Schistosomatium douthitti: Effects of Lymnaea catascopium age on susceptibility to infection

EXPERIMENTAL

PARASITOLOGY

45, 65-73

(1978)

Schistosomatium douthitti: Effects of Lymnaea Age on Susceptibility to Infection ERIC S. Department

of Zoology, (Accepted

Iowa

catascopium

LOKER

State University,

for publication

Ames, lowa

15 March

50011, U.S.A.

1978)

LOKER, E. S. 1978. Schistosomatium douthitti: Effects of Lymnaea catascopium age on susceptibility to infection. Experimental Parasitology 45, 65-73. Laboratory-reared Lymnaea catascopium snails (l-269 days old) were exposed individually to different numbers of Schistosomatium douthitti miracidia. Increasing the exposure dosage from 3 to 10 miracidia generally increased infection rates, in some age classes up to 100%. Successful reexposure of snails not infected after a primary exposure was possible. Neonatal snails were least likely to become infected, primarily because miracidia were not attracted to them. Snails 12-55 days old were most susceptible to infection. Miracidia were readily attracted to these snails, and many were ingested and subsequently penetrated the host esophageal wall. Miracidial penetration of external snail surfaces was rare. Susceptibility of older snails (65-269 days) progressively declined with age. Many miracidia were entangled and immobilized in mucus produced by these snails, and fewer were ingested. No conspicuous host cellular responses to mother sporocysts were observed in any of the snails sectioned, A comparison of susceptibility of deliberately stunted snails and comparably aged controls of normal size indicated that the former were more susceptible. INDEX DESCRIPTORS: Schistosomutium douthitti; Trematoda; Miracidia; Miracidial beLymnaea catascopium; Mollusca; Snail; Snail esophagus; havior; Miracidial penetration; Snail mucus; Host age; Host susceptibility; Host-parasite relationships.

of size of Lymnuea catascopium on susceptibility to S. douthitti.

There are relatively little published data documenting the interactions between Schistosomutium dcuthitti and its molluscan hosts under controlled laboratory conditions. This paucity extends to schistosomelymnaeid interactions in general. One aspect demanding further clarification is the effect of snail age on susceptibility to infection. As indicated by the general reviews of El-Gindy (1950) and Lim and Heyneman (1972), the effect of host age is variable ,and dependent upon the hostparasite combination under study. The present study was therefore undertaken to examine the effect of age and, secondarily,

MATERIALS

AND

METHODS

Lymnaea cata.scopium snails naturally infected with Schistosomutium douthitti were originally collected from a spring-fed roadside ditch near Alanson, Michigan, by Dr. Harvey D. Blankespoor. S. douthitti has been maintained in this laboratory for approximately 3 years, using laboratoryreared L. catascopium and hamsters as hosts. Snails were maintained at 22-26 C in artificial spring water (Ulmer 1970) supplemented with crushed oyster shells. They were fed leaf lettuce and commercial flaked 65 0014-4894/78/0451-0065$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

66

ERIC S. LOKER

fish food. Sexual maturity was normally attained between 57 and 71 days under these conditions. The conclusions of Walter (1969) and Clarke (1973) were followed in designating these snails L. catascopium rather than L. emarginata. S. douthitti eggs were harvested from the livers of hamsters exposed 1 to 2 months previously to approximately 60 cercariae each. Actively swimming miracidia were collected within 1 to 2 hr posthatching. Snails were exposed to infection individually at room temperature for at least 6 hr in clear plastic vials containing l-2 ml of artificial spring water. Because growth rates of individual snails are not uniform, age (in days posthatching) and size (the distance from the apex to the basal lip of the shell, to the nearest 0.5 mm) were recorded and monitored at regular intervals thereafter. Snails of a particular age and size at the time of exposure were maintained separately to observe the effects of these two factors on susceptibility. Snails utilized for histological studies were fixed in warm Bouin’s fixative for approximately 48 hr and, following removal of the decalcified shells, were dehydrated in ethanol and tertiary butyl alcohol, embedded in paraffin, sectioned at 7-15 pm, and stained with Harris’ hematoxylin and eosin. The P values in parentheses refer to the results of pairwise x2 tests unless otherwise indicated. Primary and seconday exposure experiments. It was not feasible to expose snails from more than two age classes to miracidia from the same hamster; therefore, variation in miracidial viability between hamsters was checked by replication of primary exposure experiments in the following representative age classes: 1-3, 6571, and 136-143 days. Secondary exposure experiments in the 63- to 72-day age group were also replicated. Snails exposed to S. douthitti for the first time were isolated and examined for

