Effect of exposure to Echinostoma itliei miracidia on growth and survival of young Biomphalaria glabrata snails

Internafional Journal for Parasitology, Vol. IO, pp. 303-308. Pergamon Press Ltd. 1980. Printed in Great Britain. 0 Australian Society for Parasitology.

EFFECT

OF EXPOSURE ON GROWTH

TO ECHINOSTOMA LIEI MIRACIDIA AND SURVIVAL OF YOUNG BIOMPHALARIA GLABRATA SNAILS ARMAND M. KURIS

Department

of Biological Sciences and Marine Science Institute, University of California, Santa Barbara, CA 93106, U.S.A. (Received 2 1 November

1979)

Abstract-Kurus A. M. 1980. Effect of exposure to Echinostoma liei miracidia on growth and survival of young Biomphalaria glabrata snails. International Journal for Parasitology 10:303-308. Exposure to miracidia of Echinostoma liei resulted in increased mortality and reduced growth of l-2 mm albino Biomphalaria glabrata snails whether or not the snails became infected. Growth rates for infected and exposed but uninfected snails were significantly more variable than growth rates of unexposed snails. Retarded growth and increased mortality were detected as rapidly as seven to nine days after exposure. Neither growth nor survivorship of 46 mm snails was altered upon exposure to or infection by E. liei. INDEX KEY WORDS:

Echinostoma liei; Biomphalaria glabratu;

miracidia;

growth; mortality.

INTRODUCTION & Zischke, 1965). In contrast, for Biomphalaria glabrata and B. pfeifleri infected with S. mansoni LARVAL trematode infections frequently have been growth was accelerated during thz prepatent period shown to affect growth and survivorship of their (Chernin, 1960; Pan, 1965; Sturrock, 1966; snail hosts. Larval trematode infection is associated Meuleman, 1972; Etges, Carter & Webbe, 1975). with snail mortality prior to the emergence of the growth of the infected snails was cercariae (i.e. prepatent snail mortality) in LyCymnaea Ultimately retarded and the unexposed control snails exceeded gedrosiana infected with Ornithobilharzia turlcestanithe uninfected snails in size (Pan, 1965). Sturrock & cum, Biomphalariapfeifleri infected with Schistosoma Sturrock (1970) confirmed these results for snails mansoni, Oncomelania hupensis quadrasi and 0. h. 10 mm or larger upon exposure to infective nosophoru (female snails only) infected with S. miracidia. However, for young snails (two weeks old, japonicum, and L. rubiginosa infected with Echino2 mm diameter) growth was consistently retarded. stoma audyi (Massoud, 1974; Sturrock, 1966; For L. stugnalis infected with Trichobilharzia Pesigan, Hairston, Jauregui, Garcia, Santos, Santos ocellata enhanced growth only is evident after & Besa, 1958; Moose, 1963; Sullivan & Palmieri, patency (McClelland & Bourns, 1969). 1979). In some instances (e.g. Massoud, 1974) snail Meuleman (1972) noted that variation in size of mortality was associated with exposure to high B. pfeifiri infected by S. mansoni was similar to miracidial densities. Other studies show no signifithe variation about the mean size of the uninfected cant increase in prepatent mortality due to parasitism controls. However, Sturrock (1966) found growth (e.g. Sturrock, 1965; Pan, 1965; McClelland & of the infected snails to be much more variable Bourns, 1969; Meuleman, 1972). Further, some of than the controls. the parasites which did not cause prepatent host The present study was initiated because Echinomortality were associated with increased host stoma liei is a potential biological control agent for mortality upon patency (Sturrock, 1965; Pan, 1965; Schistosoma mansoni (Lie, 1967; Kuris, 1973) and Meuleman, 1972). In contrast, McClelland & in double infections E. liei is a strong intrinsic Bourns (1969) showed that survival of infected dominant competitor over S. mansoni. Echinostoma snails following patency was significantly higher liei is highly infective to small (2 mm) B. glubrata than for the unexposed control snails. (Jeyarasasingam, Heyneman, Lim & Mansour, Infections with larval trematodes have variable 1972; Kuris, 1980). Infectivity gradually decreases effects on the growth of the snail hosts. Pesigan with increasing snail size. et al. (1958) showed that the growth of Oncomelania Long-term laboratory competition experiments infected with S. japonicum was retarded, the differinvolving multiple exposure of populations of B. ence being greatest for very young snails. Moose (1963) confirmed these results for female snails glabrata to S. mansoni and E. liei may establish the conditions under which the echinostome can only. Echinostoma revolutum also slowed the growth competitively eliminate the schistosome populations. of its host Lymnaea (= Stagnicolu) palustris (Zischke 303

