Egg laying in vitro of Echinostoma caproni (Trematoda) in nutritive and nonnutritive media

Egg laying in vitro was studied in Echinostoma caproni adults placed in 10 ml of nutritive or nonnutritive media for 48 h in petri-dish cultures maintained at 37°C in an atmosphere containing 7.6% CO2. Maximal egg laying occurred within 24 h in the defined medium RPMI 1640. Egg laying was significantly greater in this medium than in McCoy’s or Locke’s solution. Eggs released into the RPMI medium were capable of producing miracidia that were infective to Biomphalaria glabrata snails. Fried and Huffman (1996) referred to a technique used to obtain eggs of Echinostoma caproni in the defined medium RPMI 1640, but details of the study were not given. No information is available on egg laying of echinostomes in vitro. Such information could contribute to a better understanding of egg release in digeneans and would also be helpful in the acquisition of eggs for biology and chemistry studies. Current techniques used to obtain echinostome eggs involve worm homogenization, teasing of eggs from the worms’ uteri, or recovery of eggs from feces (see Idris and Fried 1996 for details). The purpose of this communication is to report on an efficient procedure for the acquisition of eggs of E. caproni after the placement of adult worms in the defined medium RPMI 1640. E. caproni adults were grown in ICR mice for either 17 (young worms) or 112 days (old worms) as described previously (Ursone and Fried 1995a). Worms were removed from the small intestines and rinsed rapidly in three changes of sterile Locke’s solution containing penicillin (200 IU/ml) and streptomycin (200 μg/ml; Fried and Contos 1973). Worms were placed in culture media within 30 min of their removal from hosts. Nutritive media consisted of RPMI 1640 and McCoy’s medium (Sigma, St. Louis, Mo.). Non-nutritive media consisted of Locke’s or Locke’s 1:1 (Ursone and Fried 1995b). All media contained antibiotics as described above. A total of eight experiments were done with four different media (Table 1). Half the experiments used young worms (17 days old) and the other half involved old worms (112 days old). Each experiment was done with three plastic petri-dish cultures (6-cm diameter) containing 10 ml of medium/culture. Either one, three, or five worms were placed in each dish and three replicates of each dish were used. Cultures were maintained at 37~ and the gas phase was 7.6% CO2. At 24 h postincubation the worms were removed to new cultures and the eggs in the petri dishes were counted. Some of these eggs were also used to obtain miracidia as described below. Worms in the new cultures were maintained for an additional 24 h and were then removed and dissected with needles to determine the residual number of eggs/worm (column 7, Table 1). Eggs released into the cultures on the 2nd day were also counted (column 6, Table 1). Data were obtained on the number (mean ± SE) of eggs released during the first 24 h in each of the 24 cultures (column 5, Table 1). Student’s t-test was used to compare significant differences (P < 0.05) in egg release noted between experiments on the 1 st day. All worms were alive and active in RPMI 1640 and McCoy’s for 48 h. Some worms were sluggish or moribund by 48 h in Locke’s or Locke’s 1:1. The egg release per worm was greatest in RPMI at 24 h and was similar in each experiment regardless of the number of worms used per culture (experiments A and B, Table 1). The egg release of old worms at 24 h was significantly greater in RPMI (experiment A) than in McCoy’s (experiment C), Locke’s (experiment E), or Locke’s 1:1 (experiment G). Likewise, the egg release of young worms at 24 h was significantly greater in RPMI (experiment B) than in McCoy’s (experiment D), Locke’s (experiment F), or Locke’s 1:1 (experiment H). There was a significant increase in the egg release of worms in Locke’s versus Locke’s 1:1 (experiment E versus experiment G; experiment F vs experiment H). According to Ulmer (1970), egg release of digeneans in vitro is thought to be inhibited by high salt concentrations. Our data, at least those obtained on E. caproni egg release in Locke’s versus Locke’s l: 1, contradict Ulmer’s statement. Data on residual egg counts (column 7, Table 1) show that by 48 h, worms in RPMI released most of their eggs (experiments A and B); however, this was not the case for worms in the other media (experiments C-H). Old worms just removed from hosts and dissected with needles averaged 300 ± 35 eggs/uterus (n = 10), whereas young worms treated in the same way averaged 200 ± 25 eggs/uterus (n = 10). That old and young worms released about 800 and 550 eggs, respectively, within 24 h in RPMI suggests that egg production continues in vitro in this RPMI medium. The mechanism of egg production in digeneans is poorly understood and merits further study. To test the viability of eggs laid in the RPMI medium, about 1000 eggs collected at 24 h postincubation from old worms were incubated in artificial spring water (Idris and Fried 1996) at 22–24° for 15 days; more than 99% of these eggs were either fully developed or had released their miracidia by this time. Some of these miracidia were used to expose Biomphalaria glabrata snails, and patent redial infections were attained at 5–7 weeks postincubation. In summary, eggs laid by E. caproni adults in RPMI 1640 medium provide an excellent source of larval material for studies on the biology and chemistry of this echinostome.