The Protective Action of Chaetogaster Limnaei on Snails Exposed to Schistosma Mansoni.

Experiments demonstrated that infestation with the oligochaete worm, Chaetogaster limnaei, afforded a degree of protection to the snail Australorbis glabratus when the snail was exposed to Schistosoma mansoni miracidia. To a lesser extent, the oligochaetes protected snails exposed to echinostome cercariae. The presence of C. limnaei within the kidney of the snail Physa heterostropha is reported. An intimate association between the oligochaete worm Chaetogaster limnaei von Baer and various species of aquatic snails has long been recognized (von Baer, 1827), yet the nature of this relationship has not been adequately defined. Early naturalists considered C. limnaei to be a true parasite which was thought to feed upon the "slime" produced by the host. However, Wagin (1931) and subsequent investigators demonstrated that the oligochaete fed principally on microorganisms, and thus the worm has been considered a commensal. Wagin also observed that the oligochaete ingested cercariae and suggested that C. limnaei might be of value in controlling trematode transmission. While Krasnodebski (1936) confirmed Wagin's field observations and experimentally demonstrated that the oligochaete would ingest various types of cercariae, he did not believe that Chaetogaster could play a significant role in the control of trematodes. Backlund (1949) observed that cercariae of Fasciola hepatica were ingested by Chaetogaster, and Ruiz (1951) later reported a similar observation with respect to Schistosoma mansoni. Coelho (1957) noted that Chaetogaster frequently ingested S. mansoni miracidia, but commented that the worms did not protect snails from infection. HowReceived for publication 3 March 1964. * These studies were supported in part by Grants AI-00513 and 5 TI AI 46 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Public Health Service, Bethesda, Maryland. 441 ever, Khalil (1961) presented circumstantial evidence suggesting that snails infested with C. limnaei were refractory to infection with Fasciola hepatica under laboratory conditions. The present study was initiated to determine quantitatively whether Australorbis glabratus infested with C. limnaei were protected against invasion by miracidia of S. mansoni and by cercariae of an echinostome. MATERIALS AND METHODS Specimens of C. limnaei were obtained from Physa heterostropha collected in Boston. The oligochaetes were freed by immersing the snails in 1% Urethan (ethyl carbamate) for 3 to 5 min. Anesthetized worms were collected by pipette, allowed to recover in charcoal-filtered tap water, and used immediately or colonized for later use (Brandwein, 1937). A Puerto Rican strain (PR-1) of Australorbis glabratus was used in all experiments. This snail strain has been maintained in our laboratory for several years and has been free from infestation with C. limnaei. Miracidia were hatched from eggs obtained from the livers of mice infected with a Puerto Rican strain of S. mansoni and were used within 30 min of emergence (Michelson, 1964). Cercariae of an unidentified echinostome were obtained from Physa heterostropha collected from local ponds. Five C. limnaei were added to individually isolated A. glabratus in 5-ml beakers containing 3 ml of charcoal-filtered tap water. The snail was confined with the oligochaetes for 1 hr or until all worms became attached. At this time and depending upon the experiment, one to five miracidia or ten cercariae were added to each beaker. After an exposure of approximately 16 hr, the snails from any one experiment were maintained together in a battery jar containing 2.5 liters of water. The battery jars were continually aerated, maintained at 25 ?