304

ARMAND

Before such experiments can be undertaken it is necessary to evaluate the infection dynamics of E. Iiei in the heterogeneous habitats needed for longterm maintenance and reproduction of the laboratory snail populations. The infection rate of E. Iiei in such a complex experimental habitat is not diminished when compared to individual exposures in glass shell vials (Kuris, 1980). Since snail reproduction will occur in a long-term population study it also is necessary to describe the effect of exposure to LC liei on the survival and growth of young snails. This study compares the effect of E. fiei parasitization on one-week-old (l-2 mm diameter) snails with exposure of larger snails to the same

miracidial densities. MATERIALS AND METHODS The Ethiopian strain of Echinosfome liei and the NIH albino strain of Biomphalavia glubruta have been maintained in my laboratory for two years. Both colonies originated from stocks maintained at the George W. Hooper Foundation, University of California, San Francisco. Eggs of E. liei were filtered from fresh pigeon feces and incubated at 27°C in a Petri dish with deionized water for IO-25 days. If older eggs were used, infectivity sharply declined (Kuris, 1980). Miracidia emerged between 1000 and 1400 h when the dishes were placed under a loo-watt lamp between 0800 and 0900 h. Only active, rapidly swjnlnling miracidia were used in aquaria under a loo-watt lamp (water temperature 26-30°C) for four hours. Each aquarium contained 25 snails in I liter of warm, recently aerated, deionized water, into which I25 miracidia were added in all experiments. Control aquaria, contained the same number of snails handled identically to experimental aquaria. Three experiments were performed and all treatments were replicated. The first experiment compared growth and survival of 4.05.9 mm snails exposed to E. liei with a similar group of

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M.KURIS

unexposed control snails. The second experiment compared growth and survival of I-O-1.9, 2.0-3.5 and 4.0-5.9 mm snails exposed to E. fiei with unexposed controls. Hereafter these sizes will be termed l-mm, 2-mm and 5-mm snails. Since the outcome of the second experiment was quite unexpected, the l-mm treatment was replicated in experiment three. The critical aspect of the second and third experiments involved assessment of the effect of parasitism on growth and mortality of very young snails. To clarify the effect of parasitism on these fragile snails, experimental procedures must be such that mortality of the unexposed controls is minimized and the thin apertural edge of the shell is not damaged. This was achieved by handling the snails with two small paintbrushes manipulated as chopsticks. Prior to exposure all snails were inspected and any damaged individuals were replaced. After exposure all aquaria were placed on adjacent shelves in the snailrearing facility. The order of aquaria on the shelves was randomized. Red leaf lettuce (Lactucu mtivu) was provided for food and a piece of chalk added to maintain snail shell quality. Lettuce was added ad Iibifum and the water was changed every seven days starting 14 days after exposure. On day 7 post-exposure all snails were examined with transmitted light through the shell for the presence of E. lid mother sporocysts in the aorta, adjacent major arteries and the ventricle of the heart. Control snails were similarly manipulated in a mock examination for parasites. Infections were confirmed on day 30 by the presence of rediae in the ovotestis. On day 30 (experiment 2) and days 9 and 38 (experiment 3) all surviving snails were measured with an ocular micrometer to 0.1 mm. Statistical comparisons of snail mortality and of the snail size frequency distributions were based on the G-test (Likelihood Ratio test). Size classes in the tails of the size frequency distributions were combined until the number of snails in these size classes exceeded five (Sokal & Rohlf, 1969). Bartlett’s test was used to compare homogeneity of variances of growth among the treatments prior to f-tests of mean sizes (Sokal & Rohlf, 1969).

TABLE I-SNAIL SIZE, INFECTIVITY AND SURVIVORSHIP FOR R. glabrntu POPULATIONS MASSEXPOSED TO FIVE ,8?. &'iMIRAClDlA PERSNAIL IN I LITEROF WATER.NREPRESENTSTHENUMBEROFSNAlLSATTHESTARTOFTHEEXPERlMENTS

Initial snail size (mm) Experiment I .0-l .9

Unexposed controls Percent survival Day N

2

2.0-3.5

0 7 30

51 49 48

96 94

50 37 34

74 68

-

0

98 96

50 46 42

92 84

-

30

50 49 48

0 7 30

50 49 49

98 98

51 50 49

98 96

-

0 9 38

50 50 48

100 96

75 75 64

100 8.5

7

4.0-5.9 Experiment

L GI -9

N

Exposed Percent Percent survival positive

3

57 59 63 62 54 43

69 73

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Effect of Ef~~~~~sf~~~on snail growth and survival RESULTS

