M.S.O. Leaflet No. Dd 139779 K36 12/69. SOUTHGATE V. R. 1971. Observations on the fine structure

369

of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria. Zeitschrifr fiir

Zellforschung und Mikroskopische Anatomie 120: 420449. YAGER C. M. & HARRY M. W. 1966. Uptake of heavy metal ions by Taphius glabratus, a snail host of Schistosoma mansoni. Experimental Parasitology 19: 174 182. YE~COTT R. E. & HANSEN E. L. 1976. Effect of manganese on Biomphalaria glabrata infected with Sch&tosoma

mansoni. Journal of Invertebrate Pathology 28: 315-320.