Excystation of the Metacercariae

In vivo and ectopic encystment of the cercariae of Echinostoma revolutum from Lymnaea elodes snails in Indiana and chemical excystation of the metacercariae were studied. In vivo encystment occurred in adults of Biomphalaria glabrata and Helisoma trivolvis (Colorado strain) snails and in neonatal and juvenile L. elodes snails. These results were expected because 37-collar-spined Echinostoma species show broad specificity in their second intermediate gastropod hosts. Encysted metacercariae of E. revolutum and Echinostoma trivolvis removed from experimentally infected snails and treated in a trypsin-bile salts excystation medium at 39 C showed 30.3% excystation for the former and 55.7% for the latter at 4 hr. The ducts and openings of the paraesophageal glands of excysted metacercariae of E. revolutum from cysts formed in snails did not stain with neutral red. Abnormal ectopic cysts with distorted outer walls and granular inner walls were obtained within 48 hr of placing E. revolutum cercariae in Locke's 1:1 plus 1% dextrose. These metacercariae excysted rapidly in the excystation medium and their paraesophageal gland ducts and openings stained with neutral red. Differences in ectopic encystment and chemical excystation in vitro can be used to distinguish these closely related species in the E. revolutum complex. Sorensen et al. (1997) described a strain of Echinostoma revolutum from Lymnaea elodes snails in Indiana. Cercariae released from the lymnaeids encysted in various pulmonate snails and the cysts when fed to domestic chicks yielded ovigerous adults. This was the first unequivocal report of E. revolutum in North America. The identity, systematic status, and biology of the larval and adult stages of E. revolutum were reviewed by Kanev (1994). Experimental studies on larvae and adults of E. revolutum are sparse compared to those on Echinostoma trivolvis, a related 37-collar-spined species from Helisoma trivolvis snails in North America. The identity of this species and a redescription of larval and adult stages have been reported by Kanev et al. (1995). The E. trivolvis-H. trivolvis model has been used extensively in the U.S.A. (see Huffman and Fried, 1990, for review) and information is available on cercarial longevity (Pechenik and Fried, 1995), cercarial encystment in pulmonate snails (Anderson and Fried, 1987; Fried et al., 1995; Schmidt and Fried, 1996a), on chemical excystation of the metacercariae (Smoluk and Fried, 1994), and on ectopic encystment of the cercariae (Fried and Bennett, 1979); however, similar studies on E. revolutum are not available. To increase our knowledge of the biology of 37-collar-spined echinostomes in the E. revolutum complex, studies on in vivo and ectopic encystment and chemical excystation of the metacercariae of E. revolutum were done and compared with those on E. trivolvis. MATERIALS AND METHODS Cercariae of E. revolutum were obtained from 8 experimentally infected L. elodes snails, 20-30 mm in shell length. Each snail was isolated in a Stender dish with 5 ml of artificial spring water (ASW) as described in Schmidt and Fried (1996b). From 100 to 500 cercariae per snail were obtained within 4 hr. Cercariae were pooled from at least 2 snails and used within 4 hr postemission. To obtain in vivo encysted metacercariae, 10 Biomphalaria glabrata and 20 H. trivolvis (Colorado [CO] strain) (both labreared and 6-8 mm in shell diameter) were each exposed to 25 cercariae in multiwell chambers (Schmidt and Fried, 1996a), necropsied 24 hr later, and the cysts were counted. In 1 experiment, 10 lab-reared juvenile L. elodes snails, 1-2 mm in shell length, were each exposed to 25 cercariae. In another Received 25 June 1996; revised 4 October 1996; accepted 4 October 1996. * Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907. experiment (en masse exposure), about 25 newly hatched (neonatal) L. elodes, 1 ? 0.2 mm in shell length, were maintained for 2 days in 800 ml of ASW with 2 adult L. elodes that were emitting cercariae. Cercarial encystment in a representative sample of 10 of the newly hatched lymnaeids was determined on the second day. Cercarial longevity studies were done in ASW or ASW with 1% dextrose at 22-24 C and 4 C. Longevity was based on cercarial movement or response to a needle. The infectivity of aged cercariae was tested by placing them at room temperature in multiwell chambers containing 1.5 ml of ASW with H. trivolvis (CO strain, 5 mm in shell diameter). Three snails per solution were exposed individually to 25 cercariae and necropsied 24 hr postinfection (PI). Ectopic encystment on 25 E. revolutum cercariae/dish was studied in 6-cm diameter petri dishes containing 15 ml of either Locke's, Locke's plus 1% dextrose, 1:1 Locke's, 1:1 Locke's plus 1% dextrose, ASW, or ASW plus 1% dextrose. Ectopic encystment was also tested in the presence of mucus from B. glabrata, H. trivolvis (Pennsylvania and CO strains), and L. elodes. Mucus was collected by placing 2 snails of each species in 0.5 ml of ASW in a 6-cm diameter petri dish for 1 hr. To examine excystation of ectopic cysts, 75 were treated in the chemical excystation medium of Fried and Roth (1974) as described below. To examine chemical excystation of cysts formed in vivo, a total of 300 encysted metacercariae of E. revolutum was treated in the alkaline trypsin-bile salts medium of Fried and Roth (1974) at 39 C and similar experiments were done with E. trivolvis cysts. In the present study, cysts of both species were used within 2 wk of cercarial encystment in experimentally infected H. trivolvis (CO) snails. In each of 3 trials, 100 E. revolutum and 100 E. trivolvis cysts were placed in 4 ml of the medium, incubated at 39 C for 4 hr, and the number of activated, breached, and excysted metacercariae was determined.