Mass exposure of 5-mm snails to five miracidia per snail in experiment 1 yielded a 58% infection rate. Exposed snails did not differ from unexposed control snails in survival (96% controls, 94% exposed), nor in mean size or standard deviation (unexposed controls, X= 12.3 ~fr1.83 mm; exposed positive, 2 = 12.1 rt 2.29 mm; exposed negative, T = 12.7 + 2.01 mm) on day 30. Survival and infection of control and exposed snails in experiments 2 and 3 are summarized in Table 1. Unexposed control snails suffered negi~gible mortality (6% or less) for all size classes throughout the study period. Exposed snails were 5469% parasitized when checked on day 7 (or 9). Frequency of parasitization did not appear to be associated Exposed

Positive 9.15 i 2.23

,

5

Exposed

Negative

F5

8.79 t 2.58

I I zj 15 z

Unexposed

5

6

h

10 11 9 7 8 SNAIL SIZE (D!ame:er

:2 13 m mm)

14

15

Fro. 1. Size frequency histogram of (bottom) unexposed snails, N=48; (middle) exposed but uninfected snails, N= 14; (top) exposed infected snails, N=20. Exposed I -mm snails were presented five E. lid miracidia per snail (total 125 miracidia) in 1 liter aquaria of 25 snails, 30 days prior to measurement; results are fromexperiment 2. Numbers above the arrows indicate means and standard deviations. with snail size over the limited range of sizes used in these experiments. As in experiment 1, the 5-mm snails did not suffer increased mortality upon exposure and parasitization. However more of both the exposed 2-mm and l-mm snails died than their unexposed counterparts. The l-mm snails of experiment 2 showed significantly higher mortality for the exposed group by day 7 (PC: 0.01). Increased mortality of the 2-mm snails and of the l-mm snails of the third experiment was only significant (0.05, P>O.Ol) on the day 30 (or 38) examination. A

305

comparison of the percentage positive observed on day 7 (or 9) with the percentage positive on day 30 (or 38) for the l-mm and 2-mm snails showed essentially no change between sample dates. This suggests that mortality observed over this time span for the small snails was not necessarily associated with infection [at least as detected by day 7 (or 9)]. The decrease in the percentage of 5-mm snails positive by day 30 was due to apparent loss of infection in several formerly infected snails between days 7 and 30. Figure 1 shows the size frequency distribution of the exposed and control l-mm snails on day 30. Control snails averaged 11.155 1.49mm. In contrast, the infected snails were significantly smaller (t-test, P P> 0.01). The standard deviation of the unexposed snails fell between the exposed negative and positive snails. Some of the unexposed snails exhibited varying degrees of growth retardation. This presumably accounted for the high standard deviation in the size of the control snails in experiment 3 when compared with the control snails of experiment 2.

306

ARMAND

Exposed Positive

Unexposed Controls 20 15 IO 5 0 SNAIL SIZE

(mm)

Day 9

Day 3%

2. Size frequency histogram of unexposed snails (bottom), exposed but uninfected snails (middle) and infected snails (top) on days 9 and 38 after exposure; results are from experiment 3. See Table 1 for sample sizes and Fig. 1 for additional explanations. FIG.

DISCUSSION That trematode infections cause significant mortality and reduced growth of young (l-mm) snails is not surprising. As noted above, several other studies report similar findings. What is unusual in the present case is that the exposed negative snails experienced mortality and growth retardation similar to the exposed positive snails. Moose (1963) obtained similar results for female Oncomelnnia hupensis nosophora (but not for males) infected with ~chistosomff j~ponic~lm. The mean size of the exposed but negative female snails was 5-7 mm, significantly less than the unexposed control snails (6.3 mm) and significantly greater than the positive snails (5.0 mm). Also, the variance about the mean size of the exposed but uninfected snails was significantly greater than for the unexposed control snails. The O~~corne~~niustudy used relatively old (3-4 mm) snails individually exposed to two miracidia each and snails were not examined for trematode infection until 16 weeks after exposure. In the only other study investigating the growth of exposed but negative snails McClelland & Bourns (1969) found no significant difference between the growth of these snails and their unexposed counterparts. Both groups were significantly smaller than the infected snails. The findings of the present study that growth of infected B. gluhratcc is more variable than for

M.