cercarial shedding 25 days postexposure and at weekly intervals thereafter. In some instances, those snails not shedding cercariae within 50 days after their initial exposure to three miracidia were reexposed to the same number of miracidia. Snails so treated were examined for cercarial shedding at weekly intervals thereafter to insure that no primary infections had been overlooked. Exposed snails were eventually crushed and examined for sporocysts, except those retained for longevity or histological studies. Miracidium-snail interactions. A total of 122 snails from three different age classes ( 1-3, 12-16, and 160 days ) were individually exposed to a known number of S. douthitti miracidia (one, three, or five) derived from one hamster. A total of 94 miracidia were initially placed with snails l-3 days old, 150 with snails 12-16 days old, and 120 with snails 160 days old. The number of ‘actively swimming miracidia in each vial was recorded hourly for 5 hr after initial exposure. Some observations on the interactions between snails and miracidia were made at this time and were supplemented with additional observations on comparably aged snails and miracidia derived from a different hamster. Penetration of miracidia. Four snails (4O45 days old) were exposed to miracidia (two snails to 50 miracidia each and two snails to 100 miracidia each), fixed within 30 min ‘after initial contact with miracidia, and sectioned to locate the areas most frequently penetrated by miracidia. Four snails 215-220 days old were similarly treated. Neonatal snails were not utilized because miracidia were rarely attracted to or came in contact with them. Miracidia used were obtained from a single hamster. To determine the relative penetration success of miracidia in snails of different ages (1, 12-21, and 100-110 days), snails were exposed to 20 miracidia each, fixed at 1248 hr postexposure, and sectioned. Miracidia utilized in this experiment were de-

Schistosomatium

douthitti:

rived from one hamster. Mother sporocysts stained intensely and thus could be readily identified in sectioned material and enumerated. Exposure of stunted snails. One cohort of young snails of similar age and size was split into two groups; one group was reared under normal culture conditions with standard precautions taken to prevent overcrowding. The second was maintained under deliberately overcrowded conditions. Food was given ad libitum to both groups. Snails from the latter group became stunted in size. Snails in both groups were then exposed to S. douthitti (three miracidia per snail) when 73-80 days old. All miracidia were derived from a single hamster.

RESULTS

Primary Exposure to Three Miracidia per Snail The proportion of snails that became infected with Schistosomutium douthitti did not differ significantly between replicates in ‘any case (P < 0.75). All replicates are lumped in Table I. With the exception of the youngest and oldest age classes, prepatent mortality was low (Table IA). The high mortality of exposed snails in the l- to 3-day age class did not differ significantly from unexposed control snails of comparable Iage sand size (P < 0.25). Significant differences in susceptibility (P < 0.05) were obtained with

TABLE Exposure

and Reexposure Number of snails exposed

Snail age (days)

A. Primary

100

27-32 45-55 65-71 94-103 136-143 262-269

56 52 44 33 79 23

70

B. Reexposure

C. Primary 26-33 60-64 72-82

100-109 143-153

I

Age Classes of Lymnaea

Average shell length f SE (mm)

catascopium

Range of shell length (mm)

