Effects of Trichobilharzia ocellata on growth, reproduction, and survival of Lymnaea stagnalis

EXPERIMENTAL PARASITOLOGY 24, 137-146

Effects

(1969)

of Trichobilharzia

ocellata

Reproduction, and Survival Lymnaea sfagnalis’ Gary Department

of Zoology,

McClelland University

(Submitted

and

of

T. K. R. Bourns

of Western

for publication,

on Growth,

Ontario,

London,

Ontario,

Canada

21 June 1968)

MCCLELLAND, CARY,~ AND Bounds, T. K. R. 1969. Effects of Trichobilharzia ocellata on growth, reproduction and survival of Lymnaea stagnalis. Experimental Parasitology 24, 137-146. Juvenile Lymnaea stagnalis were exposed to single miracidia of Trichobilharzia ocelluta after which shell lengths, numbers of eggs, and mortality of parasitized and unexposed control snails were recorded weekly. No egg-laying occurred and no significant difference in shell growth or mortality was observed during most of the prepatent period. From week 12 after exposure, infected snails laid about 1 egg/snail/week while controls laid 35-85 ( mean 57 ) eggs/snail/week. Shell growth of infected snails continued at rates of 1.5 to 2 mm/week tapering off as maximum size was approached. Controls, on the other hand, showed a depression of growth rate to 0.5 to 1 mm/week. At 28 weeks of age all controls were dead but 90% of infected snails were alive. Histological study revealed that the ovotestis regressed and the reproductive tract failed to develop in parasitized snails but there was no evidence of mechanical damage to the gonad or digestive gland. INDEX DESCRIPTORS: Lymnaea stagndis; Trichobilharzia ocellata; Trematode-snail relationships; Schistosome-snail relationships; Snail-schistosome relationships; Growth of parasitized Lymnaea stagnabs; Survival of parasitized Lymnaea stagnalis; Fecundity of parasitized Lymnaea stagnalis.

Field data concerning Lymnaea stagnalis and its trematode parasites at Peterborough, Ontario (Bourns, 1963 and unpublished) indicate that some parasitic species harm their molluscan host to the extent of inhibiting its growth and shortening its life. Such data do not, however, permit one to conclude with confidence that specific cause and effect mechanisms have been at work nor do they provide information concerning the effects of parasitism upon the host’s fecundity. With a view to establishing the existence of specific mechanisms the authors have, as a preliminary step, examined the effects of 1 This study was supported by the Ontario Department of University Affairs. 3 Present address: Department of Zoology, University of Guelph, Guelph, Ontario, Canada.

monomiracidial infections of Trichobilharzia ocellata in juvenile L. stagnalis on the subsequent growth, fecundity, and survival of the snails under controlled conditions. MATERIALS

AND METHODS

Snail Rearing and Maintenance Snails used in this study were 2nd and 3rd generation L. stagnalis derived from a single field-collected egg mass. Juveniles were kept at room temperature until the beginning of the experiment when they were moved to 3.5-gal aquaria under constant conditions of fluorescent lighting at 20°C. Population densities of 25 juveniles or 10 adults per tank were maintained. Fresh lettuce, chalk, and sand were avail137

MC CLELLAND AND BOURNS

138

able at all times, water was changed weekly, and charcoal-glass wool filters replaced monthly. Plastic bird bands (Bourns and Scott, 1964) were used to identify individual snails in one experiment. Exposure to Parasites Black ducks, Anas rubripes, which had been exposed in the laboratory to cercariae of T. ocellata were kept on wire over 0.857c saline. ‘Miracidia were obtained by decanting the salt solution, mixing the sediment with distilled water, and transferring the fecal suspension to 3-liter side-arm flasks (McMullen and Beaver, 1945). A single miracidium in a droplet of water was deposited into each of a number of 10 X 35-mm plastic petri dishes and a single snail was placed directly into each droplet. After a few minutes the dishes were partly filled with distilled water and were left overnight. Most of the snails were exposed when 6 weeks old with shells 8-15 mm long. Ten-week-old snails with shell lengths of 15-25 mm were used when it was necessary to identify individuals by affixing numbered bands to their shells. Control snails received the same treatment throughout except that no miracidium was placed in the original drop of water. Data Collection Commencing 42 days after exposure, the minimum incubation time at 20°C each snail was checked daily for cercarial shedding. Individuals were placed in distilled water for 2-3 hours, then the water was examined microscopically for the presence