+ 1 C, and the snails fed Romaine lettuce. Con442 THE JOURNAL OF PARASITOLOGY, VOL. 50, NO. 3, JUNE 1964 TABLE I. Influence of Chaetogaster limnaei infestation on the rate of infection of snails exposed to S. mansoni miracidia and to echinostome cercariae. Snails with C. limnaeil Control snails Group no. No. positive/ No. positive/ no. exposed no. exposed 5 miracidia of S. mansoni per snail 12 2/7 4/8 22 7/18 14/20 32 1/5 5/6 4 5/10 8/10 5 2/10 10/10 Total 17/50 (34%) 41/54 (76%) 1 miracidium of S. mansoni per snail 6 1/10 5/10 7 2/15 7/15 Total 3/25 (12%) 12/25 (48%) 10 cercariae of echinostome sp. per snail 8 7/10 10/10 9 7/10 10/10 Total 14/20 (70%) 20/20 (100%) 1 All snails infested with 5 worms each. 2 Test groups 1, 2, and 3 had one, two, and one snails die before they could be examined for the presence of infection; these have not been included with the data. trol snails, free of C. limnaei, were exposed to miracidia or cercariae in the same manner. Two weeks postexposure, control and Chaetogaster-infested snails were examined for the presence of S. mansoni sporocysts by the technique of Chernin and Dunavan (1962). Snails exposed to echinostome cercariae were examined 7 days postexposure for the presence of metacercariae. For this purpose the snail was removed from its shell, the mantle cut along its left margin and reflected to the right, and the pallial cavity examined under a 13 X dissecting microscope. Experimental results In five experiments a total of 54 snails, each infested with five Chaetogaster limnaei, were exposed individually to five S. mansoni miracidia and 17 (34%) of 50 snails which survived were found infected (Table I). By comparison, in the control groups of snails 41 of 54 snails (76%) were infected. Thus, a mean reduction in the rate of infection of 42% was associated with the presence of C. limnaei. In two additional experiments (Table I), snails harboring five C. limnaei were exposed individually to a single miracidium of S. mansoni. Of a total of 25 snails so exposed, three (12%) became infected. An infection rate of 48% (12/25 snails) occurred, however, in the two groups of control snails. A mean reduction of 36% in the infection rate was again observed to be associated with the presence of C. limnaei. Snails were exposed to echinostome cercariae in two experiments (Table I), and all the control snails were later found infected. However, only 70% (14/20) of the snails infested with C. limnaei had metacercarial cysts. In the first experiment 38 and 58 metacercarial cysts were recovered from the Chaetogaster-infested and control snails respectively, and in the second experiment 22 and 69 cysts respectively. Infected snails were found to have from three to eight cysts each. Miscellaneous observations The intensity of Chaetogaster infestation in snails under field conditions would appear to vary considerably. Backlund (1949) observed that no more than ten Chaetogaster were found on specimens of Lymnaea stagnalis and L. ovata; however, Krasnodebski (1936) noted as many as 300 worms on a single specimen of L. stagnalis, up to 60 worms on Physa fontinalis, and a mean of 1.3 worms on specimens of Ancylus lacustris. In our own experience, as many as 25 C. limnaei have been recovered from a single Physa heterostropha. In one collection of 105 P. heterostropha there was a mean of nine worms per snail. The heaviest infestations were found on older and larger snails, particularly those which had overwintered. Quantitative data concerning natural infestations of Chaetogaster on the snail Auistralorbis glabratus are not available, although infestations of field populations have been reported (Coelho, 1957). Natural infestations of South African populations of Bulinus tropicus, Bulinus africanus, and Biomphalaria pfeifferi have also been noted by Bayer (1955). Likewise, laboratory colonies of A. glabratus, as well as Bulinus truncatus and B. tropicus, have been observed to be infested with Chaetogaster (Khalil, 1961). Although it has generally been thought that C. limnaei acts as a commensal of snails, feeding principally on aquatic microorganisms, there is some evidence to suggest that this oligochaete may also be a parasite. The observation of Lankester (1870) of C. limnaei in the kidney of a single L. stagnalis has been overlooked. Our examination of both field and laboratory populations of P. heterostropha MICHELSON-PROTECTIVE ACTION OF CHAETOGASTER ON SNAILS EXPOSED TO SCHISTOSOMA MANSONI TABLE II. Distribution of Chaetogaster limnaei in a collection of Physa heterostropha. Number C. limnaei present Snail no.1 On body surface and Inside in mantle cavity kidney