Number of snail deaths during prepatent period

1.0 1.0-3.0

36 (51p 8 (8) 3 (5) 0 (0) 2 (5) 0 (0) 6 (8) 7 (30)

to Schistosomatium

douthitti

Number of survivors shedding

Pb

exposure to 3 miracidia/snail

l-3 12-16

63-72 124-133 199-206

of Different

67

SNAILS’ AGE SUSCEPTIBILITY

1.0 2.19 4.67 9.73 13.32 14.77 17.68 22.04

to 3 miracidia/snail 81 29 40

f 0.0 f 0.07 f 0.16 f 0.30 f 0.32 f 0.70 f 0.29 f 0.63

2.5-7.0 4.0-14.0 9.0-17.0 7.5-21.5 8.5-23.0 16.5-28.5

after unsuccessful

12.89 f 0.29 18.29 f 0.51 20.50 f 0.36

primary

2 W’ 46 49 41 17 9 18 3

(50) (92) (79) (40)

(27) (25) (1%

exposure

6.e18.5 10.0-22.5 18.0-27.0

10 (12) 9 (31) 4 (10)

34 (48) 6 (30) 13 (36)

<0.50 <0.75 <0.25

4.0-11.0 8.0-14.0 9.0-16.5 10.5-21.0 13.0-22.5

3 (10) 4 (11) 2 (10) 2 (11) 0 (0)

26 (100) 29 (91)

<0.25 <0.005 <0.005 <0.25 <0.50

exposure to 10 miracidia/snail 29 36 21 18 15

7.34 12.24 13.05 17.06 18.30

f 0.26 f 0.23 f 0.51 f 0.67 f 0.71

0 Numbers within parentheses are corresponding percentages. * P values refer to the results of pairwise x2 tests comparing the susceptibilities If comparable age in B or C.

19 (100) 7 (44) 2 (13)

of snails in A with snails

68

ERIC

S. LOKER

the following age class combinations: I-3 and E-16, 12-16 and 27-32, 27-32 and 4% 55, and 45-55 and 65-71 days. Snails of a given age class varied considerably in size at the time of exposure, and consequently some snails of different ages were of similar size at the time of exposure. Despite individual differences in snail growth rate, there was a strong correlation between age and size in snails used in this experiment (correlation coefficient: 0.895). Snail age and size were normally too positively correlated to demonstrate any relative differences in their effects on susceptibility. The one exception to this result involved snails 12-16 days old, where I-O-mm-long individuals were more refractive to infection than larger snails in this age group (2’ < 0.001). Reexposure Experiments Patent infections were obtained in snails from all three age classes reexposed to S. douthitti ( Table IB ) . Reexposed snails showed no significant differences in susceptibility when compared with snails from the primary exposures (Table IA). Likewise, no difference in overall prepatent mortality rates between the two groups was noted (P < 0.25).

A

12-16 DAYS

FIG. 2. Schistosomatium douthitti miracidium penetrating the esophageal wall of Lymnaeu catascopiunz (15 min after initial contact with the snail; snail age, 45 days). X 480.

Primary Exposure to 10 Miracidia Prepatent mortality rates among snails of different ages were not significantly higher than those of snails of comparable age exposed to three miracidia (P < 0.25). The percentage of infected snails was greater among snails exposed to 10 miracidia than among comparably aged snails exposed to 3 miracidia, with the exception of the oldest age group (143-153 days) (Table IC ) . Significantly higher proportions of snails in the age groups 60-64 and 72-82 days became infected than did snails of similar age exposed to three miracidia. Miracidium-Snail

HOURS

FIG. 1. Percentage of Schistosornutium douthitii miracidia actively swimming at hourly intervals in the presence of Lymnaea catuscopium of different ages.