of cercariae. Each week the shell length of each snail was measured to the nearest 0.02 mm with a Vickers vernier microscope and snail egg counts and egg mass measurements were made. Dead snails were removed daily but were tabulated on a weekly basis. Histology

and Anatomy of Snails

Entire snails removed from their shells were fixed in Davidson’s fluid after which the gizzards were removed to protect the knife from gravel. Sections cut at 7 ~1were stained in Ehrlich’s alum hematoxylin, Bowie’s eosin, and orange G. Accessory reproductive organs for gross anatomy or embedded in paraffin were softened in a solution of 1 part glycerine to 1 part 60% alcohol ( Holm, 1946). RESULTS

Efect

of Parasitism on Growth

A. During the prepatent period in juvenile snails. Thirty-seven banded snails developed infections and survived to patency following exposure to’ single miracidia when 10 weeks old and 35 unexposed control snails survived the 6-week incubation period. Increases in shell length did not differ during the first 4 weeks of incubation (Table I) but during the fifth and sixth weeks, when the snails were lP16 weeks old, the infected snails showed significantly greater growth (11 < .025) than did the controls. B. During the patent period in adults. Two hundred infected snails and 200 un-

TABLE

I

Shell Length Increments in a-Week Intervals Shown by 37 Lymnaea stagnalis Juveniles with Prepatent Trichobilharzia ocellata Infections and by 35 Uninfected Controls

Age of snails ( weeks ) lo-12 12-14 14-16

Infected ?7, +- s1 6.55 c 2.49 4.27 ” 1.54 2.41 2 1.20

Control x2 t SL’ 6.26 ” 3.15 4.23 k 2.25 1.76 ‘- 0.99

P

> 0.500 > 0.500 < 0.025

GROWTH,

;

REPRODUCTION,

2%

AND

--INFECTED -CONTROL

,

, ,

15

20

,.,,., 25

AGE OF SNAILS

(WEEKS)

90

Relationship between mean shell length of infected and control L. stagnalis and age in weeks. FE.

139

incubation of the parasite prior to the onset of egg-laying by the host. Counts of eggs from adults, however, show (Fig. 2) that after a modest beginning when 16 and 17 weeks old, control snails laid 35-85 (mean 57) eggs/snail/week in successive waves with increasingly higher peaks cverv three weeks while infected snails’ output was low and irregular with a mean number of 0.9 eggs/snail/week. The egg masses which were laid by infected snails contained numbers of eggs similar to those laid by controls (Table II), but the masses

20

1

suR\wAL 0F Lymnaea stagnalis

1.

exposed controls were measured weekly beginning 9 weeks after exposure, when this number of infections had become patent. The snails, 15 weeks old at this time, showed a slight difference in size, a disparity which became increasingly apparent in subsequent weeks (Fig. 1). Analysis of covariance of the shell length-age regression indicated that infected snails had grown at a significantly greater rate (1’ < .OOl) than had the controls. Similarly, 400 exposed snails which were crushed at week 16 and found to be uninfected, were indistinguishable from controls (11 > .500) but significantly smaller than infected snails ( 11< .OOl ) .

80

70

60 1 2 v) \ $ (3 w

50

4o

30

20

10

Efect of Parasitism on Fecundit!y Virtually no data are avaiIable concerning fecundity during prepatency, our experiment having been designed to permit

16

17

18

19

20

21

22

AGE OF SNAILS FIG. 2. by infected

23

24

25

.. 27

26

(WEEKS)

Mean numbers of eggs laid per week and control L. stag&is.