KURIS

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unexposed control snails parallels the results of Sturrock (1966) for this snail when infected with S. munso~i. In contrast, growth of B. pj>ifir-i parasitized by S. munsoni (Meuleman, 1972) was no more variable than for unexposed control snails. Moose (1963) noted that growth of female Oncomelania exposed but not infected by S. ~Yzponielrm was significantly more variable than for unexposed controls. Inspection of the size distribution for infected female O~eome/ffniu indicated that growth of infected snails was no more variable than for unexposed control snails. The effect of this variability in response to parasitism (or to exposure without infection) in the present study is such that about half of the exposed groups grew as large as the unexposed control snails. I suggest three hypotheses to account for the observed results. (1) The presence of numerous parasitized snails together with Llnparasitized snails inhibited the growth of the latter in some manner. (2) Most of the exposed negatives suffered trauma during the brief period of miracidial attack and unsuccessful penetration. Their growth performance is permanently impaired as a result of this early trauma. (3) Most of the exposed negatives were parasitized, but lost the infections. Growth of these temporarily parasitized animals is permanently reduced. The third hypothesis is weakened by several observations. The similarity of the results on the day 7 (or 9) examination for the presence of mother sporocysts with the day 30 (or 38) examination for rediae suggests that if the exposed negative snails had ever been positive they had lost the infection before day 7 (or 9). In fact, if these snails ever were infected, the infections apparently were lost very soon after infection (hypothesis 2). Echinostoma lindoense, a very close relative of E. iiei (Jeyarasasingam et ul., 1972) required only 40 h for the mother sporocyst to reach the ventricle of the heart (Lie, 1969). When mother sporocysts were killed shortly after they had reached the ventricle a dense aggregation of amebocytes surrounded the dead or dying parasite (Lie & Heyneman, 1975, 1976; Kuris & Boss, personal observations). Although in larger snails (greater than 4 mm) the aorta was the most common site for the mother sporocyst of E. liei (Jeyarasasingam et nl., 1972), in l-mm snails most mother sporocysts occurred in the ventricle. Of the 73 snails positive for E. liei on day 7 (or 9) (experiments 2 and 3) 65 (89%) had at least some mother sporocysts in the ventricle. By comparison only 22 of 29 (76%) of the 2-mm snails and 5 of 27 (19%) of the 5-mm snails had mother sporocysts in the ventricle (day 7, experiment 2). Amebocytic aggregations also are apparent around dead or dying E. liei. Five positive snails in the 5-mm exposed group of snails in experiment 2 showed such an amebocytic response on day 7.

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These five snails lost their infections between days 7 and 30 and were recorded as negative on day 30 (Tabfe 1). No amebocyte aggregations were observed surrounding E. Iiei in the l-mm and 2-mm snails.

307

KURIS A. M. 1973. Biological control: Implications of the analogy between the trophie interactions of insect pest--parasitoid and snail-trematode systems. .&et+ mental Farasitoiog~~ 33: 365-379.

Apparently small snails less frequently mount such a defensive response. Thus, it is unlikely that the exposed negative snails included individuals which

KURIS A. M. 1980. Echitzostoma liei miracidia and Biomphaluria glabrata snails: Effect of egg age, habitat heterogeneity, water quality and volume on infectivity.

successfully penetrated, reached the ventricle of the heart and then died. If penetration occurred, encapsulation presumably took place in the tissues and migration to the heart was never completed. The two remaining hypotheses cannot be distinguished with the data at hand. A transfer experiment on day 7 in which exposed negative snails are separated from the exposed positives, replaced with unexposed controls and then reared alone could test

LIE K. J. 1967. Antagonism of Paryphostomum segregatum rediae to Sehistosoma mansoni sporocysts in the snail Biomphalaria glabrata. Journal of Parasitology

E. liei

hypothesis 1. The implication

of this study for the biological control of Schistosoma mnnsoni and its intermediate host Biomphaluria glabrata is that Echinostoma liei and other echinostomes may have some potentially important unforeseen population effects. In addition to a modest contribution to juvenile snail mortality, and its high infectivity coupled with its competitive dominance, E. liei causes a pronounced decrease in the growth rate of young snails. In this Iaboratory B. giabrata reaches reproductive maturity at about 9 mm. Thus 26”/, of the exposed negative snails in experiments 2 and 3 at termination were less than 9 mm, whereas only 7% of the unexposed controls were still below the mean size at maturity. Since the average life expectancy of B. giabrata in nature is very low, on the order of a few weeks (Sturrock, 1973; Sturrock & Webbe, 1971), any factor that prolongs the time to reach adulthood may have serious population consequences.

International

53

Journalfor

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A&zowIedgements-I thank Linda Hagen, Daniel Clause and Steven Mertens for able technical assistance; Donald Heyneman, Daniel Morse, Jack O’Brien, Robert Trench and Kenneth van der Laan for helpful comments on the manuscript and the University of California, Santa Barbara, for financiat support.

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