After douthitti trapped activiely miracidia

Interactions

1 hr, approximately 50% of all S. miracidia were ingested, became in snail mucus, or were no longer swimming (Fig. 1). More active remained at all times in those

Schistosomatium TABLE

douthitti:

II

Penetration Success of Schistosomutium douthitti Miracidia in Lymnaea catascopium of Different Agesa Snail age (days)

Number of snails exposed

Total number Average. of mother number of mother sporocysts observed sporocysts per snail

1 12-21 106-110

9 15 10

1 (0.6)b 112 (37) 33 (17)

0.1 7.5 3.3

Total

34

146 (21)

4.3

mAll snails were exposed individually to 20 miracidia. b Numbers in parentheses are the corresponding percentages and are based on the total number of miracidia placed in contact with the snails of a particular age group.

SNAILS'

AGE SUSCEPTIBIL~Y

the odontophore was extended. Some miracidia were not attracted to these snails. Interactions between miracidia and 160day-old snails were generally similar to those observed for snails 12-16 days old, but protrusion and retraction of the odontophore was observed less frequently in older snails, and fewer miracidia were ingested, particularly by the largest snails observed. In addition, older and consequently larger snails produced copious amounts of mucus which covered not only the soft parts of the animal, but parts of the shell as well. On several occasions, miracidia were trapped and immobilized by this substance. Mucus was apparently produced at an accelerated rate when these snails were confined to relatively small volumes of water. Penetration

vials containing snails l-3 days old, than in those containing snails 1216 days old (P < 0.01). At Hours 2 and 4, there were significantly more miracidia present in the vials containing l- to 3-day-old snails than in those containing 160-day-old snails (P < 0.025). No significant differences were seen between vials containing snails 12-16 and 160 days old. Miracidia showed little evidence of attraction toward l- to 3-day-old snails and infrequently were observed swimming near, or attached to, them. Miracidia were rarely observed to be ingested by these snails. Many miracidia were attracted to 12- to Is-day-old snails and were frequently in close contact with, or attached to, them. Initially it was thought that this attachment preceded successful penetration, but further observation indicated that most of these miracidia eventually detached. Snails of this age would often quickly retreat into their shells in apparent attempts to dislodge attached miracidia. Many miracidia congregated at the anterior end of the snail, near the mouth, and were subsequently ingested as the mouth opened and

69

of Miracidia

Examination of snails 40-45 and 215220 days of age, fixed within 30 min postexposure, indicated that out of 300 total miracidia placed in contact with each group, 86 ( 29% ) and 57 ( 19% ) had been ingested, respectively. In both groups, the majority of miracidia had already penetrated the esophageal wall or were in the process of doing so (Fig. 2). Some penetrated the buccal mass. Many were observed adjacent to the salivary glands and the esophageal wall. Penetration of external surfaces was rarely successful despite the frequent

attachment

of some mir-

acidia to these surfaces. Significantly more miracidia were ingested by the younger snails (P < 0.01). No substantial differences in the thickness of the esophageal wall were noted in snails of the two age groups, but the muscular layers of the wall in older &ails were more distinctly organized and more dense. A small number of lysed miracidia were observed within the stomach and intestine of snails in both age groups. Relative numbers of S. douthitti miracidia successfully penetrating and trans-

70

ERIC S. LOKER

forming into mother sporocysts in snails of different ages are presented in Table II. In contrast to the l-day-old snails, in which only a single mother sporocyst was observed, 12- to 21-day-old snails were readily penetrated, and as many as 15 mother sporocysts were noted within a single snail. All 15 snails in this age group were penetrated and at least three mother sporocysts were observed in each snail. Snails 100-110 days old were intermediate, with the maximum number of mother sporocysts observed in one snail being five. Two snails from this group did not contain mother sporocysts. Significantly fewer mother sporocysts were observed in neonatal snails than in snails 12-21 and 100-110 days old (t test, P < O.OOl), but the latter two groups were not significantly different (P < 0.10). Essentially all mother sporocysts were located between the tubules of the salivary gland or adjacent to the external esophageal wall. Sporocysts appeared to be developing normally without evidence of any host cellular responses. Exposure of Stunted Snails Snails grown under normal conditions were on the average more than twice as large ‘as stunted snails at the time of exposure (Table III). Both groups suffered little prepatent mortality, suggesting that stunting did not materially affect the capacity to withstand infection. A significantly higher proportion of stunted snails became infected in comparison with controls of normal size (P < 0.005), but stunted snails and younger snails of comparable size exposed to three miracidia and grown under normal conditions were not significantly different with respect to their susceptibility to S. douthitti. DISCUSSION