TABLE II Relationship between Numbers of Eggs and Lengths of Egg Masses in 37 Masses Laid over 31 Weeks by Infected Snails and 907 Masses Laid over 12 Weeks by Control Snails

No. eggs/Mass Length of Mass

Eggs/mm

Infected

Control

x,

x, 2 s,

es,

47.43 t 17.81 23.58 -c- 5.56 1.95 & 0.54

48.11 2 18.55 20.08 -r- 4.34 2.33 3~ 0.63

x1 3, -0.68 +3.50 -0.38

P > 0.500 < 0.001 < 0.001

140

MC CLELLAND

AND

BOURNS

themselves were longer. In effect, the control snails packed more eggs into each millimeter of egg mass. Further, this tendency of control snails showed a progressive increase, the mean number of eggs per millimeter of egg mass being 1.54 f 0.31 at 15-16 weeks of age, 2.40 + 0.44 at 20-21 weeks, and 3.45 i 1.52 at 26-27 weeks. The prevalence of polyembryony was also seen to increase with time.

other hand, 90% of the infected snails were surviving at 28 weeks and nearly 50% at 46 weeks. Three individuals were still alive and shedding cercariae 19 months after infection. Analysis of variance substantiates the existence of lower mortality in the infected snails (p < .OOl) and also indicates that mortality in infected adults did not differ significantly from that in exposed and control juveniles (p > ,250).

Efects

Effects of Parasitism on Anatomy Histology

of Parasitism on Survival

A. During prepatency. Twelve percent of 300 controls and 9% of 800 snails exposed when 6 weeks old, died during the prepatent period. Analysis of variance following an arcsin conversion indicated that no significant difference existed between the weekly percentage mortalities of the two groups ( p > .500). It must be noted, however, that only 240 or 33% of the surviving exposed snails had actually become parasitized. B. During patency. Weekly mortality data for adult snails (Fig. 3) show that controls died rapidly, all having expired before they were 28 weeks of age. On the loo-

20

25 AGE

FIG.

control

3.

Cumulative

L. stagnalis.

30 OF

35 SNAILS

mortality

40

.

45

50

of infected

and

(WEEKS1

and

Histological examinations were conducted on two or three infected and control snails each week commencing 2 weeks after exposure, when the snails were 8 weeks old. A. Effects on the ovotestis. Sporocysts were not apparent in the connective tissue sinuses of the visceral sac until the third or fourth weeks after exposure. During the early stages of cercarial development the gonadal follicles appeared to be of normal size and condition for the age of the snail. However, as the size and number of developing cercariae increased, the numbers of germ cells and the size and number of gonadal follicles themselves diminished. The distal branches of the ovotestis, including those near the tip of the spire and those forming ramifications among the digestive gland tubules, were the first to be affected. Ultimately, the follicles in the columellar region, normally round and swollen with generative cells, became relatively empty, the partially collapsed walls presenting an irregular outline (Figs. 4, 5). There was no evidence of predation or of mechanical pressure on the gonadal follicles. Although the ovotestis had all but disappeared shortly after cercarial shedding began, close inspection revealed that activity continued at a low level. Sperm and rather shrivelled Sertoli cells were still present (Figs. 6, 7) as well as developing oocytes and their nurse cells. These were

GROWTH,

REPRODUCTION,

AND

SURVIVAL

OF

~~~~UeU

h~~?lU~iS

141

FIG. 4. Conadal follicle of 16-week uninfected snail. FIG. 5. Visceral sac of 16-week infected snail. Key: D, digestive gland; 0, ovotestis; S, sporocyst. FIG. 6. Spermatogenesis in 16-week uninfected snail. Key: lS, primary spermatocytes; 2S, secondary spermatocytes; Se, Sertoli cell; Sp, spermatozoa; T, spermatids. FIG. 7. Spermatogenesis in 16-week infected snail.

142

MC CLELLAND

reduced in size and abundance and some ooplasm showed vesicular individuals (Figs. 8, 9). No oocytes or ova were found in specimens infected 12 or more weeks previously, but low-grade spermatogenesis was observed in specimens with infections over 9 months old.

B. Efects

on the reproductive

tract.

As uninfected snails attained sexual maturity, the accessory reproductive organs became large, uninfected snails being readily identifiable by the pink albumen gland which was visible through the shell. The reproductive tract of infected snails, on the other hand, remained rudimentary (Fig. lo), the albumen and muciparous glands often being indiscernible. The secretory epithelium of infected snails sometimes presented a normal histological picture but more frequently did not develop the extensive folding which normally obscures the lumena of the glandular elements. Further, although the cells contained recognizable secretory products they did not attain col-

AND

BOURNS

umnar stature but remained cuboidal form. Of special interest was the that two lo-month-old snails lost their infections, developed ductive tracts and produced egg masses each week.

in the low observation which had large reproone or two

C. Effects on the digestive gland. The proliferation of parasites was attended by a widening of the connective tissue sinuses between the hepatic tubules whose lumena were broad and fret from obstruction. There was no evidence of direct attack or of mechanical pressure on the gland and, indeed, the visceral sac seemed to be occupied on an equal-sharing basis between parasites and digestive gland; (such was not the case in uninfected snails whose digestive gland tubules were crowded into the outer regions of the visceral sac bv the ovotestis). Most of the digestive gland epithelium appeared to be capable of functioning normally (Fig. 11) although regional squamatization occurred, this having

FIG. 8. Oocyte of 16 week uninfected snail. (N, nurse cell). FIG. 9. Oocyte of 16 week infected snail. (N, nurse cell. )

GROWTH, REPRODUCTION, AND SURVIVAL

FIG. 10. infected 3% muciparous cle; U.PR.,

OF Lymflaea stagnalis

143

Reproductive tracts of 5-month-old L. stagnalis: A from uninfected snail; B from snail months. (ALB, albumen gland; H. D., hermaphroditic duct; L.PR., lower prostate; M.G., gland; O.G., oothecal gland; P., penis sheath; S.R., seminal receptacle; S.V., seminal vesiupper prostate; UT., uterus; VAG., vagina; V.D., vas deferens.)

no apparent relationship of parasites.

to the proximity

D. Effects on the kidney. The only other vital organ in which developing cercariae were seen was the kidney (Fig. 12). Again, they occupied the sinuses, causing no apparent mechanical damage to the organ, although marked vacuolization was observed in cells of the tubule epithelium. DISCUSSION

The results of these experiments show clearly that under our conditions uninfected snails grew slowly, reproduced actively, and died early; and that infected snails grew quickly, reproduced little or not at all, and lived for longer periods. In seeking cause and effect, the authors note

that inhibition of snail reproduction is a common, if not the usual result, of parasitism by trematodes (Wright, 1966). We regard this phenomenon as primary and predisposing to discrepancies in growth and longevity ( Neuhaus, 1949). Several mechanisms have been proposed to explain “parasitic castration” and it emerges that each of these may apply in some instances but not in others. In the present case direct predation upon the gonad was not involved since no redial stage exists in the life cycle. We detected no evidence of mechanical pressure (Rees, 1936) being exerted upon the reproductive system directly or upon blood sinuses which serve it. Neither did we see any sign of connective tissue reaction to migrating cercariae (Pan, 1965). We thus conclude

144

MC CLELLAND

AND BOURNS

FIG. 11. Epithelium of digestive gland of infected snail. (I+ lumen of tally&; tubular connective tissue.) FIG. 12. Kidney of infected snail. (S, sporocyst.)