Results of primary exposures to three miracidia indicate that the age of Lymnaea catascopium does influence susceptibility to

TABLE

III

Comparison of Susceptibility of Normal and Stunted Lymnuea catascopium to Schistosomatiumdoufhittia Number of snails exposed

Normal Stunted

30 38

) Snails were each * Numbers within percentages.

Average length

f

shell SE

hmd

13.12 5.40

f f

0.17 0.39

exposed to three parentheses

Number of snail deaths during prepatent period 0 COP 2 (5) miracidia. are the

Number survivors shedding

of

12 (4OP 33 (92)

corresponding

Schistosomatium douthitti. Snails 1-3 days of age were the most difficult to infect, whereas susceptibility was highest in snails approximately 1 month old. Susceptibility declined thereafter, although at no age were snails totally refractory. This pattern is similar to the results obtained by ElGindy (1950) with S. douthitti and Lymnaea stagnalis. Kagan et al. (1954) exposed Lymnuea palustris ranging in age from 16 to 149 days to five S. douthitti miracidia each and concluded that there was no significant variation in susceptibility among the age groups employed. Malek ( 1977) exposed Pseudosuccinea columella of various sizes (and presumably different ages ) to between 6 and 10 miracidia and concluded that size (age) had little or no effect on susceptibility to infection. By increasing the number of miracidia employed in exposure experiments from 3 to 10, the percentage of infection was generally increased, in some cases up to 100% (Table IC). El-Gindy ( 1950) and Kagan et aZ. (1954) also indicated that higher rates of infection with S. douthitti could be achieved by increasing the miracidial exposure dosage. Although mature snails (55 days and older) were not readily infected after exposure to three miracidia, achievement of a 100% infection rate in snails 72-82 days old, when exposed to 10 miracidia each, implies that attainment of sexual maturity per se does not render snails insusceptible to infection. Increasing the

Schistosomatium

douthitti:

exposure dosage from 3 to 10 miracidia did not significantly increase the prepatent mortality rates. Results of the secondary exposure experiments (Table IB) suggested that the failure of some snails to become infected after primary exposure does not necessarily indicate that they were refractory to infection; they may simply not have been successfully penetrated during the primary exposure. Failure of some S. douthitti miracidia to be attracted to snails of highly susceptible age groups indicates that not all miracidia utilized in this study were fully viable. This suggests one possible explanation for the failure to obtain 100% infection rates in highly susceptible ‘age groups, particularly in those experiments employing only three miracidia. Also, some miracidia may have exhausted the contents of their penetration and apical glands attempting to penetrate external snail surfaces. Failure to consistently obtain 0 or 100% infection rates may also be a consequence of genetic heterogeneity in the outbred L. catascopium population used in this study. Miracidia were not readily stimulated to come into contact with, or attach to, neonatal snails. The relatively large number of miracidia remaining at each hour after exposure in vials containing neonatal snails (Fig. 1) and the very small number of mother sporocysts observed in neonatal snails (Table II) support this observation. High susceptibility observed in snails of intermediate ‘age ( 1255 days) was in part due to the evident attraction of most miracidia to them. Once in close contact with these snails, miracidia did not become entangled in snail mucus and were readily ingested and thereby carried to their normal penetration sites in the esophagus; consequently, relatively more miracidia were capable of penetrating these snails. The diminished susceptibility of older snails was not attributable to a failure to attract miracidia. Mucus production by