that an indirect mechanism at the chemical level is probably involved. If a toxic substance were active (Neuhaus, 1940, Rees, 1936, Szidat, 1941) one would expect cell degeneration and necrosis to be evident and the “parasitic castration” to be irreversible (Szidat, 1941). The tissues of our snails did not show these signs and in at least two cases snails which lost their infections became active egglayers. The possibility of starvation of the ovotestis (Rees, 1936) seems to be a real one. It is not known whether the actual amount of functioning digestive gland differs in infected and uninfected snails; in the former the organ is thoroughly interlaced by sporocysts, in the latter it is crowded into the outer regions of the visceral sac by the ovotestis. In any event, the sporocysts are located where they might easily intercept

S, sporocyst in inter-

nutrients which would otherwise find their way to the gonad. On the other hand, Neuhaus (1949) has shown that the gonad is the last organ to be effected in starved snails implying that only total destruction of the digestive gland or complete isolation of the gonad by parasites could result in starvation of the ovotestis. More likely, in our view, is the possibility that some substance(s) elaborated by the parasite exerts an hormonal effect (D. Fairbairn and R. E. Thorson, personal communication) upon the host with the result that the snail’s reproductive tract fails to mature. Fisher (1963) describes many cases in which animal and plant hormones are mimicked by their parasites. Continuing studies in this laboratory are designed to determine whether this is true in the present case and whether functioning snail reproductive systems regress in conjunction

GROWTH,

REPRODUCTION,

AND SURVIVAL

with parasitism contracted after the host has become mature. Given that T. oceZZuta frees its snail host from the duties of reproduction, it may be that growth and longevity are governed by the nutritional drain called forth by the production of either eggs or cercariae ( Ncuhaus, 1949). Unpublished data support the view that it is more “expensive” in nutritional terms for a snail to produce eggs than it is to produce cercariae. If this is true, the many reports that infected snails appear as giants (Linke, 1934, Wesenberg-Lund, 1934, and others, especially Boettger, 1952) would not be unexpected. The observations of Chernin (1960) that Schistosoma mansoni stimulates the growth of its snail host during prepatency, however, implies that the situation may be more complex than that suggested here. In any event, we feel that the inhibition of molluscan reproduction bv trcmatode parasites must be regarded as a major adaptation which serves on the one hand to spare snails from the double burden of producing both eggs and cercariae, and on the other hand as a resources-management device whereby one portion of a snail population serves its own kind and its parasites by reproducing, while the remainder serves the parasite by incubating cercariae over an extended period of time. Clearly, parasites which initiate this chain of events enjoy increased chances of survival as a consequence. It must be pointed out that the fortunes of our control snails may not be indicative of events in the field. Not only were our animals relatively crowded but also the diet provided for them was restrictive. Perhaps it was fortunate that we happened to choose conditions which accentuated a differential in nutritional drain that would have remained obscure under more favorable circumstances.

OF hj77lnUeU

145

StU@UZliS

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MCMULLEN,

NEUHAUS, W. 1940. Parasitire Kastration bei Bithynia tentaculata. Zeitschrift fiir Parasitenkunde 12, 65-77. NEUHAUS, W. 1949. Hungerversuche zur Frage der parasitaren Kastration bei Bithynia tentaculata. Zeitschrift fur Parasitenkunde 14, 300-319. rePAN, C. 1965. Studies on the host-parasite lationship between Schistosoma munsoni and Australorbis glabratus. American Journal of Tropical Medicine and Hygiene 14, 931-976. REES, W. J.

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The effect

of parasitism

by

146

MC CLELLAND

larval trematodes on the tissues of Littorina littorea LinnC. Proceedings of the Zoological Societtl of London 1936, 357-368. ”

I

SZDAT, L. 1941. Bemerkungen ten parasitaren Kastration fiir Parasitenkunde Zeitschrift WESENBERG-LUND,

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zur sogenannvon Mollusken. 12, 251-258. Contributions

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AND

BOURNS

the development of the Trematoda Digenea. Part II. The biology of the freshwater cercariae in Danish freshwaters. Kongelige Danske Videnskabernes Selskabs Skriftery 9th. series 5, l-223. WRIGHT, C. A. 1966. The pathogenesis of helminths in the Mollusca. Helmintholoeical Abstracts 32, 207-224.