SNAILS'

AGE SUSCEPTIBILITY

71

these snails was sufficient to trap and immobilize some miracidia. Also, rhythmic movements of the odontophore and radula were less frequent in older snails, and consequently, fewer miracidia were ingested. The more developed muscle layers observed in the esophageal wall of older snails may reduce penetration success, but additional studies are needed to confirm this. Mucus production by old snails was shown to have a deterrent effect on miracidial penetration by Newton (1953), working with Australorbis glabratus and Schistosoma mansoni, and by Lo ( 1972), with Bulinus guernei and Schistosoma haemutobium. Effects of mucus production on penetration success in nature are not known. Boray (1966) suggested that the insusceptibility of older snails may be due to their possession of thicker epithelia. The insusceptibility of those snails 1216 days old that had not grown significantly larger than neonatal snails suggests that the failure to attract miracidia, and subsequently to become infected, is specifically a function of very small body size rather than age. The substances that serve as miracidial attractants may only be produced (in sufficient quantities) when snails become more metabolically active, and growth has been initiated. With this one exception, age and size of snails grown under normal conditions were too positively correlated to separate their effects. The high percentage of infection obtained in snails deliberately stunted, in comparison with controls of normal size, again implies that size is more influential than age in determining susceptibility, and in particular, that large size tends to reduce susceptibility to infection. Malek’ (1950) found that stunted Biomphaluria boissyi were less susceptible to infection with S. munsoni than controls of normal size. All mother sporocysts observed in sectioned material appeared normal, and no evidence of cellular responses to them was noted. In numerous other L. catascopium

72

ERIC S. LOKER

snails of various age exposed to S. douthitti and fixed at longer intervals after exposure, destructive cellular responses to developing sporocysts were rarely noted (unpublished data). The relative insusceptibility of certain age classes of snails does not necessarily imply that they possess “immunity,” in contrast to the implication of El-Gindy (1950). Ingestion of S. douthitti miracidia and their subsequent penetration through the repreesophageal wall of L. catascopium sents a very different method of entry into the molluscan host, as compared with other schistosome species. Maldonado (1967) observed miracidia of S. mansoni to be ingested, but concluded that most miracidia penetrated the external surfaces of the snail. Faust ( 1924), Lengy (1962), Wajdi (19W), and Blankespoor and van der Schalie (1976) likewise concluded that the miracidia of a variety of schistosome species penetrate the external surfaces of their molluscan hosts. The ingestion of miracidia may explain the failure of Short (1952a, b) and Sudds (1960) to observe consistently penetration of the external surfaces of lymnaeid snails by S. douthitti. Recovery of S. douthitti mother sporocysts from the esophageal region of L. palustris and L. stagnulis reported by Price (1931) and Cort et al. (1944) suggests that the miracidia employed in their studies also may have been ingested. The scarcity of natural S. douthitti infec( = Stagnicola tions in L. catascopium emarginata angulata) reported by Cort et al. (1937) is perplexing because the results of Sudds (1960) and of the present study indicate that Lymwa catascopium is very susceptible to Schistosomatium douthitti in the laboratory. ACKNOWLEDGMENTS Dr. Martin J. Ulmer and Dr. Harvey D. Blankespoor provided criticism, Dr. David F. Cox assisted in the statistical analysis, the Graduate

College of Iowa State University helped financially, and Carol Gostele typed the manuscript.

REFERENCES BLANKESPOOR, H. D., AND VAN DER SCHALIE, H. 1976. Attachment and penetration of miracidia observed by scanning electron microscopy. Science 191, 291-293. BORAY, J. C. 1966. Studies on the relative susceptibility of some lymnaeids to infection with Fmciola hepatica and F. gigantica and on the adaptation of Fasciola spp. Annals of Tropical Meclicine and Parasitology 60, 114-124. CLARKE, A. H. 1973. The freshwater molluscs of the Canadian Interior Basin. Malacologia 13, l-509. CORT, W. W., AMEEL, D. J., AND OLIVIER, L. 1944. An experimental study of the development of Schistosomatium douthitti (Cort, 1914) in its intermediate host. Journal of Parasitology 30, l-17. CORT, W. W., MCMULLEN, D. B., AND BRACKETT, S. 1937. Ecological studies on the cercariae in Stagnicokz emarginata angulata (Sowerby) in the Douglas Lake region, Michigan. Journal of Parasitology 23, 504-532. EL-GINDY, M. S. 1950. “Biology of Schistosomatium douthitti (Cort, 1914) Price, 1931 (Trematoda) in Its Hosts.” Unpublished dissertation, University of Michigan, Ann Arbor. FAUST, E. C. 1924. The reactions of the miracidia of Schistosoma japonicum and S. haematobium in the presence of their intermediate hosts. 10, 199-204. Journal of Parasitology KAGAN, I. G., SHORT, R. B., AND NEZ, M. N. 1954. Maintenance of Schistosomatium douthitti (Cort, 1914) in the laboratory (Trematoda: Schistosomatidae). Journal of Parasitology 40, 424-439. LENGY, J. 1962. Studies on Schistosoma bovis (Sonsino, 1876) in Israel. I. Larval stages from egg to cercaria. Bulletin of the Research Council of Israel ZOE, l-36. LIM, H. K., AND HEYNEMAN, D. 1972. Intramolluscan intertrematode antagonism: A review of factors influencing the host-parasite system and its possible role in biological control. Advances 10, 191-268. in Parasitology Lo, C. T. 1972. Compatibility and host-parasite relationships between species of the genus BuZinus ( Basommatophora: Planorbidae) and an Egyptian strain of Schistosoma haematobium (Trematoda: Digenea). Malacologia 11, 225280. MALDONADO, J. F. 1967. “Schistosomiasis in America.” Cientifico Medica, Barcelona.

Schistosomatium

douthitfi:

MALEK, E. A. 1950. Susceptibility of the snail Biomphalaria boissyi to infection with certain strains of Schistosoma mansoni. American Journal of Tropical Medicine 30, 887-894.

MALEK, E. A. 1977. Geographical distribution, hosts, and biology of Schistosomutium douthitti (Cort, 1914) Price, 1931. Canadian Journal of Zoology 55, 661-671. NEWTON, W. L. 1953. The inheritance of susceptibility to infection with Schistosoma mansoni in Australorbis glabratus. Experimental Parasitology 2, 242-257. PRICE, H. F. 1931. Life history of Schistosomatium douthitti (Cort). The American Journal of Hygiene 13, 685-727. SHORT, R. B. 1952a. Sex studies on Schistosomatium douthitti (Cort, 1914) Price, 1931 (Trematoda: Schistosomatidae ) . The American Midland Naturalist 47, 1-54. SHORT, R. B. 1952b. Uniparental miracidia of

SNAILS'

ACE

SUSCEPTIBILITY

73

douthitti and their progeny (Trematoda: Schistosomatidae). The American

Schistosomatium

Midland Naturalist 48, 55-68. SUDDS, R. H. 1960. Observations

of schistosome miracidial behavior in the presence of normal and abnormal snail hosts and subsequent tissue studies of these hosts. Journal of the Elisha Mitchell Science Society 76, 121-133. ULMER, M. J. 1970. Notes on rearing snails in the laboratory. In “Experiments and Techniques in Parasitology” (A. J. MacInnis and M. Voge, eds.), pp. 143-144. W. H. Freeman, San Francisco. WAJDI, N. 1966. Penetration by the miracidia of S. mansoni into the snail host. Journal of Helminthology 15, 235-244. WALTER, H. J. 1969. Illustrated biomorphology of the “angulata” lake form of the Basommatophoran snail Lymnaea catascopium Say. Malacological Reuiew 2, I-102.